WO2008021552A2 - Biased ligands and methods of identifying same - Google Patents

Biased ligands and methods of identifying same Download PDF

Info

Publication number
WO2008021552A2
WO2008021552A2 PCT/US2007/018394 US2007018394W WO2008021552A2 WO 2008021552 A2 WO2008021552 A2 WO 2008021552A2 US 2007018394 W US2007018394 W US 2007018394W WO 2008021552 A2 WO2008021552 A2 WO 2008021552A2
Authority
WO
WIPO (PCT)
Prior art keywords
arrestin
disease
gpcr
receptor
disorder
Prior art date
Application number
PCT/US2007/018394
Other languages
French (fr)
Other versions
WO2008021552A9 (en
WO2008021552A3 (en
Inventor
Robert J. Lefkowitz
Keshava Rajagopal
Erin J. Whalen
Matthew T. Drake
Jonathan D. Violin
Howard A. Rockman
Scott M. Dewire
Original Assignee
Duke University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Duke University filed Critical Duke University
Publication of WO2008021552A2 publication Critical patent/WO2008021552A2/en
Publication of WO2008021552A9 publication Critical patent/WO2008021552A9/en
Publication of WO2008021552A3 publication Critical patent/WO2008021552A3/en

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

  • the present invention relates to G protein-coupled receptor ligands having a relative efficacy for stimulating ⁇ -arrestin/G protein-coupled receptor kinase (GRK) function (e.g., signaling) that is greater than their relative efficacy for stimulating G-protein signaling function.
  • GRK ⁇ -arrestin/G protein-coupled receptor kinase
  • These ⁇ -arrestin/GRK "biased ligands" can act as agonists or antagonists of G protein-mediated signaling and agonists of ⁇ -arrestin/GRK-mediated signal transduction.
  • the invention further relates to methods of identifying such ligands and to methods of using same in a variety of therapeutic settings.
  • G protein-coupled receptors also called seven transmembrane receptors (7TMRs)
  • 7TMRs seven transmembrane receptors. These receptors regulate virtually all known physiological processes in mammals. Moreover, they are the most common targets of currently used therapeutic drugs.
  • Two classical examples of drugs that target these receptors are “ ⁇ -blockers” and “angiotensin receptor blockers” (ARB 's), which block or antagonize the ⁇ -adrenergic receptor for adrenaline or the receptor for angiotensin, respectively.
  • ⁇ -blockers and “angiotensin receptor blockers” (ARB 's)
  • ARB 's angiotensin receptor blockers
  • These two classes of drugs are among the most widely used medications for the treatment of a variety of cardiovascular illnesses, including hypertension, heart failure and coronary artery disease.
  • Drugs that target GPCRs have been developed based on a signaling paradigm in which stimulation of the receptor by an agonist (e.g., adrenaline) leads to activation of a heterotrimeric "G protein", which then leads to second messenger stimulated signaling (e.g., via cAMP) and changes in physiological function (e.g., heart rate).
  • an agonist e.g., adrenaline
  • second messenger stimulated signaling e.g., via cAMP
  • physiological function e.g., heart rate
  • “Blockers” competitively antagonize these actions.
  • the stimulatory effects of agonists like adrenaline and angiotensin II on their respective receptors are also rapidly attenuated by a physiological process called “desensitization” (Koch et al, Annu. Rev. Physiol. 62:237 (2000)).
  • G protein-coupled receptor kinase GRK
  • ⁇ -arrestin G protein-coupled receptor kinase
  • the present invention results, at least in part, from the finding that the GRK and ⁇ -arrestin mechanism not only desensitizes G protein signaling but also leads to signaling in its own right.
  • the signaling pathways that have been identified include certain important enzymatic cascades, such as the ERK MAP kinases, PI3 kinase, and Akt, as well as others (Maudsley et al, J. Biol. Chem. 275:9572 (2000); Luttrell et al, Proc. Natl. Acad. Sci. USA 98:2449 (2001); Pierce et al, Oncogene 20:1532 (2001); Wei et al, Proc. Natl. Acad. Sci.
  • the invention provides compounds ( ⁇ -arrestin/GRK biased ligands) with a unique profile. They can act as agonists or antagonists of G protein-mediated signaling and as agonists of G-protein independent ⁇ -arrestin/GRK-mediated signal transduction. Thus, while these compounds can act as "blockers" in the classical sense, they can also stimulate potentially cell protective pathways.
  • the invention further provides methods of identifying compounds with this unique profile, that is, compounds that can promote ⁇ -arrestin-mediated signaling.
  • the invention additionally provides treatment methods based on the use of these biased ligands (such treatment methods being applicable to human and non- human animals).
  • the present invention relates to ⁇ -arrestin/GRK biased ligands that can act as agonists or antagonists of G protein-mediated signaling and as agonists of ⁇ - arrestin/GRK-mediated signal transduction (agonists biased for ⁇ -arrestin signaling can be either G protein independent or can show greater ⁇ -arrestin than G protein signaling).
  • the invention also relates to methods of identifying such biased ligands and to methods of using the biased ligands for the treatment of disease. Objects and advantages of the present invention will be clear from the description that follows. BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. IA Figures IA- ID. ⁇ iAR-mediated transactivation of EGFR requires GRK phosphorylation sites but not PKA phosphorylation sites.
  • HEK 293 cells stably expressing WT ⁇ ,AR (Fig. IA), PKA ' ⁇ ,AR (Fig. IB), GRK ' ⁇ ,AR (Fig. 1C) or PKA ' GRK " ⁇ i AR (Fig. 1 D) and transfected with FLAG-EGFR are treated for 5 minutes with l ⁇ M dobutamine (Dob) and lO ⁇ M ICI 118,551 (ICI, ⁇ 2AR antagonist) and compared with no stimulation (NS) or lOng/mL EGF stimulation.
  • Dob dobutamine
  • ICI, ⁇ 2AR antagonist ICI, ⁇ 2AR antagonist
  • WT ⁇ iAR (Fig. IA) and PKA " ⁇ iAR (Fig. IB) induce increases in phospho-EGFR and phospho-ERKl/2 in response to treatment with Dob, while GRK " ⁇ iAR (Fig. 1C) and PKA ' GRK ' ⁇ iAR (Fig. ID) lack this effect; * p ⁇ 0.05 versus NS.
  • EGFR internalization following Dob or EGF stimulation for 30 minutes is visualized using confocal microscopic analysis of HEK 293 cells stably expressing the above ⁇ iAR mutants and transfected with EGFR-GFP. In the absence of agonist, EGFR-GFP is visualized on the membrane in each stable cell line (Figs.
  • FIGS 2A-2C ⁇ -arrestin is required for ⁇ iAR-mediated EGFR transactivation.
  • Fig. 2A HEK 293 cells stably expressing WT ⁇ iAR, PKA ' ⁇ iAR or GRK " ⁇ iAR are transfected with GFP- ⁇ -arrestin. In the absence of agonist, GFP- ⁇ -arrestin (green) has a cytosolic distribution (panels 1, 2, 3).
  • Agonist stimulation results in redistribution of GFP- ⁇ -arrestin to the membrane in cells expressing WT ⁇ iAR and PKA " ⁇ iAR (panels 4, 5, arrowheads), whereas no redistribution is observed in cells expressing GRK " ⁇ i AR (panel 6, arrows).
  • Fig. 2B HEK 293 cells stably expressing PKA " ⁇ iAR are transfected with FLAG- EGFR alone (MOCK) or with siRNAs targeting ⁇ -arrestinl (si- ⁇ arrl), ⁇ -arrestin2 (si- ⁇ arr2), ⁇ -arrestinl/2 (si- ⁇ arrl/2) or scrambled siRNA (si-Control).
  • HEK 293 cells stably expressing WT ⁇ iAR are transfected with EGFR-GFP and si-Control or si- ⁇ arrl/2 to knock down expression (side panel).
  • EGFR-GFP is located at the membrane (panels 1, 5, arrows), while EGF stimulation induces EGFR-GFP redistribution into aggregates (panels 4, 8).
  • FIGS 3A-3F GRK5 and 6 are required for ⁇ -arrestin-mediated EGFR transactivation.
  • Fig. 3A Stably expressing WT ⁇ i AR and GRK * ⁇ iAR cells transfected with constitutively active ⁇ -arrestin, r ⁇ arrR169E, both respond to Dob stimulation by increasing association of ⁇ -arrestin and AP2.
  • Fig. 3B GRK " ⁇ iAR cells are transfected with EGFR-GFP, localized to the plasma membrane (panel 1 , arrows), and YFP-r ⁇ arrR169E, localized in the cytosol (panel 2, arrowheads).
  • EGF stimulation induces redistribution of EGFR-GFP from plasma membrane to aggregates (panel 7, arrows) with no effect on YFP-r ⁇ arrR169E (panel 8, arrowheads).
  • Dob stimulation results in redistribution of YFP- r ⁇ arrRl 69E from the cytosol to the plasma membrane (panel 5, arrowheads) with no change in EGFR-GFP distribution (panel 4, arrows).
  • Fig. 3C Transfection of GRK " ⁇ i AR cells with constitutively active r ⁇ arrRl 69E does not restore EGFR transactivation in response to Dob stimulation.
  • Fig. 3D Transfection of GRK " ⁇ i AR cells with constitutively active r ⁇ arrRl 69E does not restore EGFR transactivation in response to Dob stimulation.
  • HEK 293 cells transiently expressing PKA ' ⁇ iAR are transfected with FLAG-EGFR alone (MOCK) or with siRNAs targeting ubiquitous GRKs (siGRK2, siGRK3, siGRK5 and siGRK ⁇ ) or a scrambled siRNA sequence (si-Control).
  • Figs. 3E, 3F Summary of 6 independent experiments showing significant inhibition of EGFR transactivation (Fig. 3E) and ERK 1/2 activation (Fig. 3F) upon Dob stimulation in the cells transfected with siRNA targeting GRK5 or 6; *p ⁇ 0.001 versus Dob- stimulated Mock, si-Control, si-GRK2 and siGRK3.
  • ⁇ -arrestin2 knockout mice ⁇ arr-2 KO, Fig. 4A
  • GRK5 KO mice Fig. 4B
  • GRK6 KO mice Fig. 4C
  • WT wild-type littermate controls
  • ⁇ -arrestin2 knockout mice ⁇ arr-2 KO, Fig. 4A
  • GRK5 KO mice Fig. 4B
  • GRK6 KO mice Fig. 4C
  • WT wild-type littermate controls
  • Myocardial lysates are immunoblotted with anti-phospho- and anti-total- ERK 1/2 antibodies (lower panels).
  • Accompanying histograms show summary data of 6 independent experiments depicting the fold increase ERK 1/2 phosphorylation following Dob treatment; * p ⁇ 0.05 versus ICI.
  • Dob-mediated ERK 1/2 phosphorylation is completely blocked in ⁇ arr
  • FIG. 5A Cardiac characteristics of transgenic mice overexpressing mouse WT ⁇ , AR, GRK “ ⁇ iAR or PKA " ⁇ iAR.
  • Fig. 5B Myocardial expression levels of ⁇ AR are
  • Figs. 5D, 5E Immunoblotting of left ventricular lysates of NTG and the 3 lines of ⁇ i AR TG mice given intraperitoneal injections of Dob (lmg/kg, 10 min) or EGF (30 ⁇ g/kg, 15 min) reveals increased ERK1/2 (Fig. 5D) and Akt (Fig. 5E) phosphorylation in NTG, WT ⁇ jAR TG and PKA " ⁇ iAR TG mice. TG overexpression of GRK " ⁇ i AR prevents Dob-mediated
  • FIGS. 6A-6G Deterioration of cardiac function in GRK " ⁇ i AR-TG mice following chronic ISO treatment.
  • Fig. 6A WT ⁇ iAR TG and NTG mice show ⁇ AR downregulation in the LV membrane fraction following chronic ISO treatment (1 week) whereas GRK " ⁇ i AR TG and PKA " ⁇ iAR TG mice do not; f p ⁇ 0.01 versus vehicle in corresponding group.
  • Fig. 6B Deterioration of cardiac function in GRK " ⁇ i AR-TG mice following chronic ISO treatment.
  • Adenylyl cyclase activity following acute ISO-stimulation is enhanced in all vehicle-treated mice, but desensitized in all chronic ISO-treated NTG and the 3 lines of ⁇ i AR TG mice; *p ⁇ 0.05 versus vehicle-treated NTG ISO, JpO.05 versus vehicle-treated WT ⁇ iAR TG and PKA " ⁇ iAR TG ISO, tp ⁇ 0.01 versus vehicle-treated ISO in each corresponding group.
  • Fig. 6C Representative M-mode echocardiography pre- and post-chronic ISO in ⁇ IAR-TG mice and NTG littermates, with bar scales of 5 mm (vertical) and 1 sec (horizontal).
  • Fig. 6D Percent changes from pre-ISO treatment in LV end-diastolic dimension (Fig. 6D) and fractional shortening (FS, Fig. 6E) indicate significant LV dilatation and decreased FS in GRK ' ⁇ iAR TG mice following chronic ISO treatment; J p ⁇ 0.01 versus all TG groups, *p ⁇ 0.05 versus NTG littermates.
  • Fig. 6F Representative HE, MT and TUNEL staining following chronic ISO treatment reveal increased interstitial fibrosis (blue stain, MT panels) and apoptosis (arrows, TUNEL panels) in GRK " ⁇ iAR TG mice.
  • FIGS. 7A-7E Pharmacological inhibition of EGFR caused dilated cardiomyopathy following chronic ISO treatment.
  • Figs. 7B, 7C Pharmacological inhibition of EGFR caused dilated cardiomyopathy following chronic ISO treatment.
  • Fig. 7A NTG mice are pretreated for 1 hour with Erlotinib (20mg/kg, EGFR antagonist) or DMSO (10%, Control) intraperitoneally, following which Dob (lm
  • TUNEL staining following chronic ISO with or without Erlotinib shows increased apoptosis (arrowheads) in LV sections from NTG mice undergoing chronic ISO with Erlotinib treatment, as described above.
  • Figures 8A-8F Dob-stimulated EGFR transactivation and ERKl/2 activation requires ⁇ iAR expression and is sensitive to EGFR inhibition and ⁇ - arrestin function.
  • ⁇ iAR-expressing U2S osteosarcoma cells induce EGFR and ERK 1/2 phosphorylation upon stimulated with Dob (l ⁇ M, 5 min) which is prevented by pretreatment AG1478 (1 ⁇ M, EGFR antagonist).
  • Fig. 8B HEK 293 cells lacking endogenous ⁇ iAR expression (top panel) do not elicit phosphorylation of ERK 1/2, whereas those transfected with WT ⁇ iAR elicit a Dob-stimulated ERKl/2 response that is sensitive to EGFR inhibition.
  • Fig. 8C HEK cells transfected with WT ⁇ iAR are unable to elicit a Dob-stimulated ERKl/2 response in the presence of siRNA targeting either ⁇ -arrestinl or 2.
  • Fig. 8D Plot indicates the linear relationship between percentage knockdown of ⁇ -arrestins and the amount of EGFR transactivation following Dob stimulation of PKA ' ⁇ lAR cells.
  • Figs. 8E, 8F Upper panels, HEK 293 cells stably expressing either WT ⁇ ,AR (Fig. 8E) or PKA ' ⁇ iAR (Fig. 8F) and transfected with FLAG- EGFR and ⁇ Arrl V53D which mimics a constitutively phosphorylated, dominant negative state of ⁇ -arrestin. Lower panels, WT ⁇ iAR (Fig. 8E) or PKA ' ⁇ iAR (Fig.
  • FIGS. 9A-9C Transient ⁇ l AR expression is sufficient to enable ⁇ - arrestin- and GRK-dependent EGFR transactivation and ERKl/2 activation.
  • Figs. 9 A HEK 293 cells transiently expressing WT ⁇ iAR are transfected with FLAG-EGFR alone (MOCK) or with siRNAs targeting GRK 2, 3, 5 or 6. GRK siRNAs reduce Dob-stimulated phospho-ERKl/2 and phospho-EGFR and -ERK1/2, respectively.
  • Fig. 9B The expression of cells transiently expressing WT ⁇ iAR are transfected with FLAG-EGFR alone (MOCK) or with siRNAs targeting GRK 2, 3, 5 or 6. GRK siRNAs reduce Dob-stimulated phospho-ERKl/2 and phospho-EGFR and -ERK1/2, respectively.
  • Fig. 9B Fig. 9B.
  • HEK 293 cells transiently expressing WT ⁇ iAR and FLAG-EGFR are treated with pertussis toxin (PTX, Gi protein inhibitor), H89 (PKA inhibitor) and siRNA targeting GRK5 or GRK6.
  • PTX pertussis toxin
  • Gi protein inhibitor H89
  • PKA inhibitor H89
  • siRNA targeting GRK5 or GRK6 Dob-mediated phospho-EGFR and -ERK 1/2 are only prevented by GRK5 or GRK6 siRNA knockdown.
  • Fig. 9C HEK 293 cells stably expressing WT ⁇ i AR or WT AT, A R with or without transient transfection of FLAG-EGFR are treated with Dob, EGF or angiotensin II (All, l ⁇ M, 5 min) with or without AG 1478.
  • Dob-induced phospho-EGFR is detectable only with FLAG-EGFR transfection in WT ⁇ iAR cells, while ERK 1/2 phosphorylation occurs with either endogenous EGFR or transient FLAG-EGFR expression and is sensitive to AG 1478. All does not induce EGFR phosphorylation in WT AT 1 A R cells and All-induced ERK 1/2 phosphorylation is insensitive to AGl 478.
  • FIG. 10 In vivo transactivation of EGFR and activation of ERK1/2 requires GRK5 or GRK6 and ⁇ -arrestin2.
  • Myocardial lysates are immunoprecipitated with anti-EGFR antibody and blotted for phospho- and total-EGFR.
  • Accompanying histograms show summary data of 6 independent experiments depicting the fold increase EGFR phosphorylation following Dob treatment; * p ⁇ 0.05 versus ICI.
  • Dob-mediated EGFR phosphorylation is blocked in ⁇ arr-2 KO, GRK5 and GRK6 KO mice compared to WT littermate controls.
  • Fig. HA Schematic depicting the location of serine and threonine residues mutated in the 3 rd intracellular loop and C-terminal tail of WT ⁇ iAR in order to generate PKA * ⁇ i AR and GRK " P 1 AR, respectively, for the generation of the 3 lines of ⁇ iAR TG mice.
  • Fig. 1 IB WT ⁇ iAR TG, PKA " ⁇ iAR TG and GRK " ⁇ iAR TG mice are pretreated with Erlotinib followed by intraperitoneal injections of Dob or EGF as described in Figure 7.
  • GRK " ⁇ iAR TG mice do not elicit ERK 1/2 phosphorylation in response to Dob, while WT ⁇ iAR TG and PKA " ⁇ iAR TG do increase phospho-ERKl/2, a response that is completely blocked with pretreatment with Erlotinib.
  • Figure 12 ⁇ -arrestin translocation vs. G protein activation.
  • This graph shows the relative properties of the noted compounds for biologic responses.
  • the vertical axis shows recruitment of ⁇ -arrestin proteins, while the horizontal axis shows the generation of c AMP.
  • Compounds with ethyl substitutions on the ⁇ - carbon such as cyclopentylbutanephrine, isoetharine, and ethylNE are relatively better for GRK activity compared to their cAMP production, and thus are above the line.
  • FIGS 13A-13C Application of the GRK activity assay to high- throughput 7TMR ligand studies.
  • Figures 14A and 14B Measuring GRK function for biased ligands for the
  • FIGS 15A-15C Recruitment of ⁇ -arrestin to the ⁇ 2 AR reported by FRET.
  • Fig. 15 A P 2 AR-InCFP co-expressed with ⁇ -arrestin2-mYFP undergoes FRET upon agonist-stimulated phosphorylation of the receptor.
  • Fig. 15B P 2 AR-InCFP co-expressed with ⁇ -arrestin2-mYFP undergoes FRET upon agonist-stimulated phosphorylation of the receptor.
  • FRETc the FRET image corrected for spectral bleed-through at each pixel, is detected 15 minutes after addition of 1 ⁇ M isoproterenol, and corresponds to translocation of ⁇ -arrestin2-mYFP to membrane.
  • %F is displayed as a pseudocolor spectrum, intensity-modulated to correspond to ⁇ 2 AR-mCFP fluorescence to differentiate free ⁇ 2 AR (no FRET, blue) and ⁇ -arrestin- ⁇ 2 AR complex (high FRET, red).
  • Fig. 15C intensity-modulated to correspond to ⁇ 2 AR-mCFP fluorescence to differentiate free ⁇ 2 AR (no FRET, blue) and ⁇ -arrestin- ⁇ 2 AR complex (high FRET, red).
  • FRET is quantified as a percentage of whole-cell total CFP-excited fluorescence (%F), and shows monophasic kinetics for both isoproterenol-stimulated recruitment and ensuing dissociation by propranolol. Data is representative of 3 independent experiments.
  • FIGS 16A-16C ⁇ 2 AR-mCFP has two binding affinities for ⁇ -arrestin2- mYFP.
  • Fig. 16A At low expression of ⁇ -arrestin2-m YFP (R.F.U.: relative fluorescent units of YFP) isoproterenol-stimulated association is monophasic with a half-time of 90 seconds; at higher expression levels, association is biphasic, with half times of 2 and 90 seconds. Data is from 5 individual cells representative of 25 from 6 separate experiments.
  • Fig. 16B The expression of ⁇ -arrestin2-m YFP (R.F.U.: relative fluorescent units of YFP) isoproterenol-stimulated association is monophasic with a half-time of 90 seconds; at higher expression levels, association is biphasic, with half times of 2 and 90 seconds. Data is from 5 individual cells representative of 25 from 6 separate experiments.
  • Fig. 16B At low expression of ⁇ -arrestin2-m YFP
  • a phosphorylation-deficient ⁇ 2 AR- mCFP exhibits a rapid association similar to wild-type ⁇ 2 AR-mCFP, but is markedly impaired in the slow association, consistent with a phosphorylation- independent rapid association and phosphorylation-dependent slow association.
  • Data is average +/- s.e.m. of 3 separate experiments.
  • Fig. 16C The slow association is much higher affinity than the fast association, as described by the fit of a saturable binding curve for each phase.
  • Data are from single cells from 6 independent experiments.
  • FIGS 17A and 17B A phosphorylation-independent ⁇ -arrestin mutant displays enhanced low-affinity binding.
  • Fig. 17A ⁇ -arrestin 1/2 recruitment is monophasic at moderate expression levels, but the recruitment of the phosphorylation-independent mutant ⁇ -arrestinl-mYFP R169E expressed at similar levels is biphasic, with an enhanced rapid recruitment.
  • Figures 18A and 18B The rate and magnitude of ⁇ -arrestin recruitment depend on agonist concentration.
  • Fig. 18A Cells stably expressing both ⁇ 2 AR- mCFP and ⁇ -arrestin2-mYFP were stimulated with varying concentrations of isoproterenol. Data is from one of 3 separate experiments.
  • Fig. 18B The rate (k obs ) of ⁇ -arrestin-mYFP recruitment is less sensitive to isoproterenol than the final amount of recruitment (%F max ). Data is average +/- s.e.m. of 3 independent experiments.
  • FIGS 19A-19D High-affinity ⁇ -arrestin2-mYFP binding is kinetically limited by GRKs.
  • Fig. 19A Overexpression of GRK2 increases the rate of ⁇ - arrestin2-mYFP recruitment compared to vector alone. Data is representative of 3 separate experiments.
  • Fig. 19B siRNA silencing of GRK6 reduces the rate of recruitment of ⁇ -arrestin 1-m YFP (wt siGRK6) compared to a scrambled siRNA (wt CTL), and reduces only the slow rate of phosphorylation independent ⁇ - arrestinl-mYFP (R169E siGRK ⁇ and R169E CTL) without affecting the rapid association. Data is average +/- s.e.m.
  • the effect of GRK6 siRNA is most profound, and was noted with two siRNA sequences (GRK6-1 and GRK6-2).
  • a representative immunoblot (IB) for each GRK shows the effectiveness of siRNA silencing. Data is average +/- s.e.m. from three independent experiments performed in triplicate.
  • the effect of GRK6 siRNA is most profound, and was noted with two siRNA sequences (GRK6-1
  • FIG. 20A-20C ⁇ 2 AR immunoprecipitation reveals distinct GRK specificity for ⁇ -arrestin association, bulk phosphorylation, and phosphorylation of a single site in the absence overexpressed ⁇ -arrestins.
  • FIGS 21A-21D U2-Osteosarcoma cells utilize a different set of GRKs for ⁇ -arrestin recruitment than HEK-293 cells as measured by FRET.
  • Fig. 2 IB Immunoblotting reveals that U2-OS cells express relatively more GRK3 than HEK-293 cells and less GRK6.
  • An immunoblot is shown for GRK2 and 3 to verify silencing. All data is average +/- s.e.m. of 3 independent experiments performed in triplicate. All immunoblots are representative of 3 independent experiments.
  • FIGS 22A-22C Immunoblot analysis of ⁇ -arrestin-mYFP overexpression. Equal amounts of protein were separated by SDS-PAGE, transferred to membrane, and immunoblotted with an anti- ⁇ -arrestinl/2 antibody.
  • Fig. 22A HEK-293 cells stably transfected with ⁇ 2 AR-mCFP and transiently transfected with either vector plasmid (pcDNA3.1) or ⁇ -arrestin2-mYFP reveal that transient transfection results in approximately 30-fold over overexpression of ⁇ -arrestin2-mYFP in comparison to endogenous ⁇ -arrestin.
  • Fig. 22B Immunoblot analysis of ⁇ -arrestin-mYFP overexpression. Equal amounts of protein were separated by SDS-PAGE, transferred to membrane, and immunoblotted with an anti- ⁇ -arrestinl/2 antibody.
  • Fig. 22A HEK-293 cells stably transfected with ⁇ 2 AR-mCFP and transiently transfected with
  • FIG. 23A High-resolution FRET shows ⁇ 2 AR-mCFP: ⁇ - arrestin-mYFP complex at the plasma membrane but not in vesicles containing internalized receptor.
  • FIG. 23A Prior to stimulation (top row), ⁇ 2 AR-mCFP (cyan) is found in plasma membrane, ⁇ -arrestin2-mYFP (yellow) is found in the cytosol, and there is almost no detectable FRETc (red, FRET image corrected for spectral overlap at each pixel).
  • a %F pseudocolor scale showing no FRET as blue and high FRET as red, displayed with intensity corresponding to ⁇ 2 AR- mCFP intensity, shows no ⁇ -arrestin2-mYFP: ⁇ 2 AR-mCFP complex.
  • ⁇ 2 AR- mCFP has visibly internalized into vesicles
  • ⁇ -arrestin2-mYFP has translocated to plasma membrane and punctate.
  • FRETc is detected on the plasma membrane but not on vesicles containing internalized ⁇ 2 AR-mCFP. This is most evident in the %F pseudocolor scale, indicating that ⁇ 2 AR-mCFP: ⁇ -arrestin2-mYFP complex is only present on plasma membrane.
  • Fig. 23B A magnified section of the image from Fig. 23 A shows the plasmalemmal-limited FRET.
  • the arrow points to a vesicle containing internalized ⁇ 2 ARmCFP but not ⁇ -arrestin2-mYFP, and lacking FRET.
  • the circle-ended arrow points to a punctate structure containing both ⁇ 2 AR-mCFP and ⁇ -arrestin2-mYFP and high FRET. This is most likely an aggregation of ⁇ 2 ARmCFP: ⁇ -arrestin2-mYFP complex prior to internalization and dissociation of the complex.
  • Figures 24 A and 24B ⁇ -arrestin2-mYFP translocation. Fig. 24 A.
  • ⁇ - arrestin2-mYFP translocation was assessed by defining translocation as the ratio of m YFP fluorescence at membrane regions over mYFP fluorescence at perinuclear cytosolic regions in U2-OS cells stably expressing both ⁇ 2 AR-mCFP and ⁇ -arrestin-mYFP.
  • This measure of ⁇ -arrestin2-mYFP redistribution is independent of FRET and thus corresponds to ⁇ -arrestin-mYFP.
  • This measure of ⁇ -arrestin2-mYFP redistribution is independent of FRET and thus corresponds to ⁇ -arrestin recruitment and not to ⁇ -arrestin or ⁇ 2 AR conformational changes.
  • FIG. 25 Effects of chronic SII administration on left ventricular systolic function in a genetic model of heart failure in ice [mice with myocardium-specific transgenic (Tg) over-expression of the calcium handling protein calsequestrin (CSQ); these mice develop "spontaneous" biventricular dysfunction and heart failure with a mean lifespan of 16 weeks].
  • Tg myocardium-specific transgenic
  • CSQ calcium handling protein calsequestrin
  • FIG. 26 Mobilization of calcium in response to ANG and SII.
  • Neonatal rat atrial cardiomyocytes were loaded with the calcium-binding dye Fura-2, and stimulated either with ANG ( 100 nM), SII (IO ⁇ M), or ANG in the presence of pretreatment with the ATiR antagonist valsartan (50 ⁇ M).
  • Calcium fluorimetric traces are shown, with the 340/3 80 nm excitation ratio (y-axis) plotted as a function of time (x-axis). Results displayed are mean ⁇ standard error of the mean (SEM) of 3 independent experiments.
  • FIGS. 27A-27C Contribution of specific GRK isoforms to agonist- induced recruitment of ⁇ -arrestin2 to the ATi A R- HEK293 cells stably expressing ATi A R-mCFP and ⁇ -arrestin2-mYFP were stimulated with ANG (100 nM) or SII (10 ⁇ M), in the setting of exposure to the indicated siRNA.
  • Fig. 27 A Modest effects of GRK2 or GRK6 deficiency on recruitment of ⁇ -arrestin2 to the ATi A R- Fig. 27B. Substantial effects of GRK6 deficiency, but not GRK2 deficiency, on recruitment of ⁇ -arrestin2 to the ATi A R.
  • Fig. 27C Representative immunoblot demonstrating efficacy of inhibition of GRK2 or GRK6 expression.
  • FIG. 28A AT ⁇ R-dependent changes in systolic and diastolic cardiomyocyte function mediated by the natural agonist ANG and the biased agonist SII; effects of antagonism of PKC on changes in systolic and diastolic cardiomyocyte function in response to ANG and SII.
  • Fig. 28A Absence of effects of ANG and SII on fractional shortening of cardiomyocytes from ATi A R KO mice.
  • FIGS. 29A-29F Effects of deficiency of ⁇ -arrestin2 on changes in systolic and diastolic cardiomyocyte function in response to ANG and SII.
  • Fig. 29A Absolute values for each variable under indicated stimulation conditions.
  • Fig. 29B Absolute values for each variable under indicated stimulation conditions.
  • Figs. 29C and 29D Percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment).
  • Fig. 29C Absolute values for each variable under indicated stimulation conditions.
  • Fig. 29D Percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment).
  • Figs. 29E and 29F Percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment).
  • ⁇ -arrestinl KO cardiomyocytes exhibit equivalent positive inotropic responses to ANG and SII.
  • Fractional shortening of cardiomyocytes from ⁇ -arrestinl KO mice (n 5 animals) under conditions of pacing alone (Basal), or additional exposure to 10 ⁇ M ANG or SII as indicated.
  • Fig. 29E Absolute values for each variable under indicated stimulation conditions.
  • Fig. 29F Percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment). *p ⁇ 0.05 by one-way ANOVA with post hoc Bonferroni test relative to Basal, and between ANG and SII when relevant (Figs. 29A/29C/29E; in Fig.
  • FIGS. 30A-30F Effects of deficiency of specific GRK isoforms on changes in systolic and diastolic cardiomyocyte function in response to ANG and SII.
  • Fig. 3OA Absolute values for each variable under indicated stimulation conditions.
  • Fig. 3OB Percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment). Figs.
  • Fig. 3OE Absolute values for each variable under indicated stimulation conditions.
  • Fig. 3OF Percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment). *p ⁇ 0.05 by one-way ANOVA with post hoc Bonferroni test relative to Basal, and between ANG and SII when relevant (Figs. 30A/30C/30E; in Fig. 30C, the * for ANG thus represents significance relative to both Basal and SII, whereas in Figs.
  • FIGS 31 and 3 IB ATi A R-dependent changes in systolic and diastolic cardiomyocyte function mediated by the natural agonist ANG and the biased agonist SII.
  • Fig. 3 IA Absence of effects of ANG and SII on -dL/dt max of cardiomyocytes from ATi A R KO mice.
  • mice under conditions of pacing alone (Basal), or additional exposure to 10 ⁇ M ANG or SII as indicated.
  • Fig. 33 A (Above) absolute values for each variable under indicated stimulation conditions; (Below)5 percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment).
  • Fig. 33B ⁇ -arrestin2 KO cardiomyocytes display severely defective positive inotropic and lusitropic +dL/dt m a x .
  • Fig. 33B (Above) absolute values for each variable under indicated stimulation conditions; (Below) percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment).
  • Fig. 33C ⁇ -arrestinl KO cardiomyocytes display equivalent positive inotropic and lusitropic responses to ANG and SII, as assessed by changes in -dL/dt max and +dL/dt m ax .
  • Fig. 33C (Above) absolute values for each variable under indicated stimulation conditions; (Below) percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment). *p ⁇ 0.05 by one-way ANOVA with post hoc Bonferroni test relative to Basal, and between ANG and SII when relevant (Above in all panels; in Fig.
  • FIG. 34A (Above) absolute values for each variable under indicated stimulation conditions; (Below) percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment).
  • Fig. 34B (Above) absolute values for each variable under indicated stimulation conditions; (Below) percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment).
  • FIGS 35A and 35B Effects of intravenous bolus doses of SII on systemic arterial hemodynamics.
  • Fig. 35A SII decreases basal (post-treatmeht) MAP.
  • Fig. 35B SII is a functional competitive ARB.
  • Figure 36 Effects of SII on left ventricular (LV) systolic function - LV dP/dW.
  • FIGs 39A and 39B cAMP responses monitored by ICUE2.
  • HEK-293 cells stably expressing ⁇ 2AR and the cAMP biosensor ICUE2 were treated for 2 minutes with a panel of ligands described as ⁇ 2AR antagonists (Fig. 39A).
  • cAMP agonism was measured as the rate of change of the ICUE2 FRET ratio corresponding to the rate of cAMP accumulation.
  • Ligands that did not induce cAMP generation were tested for inverse agonism in the same cells (Fig. 39B). These cells exhibit constitutive ⁇ 2AR activity that, while too weak to generate high basal cAMP, causes a rapid increase in cAMP when phosphodiesterases are inhibited with isoxybutylmethylxanthine (IBMX).
  • IBMX isoxybutylmethylxanthine
  • Inverse agonism was measured by pretreating cells with ligand for 5 minutes and quantifying the rate of cAMP increase for 30 seconds after IBMX treatment.
  • Inverse agonists are those ligands which do not stimulate cAMP accumulation on their own, and decrease the rate of IBMX- induced cAMP accumulation caused by constitutive receptor activity. Data represent mean ⁇ S. E. from five independent experiments. **p ⁇ 0.001 vs. non- stimulated cells (NS).
  • FIGs 4OA and 4OB ERK activation in ⁇ 2AR and ⁇ 2AR ⁇ stable cells.
  • HEK-293 cells stably expressing ⁇ 2AR (Fig. 40A) or ⁇ 2AR ⁇ (Fig. 40B) were stimulated with the panel of ⁇ 2AR ligands used in Figure 39 at 10 ⁇ M for 5 minutes and cell lysates were analyzed for pERK and ERK by Western blot.
  • pERK was normalized to total ERK protein. Data represent mean ⁇ S. E. of at least three independent experiments done in duplicate. Quantification of pERK bands is as a percentage of maximal activity observed for isoproterenol.
  • Figures 41 A and 4 IB ⁇ 2AR phosphorylation stimulated by carvedilol.
  • HEK-293 cells stably expressing ⁇ 2AR were stimulated with 10 ⁇ M of ligand for 30 minutes and cell lysates were either analyzed for receptor phosphorylation by Western blot (Fig. 41A) or lysates were immunoprecipitated with anti-FLAG beads and analyzed by 32 P metabolic labeling (Fig. 41B).
  • Data represent mean ⁇ S. E. of at least five independent experiments. *** p ⁇ 0.0001 vs. NS.
  • FIGS. 42 A and 42B ⁇ -arrestin2-GFP translocation to the ⁇ 2AR-V2R and receptor internalization stimulated by carvedilol.
  • HEK-293 cells transiently expressing the ⁇ 2AR-V2R chimera were stimulated for 2 minutes with either isoproterenol (Iso), carvedilol (Carv), or propranolol (Prop).
  • Iso isoproterenol
  • Carv carvedilol
  • Prop propranolol
  • HEK-293 cells stably expressing ⁇ 2AR were stimulated with 10 ⁇ M of ligand for 30 minutes and assayed for internalization by fluorescence-activated cell sorting (Fig. 42B). Data represent mean ⁇ S. E. of 5 independent experiments done in duplicate. * p ⁇ 0.05 vs. NS, *** p ⁇ 0.0001 vs. NS.
  • FIGS 43A and 43B Carvedilol stimulated ERK 1/2 phosphorylation is abolished by siRNA targeting ⁇ -arrestin2.
  • HEK-293 cells stably expressing ⁇ 2AR (Fig. 43A) or ⁇ 2AR ⁇ (Fig. 43B) were stimulated with 10 ⁇ M of Iso or Carv in the presence of either control siRNA (CTL) or siRNA targeting ⁇ - arrestin2 ( ⁇ arr2) for 5 minutes and cell lysates were analyzed for pERK, ERK and ⁇ -arrestin 1/2 by Western blot. Data represent mean ⁇ S.E. of four independent experiments done in duplicate. Quantification of pERK bands is as a percentage of maximal activity observed for isoproterenol. **p ⁇ 0.01, ***p ⁇ 0.001.
  • FIGS 44A and 44B Carvedilol stimulated ERK 1/2 phosphorylation is not inhibited by pertussis toxin.
  • HEK-293 cells stably expressing ⁇ 2AR (Fig. 44A) or ⁇ 2AR ⁇ (Fig. 44B) were stimulated with 10 ⁇ M of Iso or Carv for 5 minutes after treatment of either DMSO or pertussis toxin (PTX) for 16 hours as indicated.
  • Cell lysates were analyzed for pERK and ERK by Western blot. Data represent mean ⁇ S.E. of five (Fig. 44A) and four (Fig. 44B) independent experiments done in duplicate. Quantification of pERK bands is as a percentage of maximal activity observed for isoproterenol. **p ⁇ 0.01 vs. NS, ***p ⁇ 0.0001 vs. NS.
  • the present invention relates to ligands for GPCR' s (7TMR' s) that have a relative efficacy for stimulating ⁇ -arrestin/GRK function (e.g., signaling) that is greater than their relative efficacy for stimulating G protein signaling function.
  • ⁇ -arrestin/GRK "biased ligands" can act as agonists or antagonists for G protein-mediated signaling and agonists of ⁇ -arrestin/GRK-mediated signal transduction.
  • the invention further relates to methods of identifying such biased ligands and to methods of using biased ligands to treat a variety of diseases/disorders including, but not limited to, pulmonary and cardiovascular disease, allergies/allergic diseases, immunological diseases, psychiatric disorders, psychological disorders, dermatological diseases, neurological diseases, autonomic diseases, inflammatory diseases, endocrine or metabolic diseases (e.g., diabetes and obesity), genitourinary disorders, and opthamological diseases (e.g. glaucoma).
  • diseases/disorders including, but not limited to, pulmonary and cardiovascular disease, allergies/allergic diseases, immunological diseases, psychiatric disorders, psychological disorders, dermatological diseases, neurological diseases, autonomic diseases, inflammatory diseases, endocrine or metabolic diseases (e.g., diabetes and obesity), genitourinary disorders, and opthamological diseases (e.g. glaucoma).
  • the relative efficacies of biased ligands of the instant invention can be readily appreciated from the graph below where the vertical axis is a measure of ⁇ -arrestin/GRK-dependent signaling (e.g., recruitment of ⁇ -arrestin to the receptor as assayed, for example, by FRET (see Examples below)) and the horizontal axis is a measure of G-protein dependent signaling (e.g., generation of cAMP).
  • GPCR ligands that fall on the dashed (diagonal) line are "unbiased" in that, upon binding to the receptor, their relative effects on ⁇ -arrestin/GRK- dependent signaling and G-protein dependent signaling functions are essentially equivalent.
  • biasing ligands of the instant invention upon binding to the receptor, have a greater positive effect on ⁇ -arrestin/GRK-dependent function (e.g., ERK signaling, etc.) than on G-protein dependent signaling function and thus fall above and/or to the left of the dashed line and in the shaded portion of the graph (biased ligands of the invention include inverse agonists of G-protein dependent signaling, that is, ligands with negative efficacy for G-protein signaling falling to the left of the vertical axis and above the horizontal):
  • tjj o ⁇ r_-(G protein activity)-] r -( ⁇ -arrestin functionh
  • e base of the natural logarithm
  • G protein activity 2nd messenger activity measurement, as a percentage of the activity of a reference agonist
  • ⁇ -arrestin function percentage of a reference agonist function.
  • the reference agonist can be an endogenous ligand for the GPCR (wherein more than one endogenous ligand for the GPCR exists, the reference agonist is, preferably, the endogenous ligand of highest potency) or an exogenous ligand for the GPCR.
  • a reference agonist for the angiotensin II type 1 receptor is the endogenous ligand
  • angiotensin II a reference agonist for the ⁇ 2AR is the exogenous ligand
  • isoproterenol a reference agonist for the ⁇ AR is the exogenous ligand
  • phenylephrine a reference agonist for the GPRl 09 A receptor is the ligand, nicotinic acid.
  • G protein activity mediated by a GPCR can be measured using any of a wide variety of assays, including those well known in the art.
  • G protein activity can be assayed by determining the level of calcium, cAMP, diacylglycerol, or inositol triphosphate in the presence and absence of the ligand (or candidate ligand).
  • G protein activity can also be assayed, for example, by determining phosphatidylinositol turnover, GTP- ⁇ -S loading, adenylate cyclase activity, GTP hydrolysis, etc. in the presence and absence of the ligand (or candidate ligand). (See, for example, Kostenis, Curr. Pharm. Res. 12(14): 1703- 1715 (2006).)
  • ⁇ -arrestin function mediated by a GPCR in response to a ligand
  • ⁇ -arrestin function recruitment to the GPCR or GPCR internalization can be assayed in the presence and absence of the ligand (or candidate ligand).
  • the ⁇ -arrestin function in the presence and absence of a ligand (or candidate ligand) is measured using by resonance energy transfer, bimolecular fluorescence, enzyme complementation, visual translocation, co-immunoprecipitation, cell fractionation or interaction of ⁇ -arrestin with a naturally occurring binding partner. (See, for example, Violin et al, Trends Pharmacol. Sci. 28(8):416-427 (2007); Carter et al, J. Pharm. Exp. Ther. 2:839- 848 (2005).)
  • GRK activity can be used as a surrogate for ⁇ -arrestin function
  • ⁇ -arrestin function mediated by a GPCR in response to a ligand (or candidate ligand) can thus be reflected by changes in GRX activity, as evidenced by changes in receptor internalization or phosphorylation.
  • While the relative efficacies for G protein activity and ⁇ -arrestin functions for a given ligand (or candidate ligand) acting on a GPCR are preferably determined by assays in eukaryotic cells (e.g., mammalian cells (e.g., human cells), insect cells, avian cells, or amphibian cells, advantageously, mammalian cells), one skilled in the art will appreciate from a reading of this disclosure that appropriate assays can also be performed in prokaryotic cells, reconstituted membranes, and using purified proteins in vitro.
  • eukaryotic cells e.g., mammalian cells (e.g., human cells), insect cells, avian cells, or amphibian cells, advantageously, mammalian cells
  • Examples of such assays include, but are not limited to, in vitro phosphorylation of purified receptor by GRXs, GTP- ⁇ -S loading in purified membranes from cells or tissues, and in vitro binding of purified ⁇ -arrestins to purified receptors upon addition of ligand (or candidate ligand) (with or without GRXs present in the reaction).
  • ligand or candidate ligand
  • the above equation (1) measures the distance from the dashed (diagonal) line in the above-presented graph, and expresses that distance, for compounds with G protein activity and ⁇ -arrestin function ranging between 0 and 1 , as a number between -0.63 and +0.63, where -0.63 is a perfectly G protein-biased ligand and +0.63 is a perfectly ⁇ -arrestin-biased ligand.
  • This range will vary for "superagonists" with activity/function greater than 1 , and/or "inverse agonists" with activity/function less than 0.
  • Full agonists, antagonists (also known as “blockers”), and partial agonists with equal efficacies for both pathways, have a value of zero (as discussed above (and further below), the number resulting from application of the equation above is relative to a reference agonist for a given GPCR). It will be appreciated that two ligands that differ significantly in their ability to stimulate each pathway (i.e., G protein activity and ⁇ arrestin function), yet he the same distance off the line, will have the same value in the above equation. As an example, using the Angiotensin II receptor: ligands AngII (full agonist), SII ( ⁇ -arrestin biased agonist), valsartan
  • the value derived using the above equation (1) for preferred biased ligands of the invention is >0.05, >0.075, >0.1, >0.2, >0.3, >0.4 or >0.5.
  • Biased ligands of the instant invention can share with standard ⁇ -blockers or ARBs the ability to block the deleterious effects of over-stimulation of either ⁇ receptors or angiotensin receptors, which are mediated through G protein activation. This is the property that is responsible for the ability of classical ⁇ - blockers and ARBs to lower blood pressure, reduce exercise-induced angina and improve cardiac function in heart failure.
  • the biased ligands can simultaneously activate cell protective or other therapeutically beneficial pathways through the ⁇ -arrestin dependent mechanism.
  • ARBs Currently available and clinically used ARBs have been tested and none have been found to possess this activity ( ⁇ -arrestin bias).
  • Biased ligands of the invention include, but are not limited to, catecholamine derivatives with ⁇ -carbon alkyl substituents. Such derivatives can be of formula I:
  • Ri is an alkyl, preferably, a Ci to C 4 alkyl, more preferably, ethyl,
  • R 2 and R 3 are the same or different and are -H, alkyl (preferably a Ci to C 4 alkyl) or cycloalkyl (preferably, cyclobutyl, cyclopentyl or cyclohexyl); alternatively, R 2 and R 3 together with the nitrogen to which they are attached can form a ring (substituted or unsubstitued), preferably, a C 3 to C 6 saturated ring, more preferable a C 5 saturated ring, and R 4 is -OH or -H, or pharmaceutically acceptable salt thereof.
  • Examples of specific biased ligands of formula I include cyclopentylbutanephrine and isoetharine. Compounds of formula I can be synthesized using standard protocols known in the art.
  • Biased ligands of the invention also include peptides, such as the mutant angiotensin II (Ang) peptide Sar l ,Ile 4 ,Ile 8 -Ang II (SII), as well as derivatives thereof and low molecular weight mimics thereof.
  • SII is a biased agonist for ⁇ - arrestin-mediated signaling (ERK activation) and an antagonist for G protein dependent signaling (see Example 7). SII acts to antagonize the G protein- dependent pressor actions of Ang II while directly activating GRK6 and ⁇ - arrestin2 dependent signaling (the dependence of this signaling on a particular GRK or ⁇ -arrestin isoform is likely tissue specific).
  • SII is a known peptide and, while the invention includes new methods of using this peptide (for example, as an anti-hypertensive, and in the treatment of acute heart failure and other conditions requiring increased cardiac contractility), it does not include this peptide per se.
  • Biased ligands of the invention additionally include carvedilol and derivatives thereof.
  • Such derivatives can be of formula II:
  • R is a substituted or unsubstituted linear or branched alkyl, a substituted or unsubstituted cycloalkyl or a substituted or unsubstituted heterocyclic group.
  • R is a C1-C6 (advantageously, C1-C4) linear alkyl or a C3-C6 (advantageously, C3 or C4) branched alkyl or a C3-C7 cycloalkyl. More preferably, R is a Cl -C4 alkyl (e.g., -C(CH 3 ) 3 or -CH(CH 3 ) 2) or R is a cyclopentyl.
  • Carvedilol and (+)-l — (carbazol-4-yloxy)-3- (isopropylamino)-2-propanol are both known compounds and, while the invention can include new methods of using these compounds, it does not include these compounds per se or method of using same in treating hypertension, heart failure or heart attack.
  • Compounds of formula II can be synthesized using standard art- recognized protocols.
  • the invention further relates to compositions comprising at least one biased ligand formulated with an appropriate carrier.
  • the composition can be in dosage unit form (e.g., a tablet or capsule).
  • the composition can also be present, for example, as a solution (e.g., a sterile solution) or suspension, or as a gel, cream, ointment, aerosol or powder.
  • Approaches suitable for delivering peptide and non-peptide biased ligands of the invention including oral, transdermal, intrathecal, inhalation, IV, IP, IM, IN, delivery, are known in the art.
  • Optimum formulations and dosing regimens can be determined by one skilled in the art and can vary with the biased ligand, the patient and the effect sought.
  • the present invention also relates to methods of identifying ⁇ - arrestin/GRK biased ligands, that is, methods of identifying a biased ligand for a GPCR.
  • Such methods can comprise: i) determining the effect of a test compound on GPCR-mediated G-protein activity, and ii) determining the effect of the test compound on GPCR-mediated ⁇ -arrestin function, wherein a test compound that has a greater positive effect on GPCR-mediated ⁇ -arrestin function than on GPCR-mediated G-protein activity, relative to a reference agonist for both GPCR- mediated G-protein activity and GPCR-mediated ⁇ -arrestin function, is a biased ligand.
  • candidate therapeutics can be identified by: i) determining the effect of a test compound on G-protein activity mediated by a GPCR relevant to the physiological process, and ii) determining the effect of the test compound on ⁇ -arrestin function mediated by that GPCR, wherein a test compound that has a greater positive effect on ⁇ -arrestin function than on G-protein activity mediated by the GPCR, relative to a reference agonist for both the G-protein activity and ⁇ - arrestin function mediated by the GPCR, is such a candidate therapeutic.
  • cardiovascular diseases/disorders including hypertension, heart failure, coronary artery disease, pulmonary hypertension, peripheral vascular disease or arrhythmia
  • pulmonary diseases/disorders such as asthma, chronic obstructive airway disease and pulmonary fibrosis
  • ophthalmologic diseases/disorders such as glaucoma
  • hematologic diseases/disorders including thrombolytic disorders
  • endocrine or metabolic diseases/disorders e.g., diabetes and obesity
  • neurological or psychiatric diseases/disorders including
  • Parkinsonism or Alzheimer's as well as other diseases/disorders including those referenced below.
  • One embodiment of this aspect of the invention comprises evaluating the relative efficacy of a test compound to stimulate G protein dependent pathways compared to its efficacy to stimulate ⁇ -arrestin/GRK function (e.g., association with the receptor or signaling), for example, to promote ⁇ -arrestin membrane translocation (the most proximal event in ⁇ -arrestin signaling).
  • a test compound that has a relatively greater efficacy to stimulate ⁇ -arrestin/GRK function is a candidate compound for use in treating cardiovascular diseases/disorders, pulmonary diseases/disorders, glaucoma, hematologic diseases/disorders, etc.
  • a fluorescence resonance energy transfer (FRET)-based assay is used to assess ⁇ -arrestin/GRK function stimulating efficacy.
  • FRET fluorescence resonance energy transfer
  • GRK/ ⁇ - arrestin efficacy can be measured as the rate of ⁇ -arrestin recruitment to a receptor in response to ligand, where the receptor/ ⁇ -arrestin interaction is measured by FRET or bioluminescent resonance energy transfer (BRET).
  • FRET fluorescence resonance energy transfer
  • This rate of FRET increase is a measure of ligand-stimulated GRK activity, which regulates ⁇ -arrestin function, and thus quantifies a ligand's ⁇ -arrestin/GRK efficacy. Details of a particularly preferred assay are provided in Example 5. This method can be adapted for use with a fluorescence plate reader for high-throughput screening of agonists and antagonists, which can thereby provide a rapid screen for ⁇ -arrestin/GRK biased ligands. ⁇ -arrestin/GRK function can be measured for all manner of 7TMRs, e.g., the angiotensin II type 1 receptor.
  • ⁇ -arrestin function examples include: receptor/ ⁇ -arrestin co-immunoprecipitation, receptor/ ⁇ -arrestin crosslinking, receptor/ ⁇ -arrestin BRET, receptor/ ⁇ -arrestin bimolecular fragmentation complementation, receptor/ ⁇ -arrestin translocation imaging, receptor internalization, receptor phosphorylation, and ⁇ -arrestin associated phosphorylated ERK (Violin et al, Trends Pharmacol. Sci. 28(8):416-422 (2007)).
  • approaches that can be used to measure G-protein mediated signaling function include assays for adenylate cyclase and/or cyclic AMP accumulation (ICUE (DiPilato et al, Proc. Natl. Acad. Sci. USA 101 :16513 (2004)), radioimmunoassays, ELISAs, GTPase assays, GTPgammaS loading assays, intracellular calcium accumulation assays, phosphotidyl inositol hydrolysis assays, diacyl glycerol production assays (e.g., liquid chromatography, FRET based DAGR assay (Violin et al, J. Biol. Chem.
  • the therapeutic efficacy of the biased ligands of the invention can be increased using modifications known in the art to improve pharmacodynamic profile (e.g., increased affinity, etc), to prevent degradation (for peptides this can include N-acetylation and C-amidation, etc), to increase absorption, to allow for different routes of administration and different dosing strategies (including the addition of polyethylene glycol (PEGylation), lipids and protective salting, etc) and to modulate excretion.
  • modifications known in the art to improve pharmacodynamic profile (e.g., increased affinity, etc), to prevent degradation (for peptides this can include N-acetylation and C-amidation, etc), to increase absorption, to allow for different routes of administration and different dosing strategies (including the addition of polyethylene glycol (PEGylation), lipids and protective salting, etc) and to modulate excretion.
  • biased ligands of the invention which can be identified using, for example, methods described above, can be used in a variety of therapeutic settings, including those described above and below.
  • biased ligands that are ⁇ i AR agonists can be used as positive inotropes in the treatment of acute and/or severe systolic ventricular dysfunction/heart failure.
  • Biased ligands that are ⁇ iAR antagonists can be used as antihypertensive agents both in acute (emergencies) and chronic settings (negative inotropic/chronotropic mechanism).
  • Such ligands can also be used as cardioprotective/reverse remodeling agents in the treatment of chronic heart failure.
  • Biased ligands that are ⁇ 2 AR agonists can be used as positive inotropes/afterload-reducing agents in the treatment of acute and/or severe systolic ventricular dysfunction/heart failure (e.g. as smooth muscle relaxants in obstetric setting as tocolytics).
  • Biased ⁇ 2AR ligands that antagonize Gs-mediated signaling while stimulating ⁇ -arrestin mediated signaling can be used in the treatment of cardiovascular disease and, in particular in heart failure (e.g., chronic heart failure) and post-acute myocardial infarction.
  • Biased ligands that are D] -dopaminergic receptor agonists can be used as antihypertensive agents with renoprotective/diuretic properties in the acute setting (e.g., emergencies) (the mechanism being arteriolar vasodilator, i.e., used as afterload-reducing agents in the acute setting with concomitant preload reduction.).
  • Biased ligands that are antagonists of this receptor have antiemetic and digestive motility stimulatory effects. Such ligands can be used, for example, in treating gastrointestinal reflux while avoiding confounding neurological effects.
  • Biased ligands that act as AT i -Angiotensin II receptor antagonists can be used as antihypertensive agents in the chronic setting, promoting arteriolar vasodilation and protection from renal dysfunction (associated with diabetes). Additionally, such compounds can be used as afterload-reducing/reverse remodeling agents in chronic heart failure.
  • SII and other ⁇ -arrestin biased ligands for the ATI angiotensin receptor can be used for the treatment of acute heart failure.
  • SII for example, can bind the ATI angiotensin receptor and prevent the binding of endogenous angiotensin II, acting as a classical antagonist.
  • SII and other ⁇ -arrestin biased ligands for the ATI angiotensin receptor block G protein mediated signaling, they can act as vasodilators to decrease total peripheral resistance (afterload), and inhibit aldosterone secretion preventing sodium and fluid retention.
  • SII stimulates ⁇ -arrestin mediated signaling, which has been shown to increase contractility in isolated cardiac myocytes, which would lead to an increase in inotropy of the whole heart.
  • stimulation of ⁇ -arrestin mediated signaling has been shown to be anti-apoptotic.
  • Patients presenting with late stage acute heart failure are typically given inotropic agents to increase cardiac performance.
  • the ⁇ AR agonist dobutamine is a commonly utilized inotropic agent.
  • the continuous infusion of dobutamine over periods as short as 24-72 hours is associated with the development of significant tachyphylaxis and, more importantly, an increase in cardiomyocyte apoptosis. Increased cardiomyocyte death can have a negative impact on long-term cardiac function and overall survival.
  • ⁇ -arrestin biased ligands for the ATI angiotensin receptor for example, SII
  • SII inotrope therapies
  • ⁇ -arrestin biased ⁇ AR ligands which are also conventional agonists or antagonists for G protein stimulation
  • Biased ligands that act as ET A /ET ⁇ -Endothelin receptor antagonists can be used as anti -pulmonary hypertensive agents both in acute (pulmonary hypertensive crisis) and chronic settings (arteriolar vasodilator mechanism) such ligands can be used, for example, as right ventricular (RV) afterload-reducing agents in the acute and chronic (also reverse remodeling) setting.
  • RV right ventricular
  • Biased ligands that act as Vi -vasopressin receptor agonists can be used as vasoconstrictors in the treatment of acute and refractory distributive shock. Such ligands may have pulmonary vasodilator effects. Biased ligands that act as V]- vasopressin receptor antagonists have investigational use as afterload-reducing agents in heart failure.
  • Biased ligands of the invention that act as A 2A -adenosine receptor agonists can be used diagnostically as coronary arterial/arteriolar vasodilators in myocardial function/perfusion pharmacologic stress studies and therapeutically in the treatment of supraventricular arrhythmias.
  • Biased ligands of the invention that act as prostaglandin Ei receptor agonists can be used to maintain patency of the ductus arteriosus and those that act as prostaglandin Ei receptor antagonists can be used to promote closure of the ductus arteriosus.
  • Biased ligands of the invention that act as prostaglandin I 2 receptor agonists can be used as anti-pulmonary hypertensive agents both in the acute (pulmonary hypertensive crisis) and chronic setting, that is, they can be used as RV afterload-reducing agents in the acute and chronic (also reverse remodeling) setting.
  • biased ligands of the invention have uses that include the following .
  • Biased ligands that act as ⁇ 2 -adrenergic receptor agonists can be used as bronchodilators (airways resistance-reducing agents) in obstructive airways/lung disease both in the acute and chronic setting.
  • Biased ligands that act as ⁇ 2 - adrenergic receptor antagonists can be used investigational ⁇ (Collaerts-Vegh et al, Proc. Natl. Acad. Sci. USA 101 :4948-4953 (2004)) in the long-term management of asthma.
  • Biased ligands that act as Mi -acetylcholine receptor antagonists can be used as bronchodilators (airways resistance-reducing agents) and mucous-reducing agents in obstructive airways/lung disease both in the acute and chronic setting.
  • Biased ligands that act as leukotriene D 4 receptor antagonists can be used as anti-inflammatory agents in the management of asthma in the chronic setting.
  • ⁇ arrestin biased ligands (agonists and antagonists) of ⁇ 2AR that can be used in the treatment of asthma and chronic obstructive pulmonary disease (COPD), as well as other respiratory and pulmonary disorders, include compounds of formula I above (e.g., cyclopentylbutanephrine) (isoetharine and ethylnorepinephrine have been previously used to treat asthma and thus methods of treating asthma using these specific compounds are not within the scope of the present invention).
  • These compounds are all partial agonists for G protein signaling on ⁇ 2AR and are thus expected to function as classic bronchodialators but with the additional benefits of augmented ⁇ -arrestin mediated signaling.
  • the benefits of ⁇ -arrestin mediated signaling could also potentially be obtained through ⁇ -arrestin biased ligands for ⁇ 2AR that are devoid of G protein signaling.
  • biased ligands of the invention that act as P2Y12 purinergic receptor antagonists can be used as antiplatelet agents in a variety of settings, most commonly post-percutaneous vascular intervention with stent placement (coronary being most common). They can also be used for thrombotic stroke.
  • Biased ligands that act as adenosine receptor agonists can be used as antiplatelet agents in a variety of thrombotic disorders, most commonly stroke.
  • Biased ligands that act as thromboxane receptor antagonists can be used as antiplatelet agents in a variety of thrombotic disorders, most commonly acute coronary syndrome and its prophylaxis.
  • Biased ligands that act as thrombin receptor agonists can be used topically intraoperatively in the treatment of local bleeding while those that act as antagonists can be used as anticoagulant agents in the setting of contraindication to (or failure of, or complications from) conventional anticoagulation with heparin.
  • biased ligands of the invention that act as Hj/H 2 - histamine receptor antagonists can be used in the acute setting in the treatment of anaphylactic distributive shock and in the chronic setting as an "anti- inflammatory" in atopic conditions.
  • biased ligands of the invention that act as opioid receptor agonists can be used for pain management and those that act as opioid receptor antagonists can be used for reversal of opioid effects (e.g. drug overdose).
  • Biased ligands that act as Gamma Amino Butyric Acid (GABA) Type B receptor agonists can be used for muscle relaxation.
  • GABA Gamma Amino Butyric Acid
  • biased ligands of the invention that act as smoothened receptor antagonists can be used investigational ⁇ in malignancies 5 that reactivate developmental pathways (e.g., pancreatic and other GI, prostate).
  • biased ligands that act as gastrin receptor agonists can be used in determining stomach acid production.
  • Biased ligands that act as secretin receptor agonists can be used intraoperatively for pancreatic duct identification.
  • Biased ligands that act as serotonin receptor (5HT) Type 3 i o receptor antagonists can be used for management of nausea and vomiting, particularly in patients receiving chemotherapy.
  • Biased ligands that act as cannabinoid receptor agonists can be used to stimulate appetite particularly in patients with cancer while those acting as antagonists can be used in development for appetite suppression.
  • Biased ligands that act as cholecystokinin receptor is agonists can be used to assess biliary dyskinesia prior to cholecystectomy.
  • biased ligands of the invention that act as Parathyroid Hormone (PTH)/Parathyroid Hormone related Peptide (PTHrP) receptor agonists can be used in the treatment of osteoporosis.
  • Biased ligands that act as Thyroid Stimulating Hormone (TSH) receptor agonists can be used in the endocrine/metabolic areas.
  • Biased ligands that act as Luteinizing Hormone (LH)/Follicle Stimulating Hormone (FSH)/Gonadotropin Releasing Hormone (GnRH) receptor agonists can be used in the treatment of infertility, endometriosis, and central precocious puberty.
  • Biased ligands that act as Adrenocorticotropic Hormone (ACTH) receptor agonists can be used in various
  • Biased ligands that act as calcitonin receptor agonists can be used in the treatment of osteoporosis, pain following fracture, and management of hypercalcemia.
  • Biased ligands that act as Calcium Sensing Receptor (CaSR) receptor agonists/modulators can be used in the management of hypercalcemia associated with renal failure and for patients with inoperable parathyroid carcinoma.
  • Biased ligands that act as glucagon receptor agonists can be used for the treatment of symptomatic hypoglycemia.
  • Biased ligands that act as growth hormone receptor agonists can be used in patients with pituitary insufficiency and for children with reduced growth.
  • Biased ligands that act as dopamine receptor agonists can be used used in the management of prolactin secreting adenomas and for the treatment of Parkinsonism.
  • Biased ligands that act as somatostatin receptor agonists can be used in the management of diarrhea, neuroendocrine GI malignancies, and growth hormone excess.
  • Biased ligands that act as melanocortin receptor antagonists can be used for the management of weight.
  • Biased ligands that act as ghrelin receptor agonists can be used for management of gastric motility.
  • Biased ligands that act as oxytocin receptor agonists can be used for induction of labor.
  • Biased ligands that act as vasopressin receptor agonists can be used in the management of diabetes insipidus.
  • biased ligands of the invention that act as dopamine receptor agonists can be used in the treatment of Parkinsonism and those that act as antagonists can be used as anti-emetics and to increase bowel motility.
  • Biased ligands that act as serotonin receptor agonists can be used in the treatment of migraine headaches and those that act as antagonists can be used as antipsychotics.
  • Biased ligands that act as metabotropic glutamate receptor agonists can be used as anticonvulsants and those that act as antagonists can be used in the management of chronic pain.
  • Biased ligands that act as acetylcholine receptor agonists can be used for the treatment of cognitive decline associated with Alzheimer's disease and those that act as antagonists can be used as a sedative prior to anesthesia and as an antispasmodic in disorders characterized by restlessness or agitation (e.g. delirium tremens, Parkinsonism).
  • Biased ligands that act as Neuropeptide Y (NPY) receptor antagonists can be used for reducing caloric intake.
  • Biased ligands that act as orexin antagonists can be used for the treatment of addiction.
  • biased ligands of the invention that act as ⁇ - adrenergic receptor antagonists can be used for the management of glaucoma.
  • biased ligands of the invention that act as melatonin receptor agonists can be used for induction and maintenance of sleep.
  • Biased ligands that act as trace amine receptor agonists (such as tyramine, phenyl ethylamine, tryptamine and octopamine) have cardiovascular effects similar to that observed with ⁇ -adrenergic receptors.
  • biased ligands of the invention that act as agonists or antagonists of the ghrelin, orexin, amylin, NPY, cannabanoid, obestatin, and/or melanin concentrating hormone receptors can be used to treat endocrine or metabolic diseases or disorders, such as diabetes and obesity.
  • ⁇ AR agonists can promote skeletal muscle growth and combat skeletal muscle atrophy associated with aging.
  • the ability of such agonists compounds to affect skeletal muscle cell function, growth and survival has been attributed primarily to the activation of ⁇ 2ARs, and a number of downstream signaling processes involving G- ⁇ -s, G- ⁇ - ⁇ and ERK.
  • the actual role of these processes has not been strictly demonstrated, and it is well known that the ⁇ 2AR can signal through additional pathways, such as those involving the ⁇ -arrestins.
  • ⁇ -arrestin signaling has been shown to be anabolic and anti-apoptotic, processes one might associate with promoting skeletal muscle growth and combating skeletal muscle atrophy associated with aging.
  • ⁇ -arrestin biased ligands for the ⁇ 2AR such as cyclopentylbutanephrine, isoetharine and ethyl-norepinephrine, are used for the treatment of skeletal muscle atrophy, sarcopenia, age related frailty and improved healing after skeletal muscle injury.
  • ⁇ -arrestin biased ligands for the ⁇ 2AR such as cyclopentylbutanephrine, isoetharine and ethyl-norepinephrine
  • ⁇ -arrestin biased ligands of the invention can also be used in the treatment of diseases/disorders associated with physiological processes mediated by one or more of the following receptors: the ⁇ -adrenergic, receptor, ADP receptor, apelin (APJ) receptor, prostaglandin receptor, neurotensin receptor, neuromedin U & B receptor, cholecystokinin receptor, chemokine receptor, endothelin receptor, orexin receptor, bradykinin receptor, GP30 receptor, mas receptor, ghrelin receptor, amylin receptor, GPRl 09a receptor, calcitonin receptor, calcitonin gene related peptide receptor, adrenomedulin receptor, rhodopsin receptor, cone opsin receptor, frizzled recpeptor, bombesin receptor, C5a anaphylatoxin receptor, FMLP receptor, interleukin receptor, prolact
  • the cells were transfected with cDNA encoding 2 ⁇ g FLAG-EGFR, EGFR-GFP, ⁇ -arrestin-GFP, ⁇ -arrestinl V53D or r ⁇ arrR169E- YFP with lipofectamine reagent (Invitrogen, CA). Transfected cells were incubated overnight in serum-free medium supplemented with 0.1% bovine serum albumin (BSA), 1OmM HEPES (pH 7.4), and 1% penicillin prior to stimulation. Under serum starvation condition, cells were preincubated with ICI-118, 551 for 30 min and then stimulated with Dob or EGF for 4-5 min.
  • BSA bovine serum albumin
  • 1OmM HEPES pH 7.4
  • Immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose membrane or PVDF (BioRad) for immunoblotting.
  • Anti- phosphotyrosine (PY20) (BD Transduction Laboratories, Lexington, KY) was used to detect tyrosine phosphorylation of EGFR at 1 :3000 and phospho-ERKl/2 (Cell Signaling) was also used at 1 :3000.
  • Immunoblotting for total EGFR (Upstate) and total ERK (Santa Cruz) was carried out at 1 :2000.
  • EGFR from the myocardial lysates was immunoprecipitated using anti-EGFR antibody (Upstate) and immunoblotted for phospho-EGFR using anti-phospho-EGFR (Tyr845) (Cell Signaling) at 1 :1000. Detection was carried out using enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech). Densitometric analysis was performed using BioRad Fluoro-S Multi-image software, ⁇ -arrestin immunoblotting was carried out using AlCT rabbit polyclonal antibody at a 5 dilution of 1 :3,000 as previously described (Wei et al, Proc. Natl. Acad. Sci. USA 100: 10782 (2003)).
  • ECL enhanced chemiluminescence
  • Anti-GRK specific antibodies (Santa Cruz, CA) were used to detect GRK 2, 3, 5 and 6 as described previously (Ren et al, Proc. Natl. Acad. Sci. USA 102:1448 (2005)).
  • ⁇ -actin and FLAG immunoblotting were carried out using monoclonal antibodies at dilutions of 1 :3000 each (Sigma).
  • i o Immunoblotting for myocardial pERK and pAkt was performed as previously described (Perrino et al, J. Clin. Invest. 116:1547-1560 (2006)).
  • Hearts were homogenized with NP -40 lysis buffer containing 137 mmol/1 NaCl, 20 mmol/1 Tris pH 7.4, 1% NP-40, 20% glycerol, 10 mmol/1 PMSF, 1 mmol/1 Na 3 VO 4 , 10 mmol/1 NaF, 2.5 ⁇ g/ml aprotinin, and 2.5 ⁇ g/ml leupeptin,
  • EGFR-GFP fluorescently labeled EGFR
  • siRNA experiments targeting ⁇ -arrestins and GRKs siRNA targeting ⁇ -arrestin 1 , 2 or both were generated by BLAST search algorithim for a unique 21 nt sequence for ⁇ -arrestins from National Center for Biotechnology Information.
  • the sequence of the 21 nt siRNA's has been previous described (Ahn et al, J. Biol. Chem. 279:35518 (2004), Ahn et al, J. Biol. Chem. 279:7808 (2004)).
  • the sequence of siRNA's targeting GRK 2, 3, 5, or 6 have been previously described (Ren et al, Proc. Natl. Acad. Sci. USA 102: 1448 (2005), Kim et al, Proc. Natl.
  • HEK 293 cells stably expressing either PKA ' ⁇ iAR or WT ⁇ iAR in 6 well dishes were transfected with 0.2 ⁇ g of FLAG-EGFR and 3.5 ⁇ g of siRNA using the GeneSilencer Transfection reagent (Gene therapy systems) as previously described (Ren et al, Proc. Natl. Acad. Sci. USA 102: 1448 (2005)).
  • WT ⁇ l AR cells underwent transfection with 1 ⁇ g EGFR-GFP and 3.5 or 7 ⁇ g of siRNA targeting both ⁇ -arrestins and control siRNA, and were replated onto 35mm glass-plated culture dishes after 60 hours.
  • HEK 293 cells were transfected with 0.2 ⁇ g of FLAG-EGFR, 4 ⁇ g of siRNA targeting either GRK 2, 3, 5 or 6 and an appropriate amount of plasmid cDNA encoding either WT ⁇ iAR or PKA " ⁇ iAR.
  • the expression of the receptors in transient transfection was 475-600 fmol/mg protein. All assays were performed 60-72 hours after siRNA transfection. Cells were serum starved for 12 hr before stimulation. Treatment protocol for mice
  • mice or ⁇ -arrestin-2 knockout, GRK5 knockout or GRK6 knockout mice were administered with ICI 118,551 (5 mg) intra-peritoneal. After 5 min of ICI pre-treatment, mice were administered with saline, ⁇ iAR specific agonist Dob (10 mg/kg) as a single intra-peritoneal dose or epidermal growth factor (EGF) (300 ⁇ g/kg) as a single intra-venous bolus. Following a wait of 5 min after Dob or EGF administration, the experiment was terminated. The heart was excised and flash frozen in liquid N 2 for biochemical assays.
  • Heart specimens were fixed with 10% neutral buffered formalin, embedded in paraffin, and sectioned at 5- ⁇ m thickness. Sections are stained with Hematoxylin-Eosin (HE) and Masson Trichrome (MT). DNA fragmentation was detected in situ using TUNEL (Perrino et al, J. Clin. Invest. 116:1547-60 (2006)). In brief, deparaffinized sections were incubated with proteinase K, and DNA fragments were labeled with fluorescein-conjugated dUTP using TdT (Roche Diagnostics Corp.). The total number of nuclei was determined by manual counting of DAPI-stained nuclei in 6 fields of each section using the *200 magnification, and the number of TUNEL-positive nuclei was counted in the entire section.
  • HE Hematoxylin-Eosin
  • MT Masson Trichrome
  • Plasma membrane and cytosolic fractions from left ventricles flash frozen in liquid N 2 were separated by centrifugation at 37,00Og as previously described (Esposito et al, Circulation 105:85 (2002); Perrino et al, J. Clin. Invest. 116: 1547- 60 (2006)).
  • Receptor binding with 20 ⁇ g of protein from plasma membrane was performed using [ 125 I] cyanopindolol (35OpM) as described previously (Esposito et al, Circulation 105:85 (2002)).
  • Receptor density (fmol) was normalized to milligrams (mg) of membrane protein.
  • Adenylyl cyclase assays were performed as described previously (Esposito et al, Circulation 105:85 (2002)) using 20 ⁇ g of membrane fraction. Generated cyclic adenosine monophosphate (cAMP) was quantified using a liquid scintillation counter (MINAXI-4000, Packard Instrument Co., PerkinElmer, Boston, Massachusetts, USA)
  • the FLAG-tagged mouse wild type ⁇ l -adrenergic receptor (WT ⁇ lAR) and two different mutants lacking either the putative GRK phosphorylation sites (GRK ' ⁇ lAR) or the putative PKA phosphorylation sites (PKA ' ⁇ lAR) were directionally subcloned into a vector downstream of ⁇ -myosin heavy chain gene promoter and upstream of the SV40 polyadenylation site (Nienaber et al, J. Clin. Invest. 112:1067 (2003)).
  • the ⁇ iAR mutants were generated by mutating the following serine/threonine amino acids to alanine at residues 412, 417, 426, 427, 437, 443, 444, 445, 448, 450, 451, 461, 462, 464 for the GRK ' ⁇ lAR mutant and the four serine amino acids to alanine at residues 295, 296, 301, and 401 in PKA " ⁇ lAR (Fig. 10).
  • Transgenic founders were identified by Southern blot analysis of tail DNA using the SV40 poly (A) as a probe. Transgenic founder mice were backcrossed into a C57BL/6 background for at least 4 generations before being used in experiments to investigate the phenotype.
  • mice were screened by PCR with sense primer, 5'-CATGGGTGTGTTCACGCTC, located in mouse ⁇ lAR coding sequence, and an antisense primer, 5'-CCTCTACAGATGTGATATGGC, located in SV40 poly (A) sites.
  • ⁇ AR radioligand binding assay was carried out on multiple generations to analyze and confirm the receptor expression. Animals were handled according to the approved protocols and animal welfare regulations of the Institutional Review Board at Duke University Medical Center. Echocardiography
  • Echocardiography was performed on conscious mice with either HDI5000 echocardiograph (Philips) or Veno770 (Visualsonics, Inc, Toronto, Canada) as described previously (Esposito et al, Circulation 105:85 (2002); Perrino et al, J. Clin. Invest. 116:1547-60 (2006)).
  • mice were implanted as described previously (Nienaber et al, J. Clin. Invest. 112:1067 (2003)). Isoproterenol (ISO) was dissolved in 0.002% ascorbic acid, and pumps (Alzet model 1007D; DURECT Corp., Cupertino, California, USA) are filled to deliver at rate of 3mg/kg/day over a period of 7 days. In control mice, vehicle (0.002% ascorbic acid) was used. In erlotinib treatment, mice were given erlotinib (20mg/kg/day) intraperitoneally for 3 weeks.
  • ISO Isoproterenol
  • pumps Alzet model 1007D; DURECT Corp., Cupertino, California, USA
  • Erlotinib were dissolved into dimethyl sulfoxide (DMSO), and diluted to 10%DMSO with 0.9% physiological saline. The control mice in erlotinib treatment were given an equal volume of 10%DMSO. Echocardiography was performed pre and post ISO treatment.
  • DMSO dimethyl sulfoxide
  • ⁇ iAR stimulation could mediate EGFR transactivation and induce activation of downstream signaling pathways.
  • Cells stably expressing WT ⁇ iARs were transfected with FLAG-EGFR and stimulated with either the ⁇ iAR specific agonist Dobutamine (Dob) or EGF following pretreatment with the ⁇ 2 AR specific antagonist ICI-118,551 (ICI), to block endogenous ⁇ 2 ARs.
  • Dob ⁇ iAR specific agonist
  • EGF ⁇ 2 AR specific antagonist
  • Stimulation with Dob resulted in a significant increase in phosphorylation of the EGFR along with activation of ERK, which was somewhat less marked than direct stimulation with EGF ligand (Fig. IA upper panel).
  • mice wild type ⁇ iAR (WT) and 3 phosphorylation site deficient mutants were used that had been previously cloned and characterized (Rapacciuolo et al, J. Biol. Chem.
  • the 3 phosphorylation defective mutants contain alanine for serine or threonine substitutions at: 1) the 4 putative PKA phosphorylation sites in the third intracellular loop and proximal carboxy tail (PKA " ⁇ i AR), 2) the 14 putative GRK phosphorylation sites within the carboxy tail (GRK ' ⁇ iAR), and 3) both sets of phosphorylation site substitutions (PKAVGRK ' ⁇ i AR) (Rapacciuolo et al, J. Biol. Chem. 278:35403 (2003)).
  • GRK " ⁇ iAR receptors with only PKA phosphorylation sites
  • GRK " ⁇ iAR receptors with only PKA phosphorylation sites
  • Fig. 1C minimal ERK activation
  • Fig. 1C absence of EGFR internalization
  • ⁇ ,ARs lacking both PKA and GRK phosphorylation sites also failed to trigger EGFR transactivation (Fig. 1 D).
  • ⁇ -arrestin-1 and -2 are required for ⁇ iAR transactivation of EGFR
  • Previous studies have shown that recruitment of activated c-Src to the ⁇ 2 AR complex by ⁇ -arrestin is needed for ERK activation (Luttrell et al, Proc. Natl. Acad. Sci USA 98:2449 (2001)), Luttrell et al, Science 283:655 (1999)).
  • HEK 293 cells stably expressing either WT ⁇ iAR or ⁇ iAR mutants were transfected with GFP- ⁇ - arrestin and stimulated with Dob.
  • siRNA was used that specifically targets either ⁇ - arrestin-1 or -2 or both (Ahn et al, J. Biol. Chem. 279:7807 (2004)).
  • Dob stimulation of PKA " ⁇ iARs resulted in intense phosphorylation of the EGFR associated with significant ERK activation in mock and scrambled siRNA transfected cells (Fig. 2B).
  • transactivation of the EGFR and associated ERK activation were significantly blocked in the presence of siRNA targeting either ⁇ -arrestin- 1 or -2 or both (Fig. 2B).
  • ⁇ -arrestins are constitutively phosphorylated proteins and become dephosphorylated at the plasma membrane upon binding of activated 7TMRs.
  • ⁇ -arrestin dephosphorylation promotes the recruitment of adapter proteins such as clathrin and AP-2, which are required for receptor endocytosis (Lin et al, J. Biol. Chem. 272:31051 (1997), Laporte et al, Proc. Natl. Acad. Sci., USA 96:3712 (1999), Naga Prasad et al, J. Cell. Biol. 158:563 (2002)).
  • ⁇ arrR169E-YFP tagged a "phosphorylation independent" ⁇ -arrestin mutant that mimics dephosphorylated ⁇ -arrestin was used.
  • ⁇ arrR169E binds with higher affinity to activated receptors and scaffold proteins (Kovoor et al, J. Biol. Chem. 274:6831 (1999), Violin et al, J. Biol. Chem. 281:20577-20588 (2006)).
  • HEK 293 cells stably expressing GRK " ⁇ iAR were transfected with ⁇ arrR169E-YFP along with FLAG-EGFR.
  • Dob stimulation of GRK " ⁇ iARs resulted in significant recruitment of ⁇ arrR 169E- YFP to GRK " ⁇ i ARs as assessed by immunoblotting for ⁇ -arrestin co-immunoprecipitating with the receptor (Fig. 3A, upper panel).
  • GRKs based on their sequence similarity and distribution, have been divided into three subfamilies. GRKs 1 and 7 are exclusively expressed in the retina, GRKs 2 and 3 interact with Gp v subunits through their pleckstrin homology domain and GRKs 4, 5 and 6 are a sub- family that are membrane associated (Pitcher et al, Annu. Rev. Biochem. 67:653 (1998), Willets et al, Trends Pharmaceol. Sci. 24:626 (2003)).
  • GRKs 2, 3, 5 and 6 are expressed ubiquitously in mammalian tissues, their role in transactivation was investigated.
  • HEK 293 cells were transfected with PKA " ⁇ iAR alone or along with siRNAs targeting GRK 2, 3, 5 or 6.
  • Dob stimulation resulted in robust phosphorylation of EGFR associated with significant ERK activation in the presence of siRNA knocking down expression of GRK 2 and 3 (Fig. 3D, 3E, 3F).
  • ⁇ i AR-mediated EGFR transactivation and downstream ERK activation were nearly completely blocked in the presence of siRNA targeting GRK 5 or 6 (Fig. 3D, 3E, 3F).
  • GRK 5 or 6 knock down blocked WT ⁇ i AR-mediated EGFR phosphorylation and ERK activation, which was insensitive to Gi inhibition by pertussis toxin or PKA inhibition by H89 (Fig. 9A, 9B).
  • angiotensin II stimulation (All, l ⁇ M) in cells expressing ATiRs did not induce EGFR phosphorylation and All-induced ERK 1/2 phosphorylation was insensitive to AG 1478 (Fig. 9C).
  • mice lacking the gene encoding ⁇ -arrestin2, GRK 5 or GRK 6 were used. Mice were pretreated with ICI and then challenged with Dob or EGF. Myocardial lysates were immunoblotted for pERK. Consistent with the cell culture experiments, Dob-mediated ERK activation was completely blocked in the ⁇ -arrestin-2 knock out mice compared to their wild type littermate controls (Fig. 4A). Moreover, ERK activation was also completely blocked in GRK 5 and 6 knockout mice (Fig. 4B, 4C).
  • EGFR was immunoprecipitated from the myocardial lysates and immunoblotted for phosphotyrosine. Although detection of phosphorylated EGFR from myocardial lysates was less consistent than from cell culture lysates, Dob-mediated EGFR phosphorylation appears to be blocked in hearts from ⁇ -arrestin-2, GRK5 and GRK6 knockout mice (Fig. 10). These data demonstrate that, in vivo, the mechanism for ⁇ iAR mediated EGFR transactivation requires both GRK 5, GRK 6, and ⁇ -arrestin recruitment to agonist-stimulated ⁇ iARs.
  • transgenic mice were generated with cardiac-specific overexpression of the mouse WT ⁇ iAR (WT ⁇ ,AR TG) and the two mutant ⁇ iARs lacking either GRK phosphorylation sites (GRK " ⁇ iAR TG) or PKA phosphorylation sites (PKA " ⁇ iAR TG) used in the cell culture experiments (Fig. 1 IA).
  • GRK GRK phosphorylation sites
  • PKA PKA phosphorylation sites
  • LVEDD LeIt venlilcular end diastolic demenslon(LVEDD), ten ventricular systolic demension (LVESD). intraventricular septum (IVS),
  • LVPW IeIt ventiicular posterior wall
  • FS fractional shortening
  • Vcfc heart rate corrected mean velocity of circumferential fiber shortening
  • mice normal mice were treated with the pharmacological EGFR inhibitor, erlotinib. Acutely, erlotinib treatment in vivo blocked myocardial ERK signaling by both ⁇ iAR mediated transactivation and direct EGF stimulation in WT mice (Fig. 7A), and in hearts of the WT ⁇ iAR TG and PKA ' ⁇ iAR TG mice (Fig. HB).
  • Cardiac function in normal C57BL/6 mice following a 2 week treatment with both erlotinib (20mg/kg, ip daily) and ISO was then measured. Cardiac function significantly deteriorated in mice treated with both ISO and erlotinib as shown by the significant increase in LV diastolic dimensions and reduced fractional shortening (Figs. 7B, 7C). Moreover, erlotinib-induced cardiac dysfunction was associated with a ⁇ 3 fold increase in apoptosis compared to ISO treated mice without the EGFR inhibitor (Figs. 7D, 7E). These data support the the hypothesis that ⁇ -arrestin mediated transactivation of the EGFR confers cardioprotection under conditions of catecholamine excess.
  • ⁇ -arrestins desensitize 7TMR signaling by sterically inhibiting G protein coupling to agonist activated receptors, they also function to bring activated receptors to clathrin-coated pits for endocytosis (Lefkowitz and Shenoy, Science 308:512 (2005)). It is now appreciated that, in the context of the endocytic process, ⁇ - arrestins serve as ligand-activated scaffolds to initiate 7TMR signaling networks,
  • 7TMRs activate MAPK signaling.
  • one pathway by which agonist- stimulated 7TMRs activate ERK signaling is to switch their G-protein coupling from Gs to Gi leading to the activation of the small GTPase protein RAS (Daaka et al, Nature 390:88 (1997), Martin et al, Cell Signal 16:1397 (2004)).
  • a second recognized pathway for the activation of ERK is via the ability of ⁇ -arrestin to recruit the non-receptor tyrosine kinase c-Src following agonist stimulation (Luttrell et al, Science 283:655 (1999)).
  • ⁇ -arrestins act to scaffold and assemble specific components of a MAPK module to enhance the activation and targeting of ERK (Luttrell et al, Proc. Natl. Acad. Sci. USA 98:2449 (2001), DeFea et al, J. Cell. Biol. 148:1267 (2000)) or JNK pathways (McDonald et al, Science 290:1574 (2000)) to specific subcellular locations.
  • ERK Littrell et al, Proc. Natl. Acad. Sci. USA 98:2449 (2001), DeFea et al, J. Cell. Biol. 148:1267 (2000)) or JNK pathways (McDonald et al, Science 290:1574 (2000)) to specific subcellular locations.
  • ERK Proc. Natl. Acad. Sci. USA 98:2449
  • JNK pathways McDonald et al, Science 290:1574 (2000)
  • ⁇ -arrestin signaling is the issue of isoform specificity.
  • Accumulating evidence suggests considerable complexity in the specificity of the ⁇ -arrestin isoform that regulates ERK signaling following ligand-activation of a 7TMR.
  • reciprocal regulation a particular receptor relies upon only one ⁇ -arrestin isoform, and the other ⁇ -arrestin isoform functions to inhibit signaling. This is seen for the AT IA R (Ahn et al, J. Biol. Chem. 279:7807 (2004)), the vasopressin V2R (Ren et
  • ⁇ -arrestins While the mechanism for differences in the requirement of ⁇ -arrestins for ERK activation is not known, a possible explanation may be preferential recruitment of ⁇ -arrestin homo- or hetero-oligomers (Storez et al, J. Biol. Chem. 280:40210 (2005)). Whether the activated ⁇ -arrestins that form signaling complexes upon binding 7TMRs are in fact homo- or hetero-dimers or even monomers will require further study. The importance of GRK 5 or 6 for transactivation is highlighted by both the in vitro and in vivo studies on hearts of GRK 5 and 6 knockout mice.
  • V2 vasopressin receptor (Ren et al, Proc. Natl. Acad. Sci. USA 102:1448 (2005)).
  • ⁇ -arrestin-mediated EGFR signaling in the heart is highlighted by the transgenic mouse studies, which demonstrate how the loss of ⁇ iAR mediated EGFR transactivation results in enhanced apoptosis and cardiac deterioration.
  • Apoptotic heart cell death has been implicated in the overall process of myocardial remodeling and the transition from cardiac hypertrophy to chronic heart failure (Kang and Izumo, Circ. Res. 86:1107 (2000), Olivetti et al, N. Engl. J. Med. 336:1131 (1997)), which is due in part to chronic ⁇ ,AR stimulation (Communal et al, Circulation 100:2210 (1999), Zaugg et al,
  • EGFR transactivation is important because many of the downstream signaling pathways stimulated by receptor tyrosine kinases have been linked with pathologic processes. For example, while there is evidence showing that HB- EGF ligands can contribute to the hypertrophic signaling in the heart (Yoshioka et al, Proc. Natl. Acad. Sci. USA 102:10622 (2005), Asakura et al, Nat. Med.
  • ⁇ -AR Three catecholamine derivatives with ⁇ -carbon alkyl substitutions have been identified that appear to have a bias for ⁇ -arrestin-mediated signaling (Fig. 12). Specifically, these compounds promote greater GRK/ ⁇ -arrestin function (as assessed by recruitment of ⁇ -arrestin to the receptor) than would be predicted based on the ability of these compounds to stimulate G protein mediated cAMP accumulation (see Fig. 12)."
  • Bias was determined by comparing efficacy for stimulating G protein signals to efficacy for stimulating ⁇ -arrestin/GRK functions. Specifically, for a panel of ⁇ 2 AR ligands, ligand-stimulated cAMP generation was quantified to measure G protein efficacy, and the ligand-stimulated rate of ⁇ -arrestin recruitment was measured by FRET between ⁇ 2 AR-mCFP and ⁇ -arrestin2-mYFP to measure ⁇ -arrestin/GRK efficacy. For most ligands, relative G protein and ⁇ - arrestin/GRK efficacies correlated extremely well.
  • ⁇ - arrestin/GRK efficacy was substantially greater than G protein efficacy, indicating that these are ⁇ -arrestin-biased ligands.
  • substitutions at the ⁇ -carbon position of ⁇ -adrenergic receptor agonists and antagonists can lead to ligands that differentially recruit cytosolic ⁇ -arrestin to the ligand-bound receptor.
  • Such receptors can signal in a biased manner, i.e., selectively via ⁇ -arrestin-dependent pathways more than via classical G protein dependent pathways.
  • Cellular outputs for this differential recruitment and signaling that can be assayed include, but are not limited to: 1) assays for ⁇ -arrestin recruitment following ligand addition; 2) assays for GRK activity/recruitment; 3) assays of G protein dependent cellular activity such as cAMP; 4) assays of MAPK activation such as pERK generation and 5) ⁇ -arrestin- dependent, ⁇ i-AR mediated epidermal growth factor receptor transactivation.
  • Fig. 13A shows a dose-response curve for the classical agonist isoproterenol in cells expressing ⁇ 2 AR-mCFP and ⁇ -arrestin2-mYFP in a 96-well plate. The assay was run in equilibrium mode, allowing the FRET signal to stabilize for 10-20 minutes before acquiring data.
  • Fig. 13B shows a dose- response curve for the classical antagonist propranolol reversing the isoproterenol-stimulated FRET signal. This assay was also run at equilibrium in a 96-well plate.
  • this assay can be run in kinetic mode to measure GRK function, as described above.
  • Fig. 13C shows the kinetics of the FRET change in response to either agonist or buffer.
  • the ⁇ -arrestinl mutant R169E was used to measure both GRK-independent FRET (fast response from receptor conformational changes) and GRK-dependent FRET (slow response), as described above.
  • GRK activity assay functions for multiple receptors.
  • GRK function for the Angiotensin II type 1 receptor has also been measured.
  • Fig. 14A shows that siRNA-mediated silencing of GRKs slows the rate of ⁇ -arrestin recruitment to the ATi A R in response to 10OnM angiotensin.
  • GRK2 silencing is more effective at slowing ⁇ -arrestin recruitment than GRK6 silencing.
  • Fig. 12 shows that siRNA-mediated silencing of GRKs slows the rate of ⁇ -arrestin recruitment to the ATi A R in response to 10OnM angiotensin.
  • GRK2 silencing is more effective at slowing ⁇ -arrestin recruitment than GRK6 silencing.
  • 73 biased ligands can select from subsets of GRK functions to determine the signaling outcome of receptor stimulation.
  • Isoproterenol was obtained from Sigma.
  • [ 125 I]-Iodocyanopindolol was obtained from Perkin Elmer.
  • H-89 was obtained from EMD Biosciences.
  • Anti- phospho- ⁇ 2 AR p355/p356) was from Santa Cruz Biotechnology.
  • Anti-beta- arrestin (AlCT) is described elsewhere (Attramadal et al, J. Biol. Chem. 267:17882-17890 (1992)).
  • Anti-FLAG beads were from Sigma. All other reagents were from Sigma.
  • Rat ⁇ -arrestinl and rat ⁇ -arrestin2 were amplified by PCR to encode Hind III and Sal I restriction sites at the 5' and 3' ends, respectively, with the terminator codon replaced with a sequence encoding a diglycine linker. These products were cut, purified, and ligated into a pcDNA3.1-mYFP vector (Violin et al, J. Cell. Biol. 161 :899-909 (2003)) to generate ⁇ -arrestinl-mYFP and ⁇ - arrestin2-mYFP. The ⁇ -arrestin-mYFP inserts were then transferred to a pcDNA3.1-zeo vector providing zeocin resistance.
  • Rat ⁇ 2 AR was amplified by PCR to encode a Hind III restriction site, flag epitope, and signal peptide sequence at the 5' end (Guan et al, J. Biol. Chem. 267:21995- 21998 (1992)), and an Xho I restriction site at the 3' end with the terminating codon replaced with a sequence encoding a diglycine linker.
  • This product was cut, purified, and ligated into a pcDNA3.1-mCFP vector (Violin et al, J. Cell. Biol. 161 :899-909 (2003)) to generate ⁇ 2 AR-mCFP. All plasmids were amplified
  • kit purified (Qiagen) and validated by capillary electrophoresis sequencing.
  • siRNA Small interfering RNA silencing of gene expression
  • Chemically synthesized double-stranded siRNA duplexes (with 3' dTdT overhangs) were purchased from Dharmacon for the following targets, as described and validated elsewhere (Ren et al, Proc. Natl. Acad. Sci. USA 102:1448-1453 (2005), Kim et al, Proc. Natl. Acad. Sci.
  • GRK2 (5'-AAGAAGUACGAGAAGCUGGAG-S')
  • GRK3 (5'- AAGCAAGCUGUAGAACACGUA-3')
  • GRK5 5'-AAGCCGUGCAAAGAACUCUUU-S')
  • HEK-293 cells were transfected with Gene Silencer (Gene Therapy Systems), and U2-OS cells were transfected with Lipofectamine 2000 (Invitrogen), according to manufacturers' instructions. Silencing was quantified by immunoblotting.
  • HEK-293 cells and U2-OS cells were maintained in modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution (Sigma).
  • HEK-293 cells were transfected with FuGene ⁇ (Roche);
  • U2-OS cells were transfected with Lipofectamine 2000
  • AU transfections used 3.0 ⁇ g of plasmid in a 10 cm tissue culture plate.
  • Cells expressing ⁇ 2 AR-mCFP were selected with either 400 ng/ml G418 (Sigma) and colonies of stable transfectants were isolated.
  • a single line of stably transfected cells was chosen as representative based on membrane targeting and isoproterenol-induced internalization of the ⁇ 2 AR-mCFP.
  • Surface expression of ⁇ 2 AR was measured by l25 I-cyanopindolol binding as described (Shenoy et al, J. Biol. Chem.
  • Imaging buffer 125 mM NaCl, 5 mM KCl, 1.5mM MgCl 2 , 1.5 mM CaCl 2 , 1OmM glucose, 0.2% BSA, 10 mM HEPES, pH 7.4
  • Imaging buffer 125 mM NaCl, 5 mM KCl, 1.5mM MgCl 2 , 1.5 mM CaCl 2 , 1OmM glucose, 0.2% BSA, 10 mM HEPES, pH 7.4
  • CFP and FRET images were obtained through a 436/20 excitation filter (20 nm bandpass centered at 436 nm), a 455DCLP (dichroic longpass mirror), and separate emission filters (480/30 for CFP and 535/30 for FRET).
  • YFP intensity was imaged through a 500/20 excitation filter, 515LP dichroic mirror, and 535/30 emission filter. All optical filters were obtained from Chroma Technologies. Excitation and emission filters were switched in filter wheels (Lambda 10-2; Sutter). Integration times were varied between 100 and 300 ms to optimize signal and minimize photobleaching. Spectral bleedthrough was determined by acquiring CFP, FRET, and YFP intensity images of samples expressing CFP only and YFP only, and was
  • FRETc FRET - 0.43 • CFP - 0.24 • YFP
  • FPvETc for all FRET images was calculated on a pixel-by-pixel basis for localization of FRET.
  • all graphs display calculations based on intensity from whole cells or sets of cells.
  • GRKs were detected with isoform-specific antibodies from Santa Cruz Biotechnologies for GRK2 (sc- 562), GRK3 (sc-563), GRK5 (sc-565), and GRK6 (sc-566), according to the manufacturer's instructions.
  • Chemiluminescent detection was performed with horseradish peroxidase-coupled secondary antibody (Amersham Pharmacia) and SuperSignal West Pico reagent (Pierce). Chemiluminescence was quantified by a charge-coupled device camera (Syngene ChemiGenius2); representative images are shown as inverted greyscale.
  • HEK 293 cells plated in 100-mm dishes were incubated at 37 0 C for 60 min in phosphate-free MEM containing [ 32 P]Pj
  • the HEK-293 cell line was chosen as a model system. These cells express the 4 ubiquitously expressed non-visual GRKs (2, 3, 5, and 6) and are readily
  • FRET Green Fluorescent Protein
  • %F differs from absolute FRET efficiency (%E) only in the absence of a term correcting for hardware-specific differences in the quantum yield of CFP and FRET emission (Gordon et al, Biophys. J. 74:2702-2713 (1998)).
  • %E absolute FRET efficiency
  • the kinetics of recruitment was tested in cells stably transfected with ⁇ 2 AR-mCFP and transiently transfected with ⁇ -arrestin2-mYFP. These cells express constant amounts of ⁇ 2 AR-mCFP (1 pmol/mg protein, data not shown) and varying amounts of ⁇ -arrestin2-mYFP. Transiently transfected cells, for which single-cell overexpression is highly variable, were used to assess the effects of ⁇ -arrestin expression on the ⁇ -arrestin: ⁇ 2 AR interaction. It was found that in cells with low to moderate expression of ⁇ -arrestin-mYFP, as measured by single- cell YFP fluorescence, isoproterenol-induced recruitment is monoexponential (Fig. 16A).
  • ⁇ -arrestin2-mYFP expression yielded increased recruitment, as measured by maximum fractional FRET.
  • ⁇ -arrestin2-mYFP recruitment is biphasic, exhibiting a rapid association (less than 5 second half-time) as well as the slow association seen with low expression.
  • ⁇ - arrestin2-mYFP was transiently expressed with either wild-type ⁇ 2 AR-mCFP (WT) or with a non-phosphorylatable mutant ⁇ 2 AR-mCFP (GRK-/PKA-) (Seibold et al, MoI. Pharmacol. 58:1 162-1173 (2000)).
  • WT wild-type ⁇ 2 AR-mCFP
  • GRK-/PKA- non-phosphorylatable mutant ⁇ 2 AR-mCFP
  • the non- phosphorylatable receptor exhibits very low agonist-induced FRET (Fig. 16B).
  • ⁇ -arrestin was highly expressed, and WT and GRK-/PKA- receptor recruited similar amounts of rapidly-associating ⁇ -arrestin.
  • the defect in GRK-/PKA- receptor is thus only in the slow phase of ⁇ -arrestin recruitment. This is consistent with conformation-dependent rapid association and phosphorylation- dependent slow association of ⁇ -arrestin2 with the ⁇ 2 AR. Indeed, as shown below, GRK activity influences only the slow phase of ⁇ -arrestin recruitment.
  • ⁇ -arrestin2 recruitment kinetics can be used to assess both agonist-induced receptor conformation and agonist-induced receptor phosphorylation
  • ⁇ -arrestin 1 is also reported to be less effective than ⁇ -arrestin2 for ⁇ 2 AR internalization (Kohout et al, Proc. Natl. Acad. Sci. USA 98:1601-1606 (2001)), so the kinetics and relative binding affinity of ⁇ -arrestin 1-m YFP and ⁇ - arrestin2-mYFP were compared.
  • the "phosphorylation-independent" mutant ⁇ -arrestin 1-m YFP R169E was tested (Kovoor et al, J. Biol. Chem. 274:6831-6834 (1999)). This mutant exhibits a disrupted "polar core" which consists of closely aligned amino acid side chains of opposite charge.
  • Phosphorylation-independent ⁇ -arrestinl-mYFP (Rl 69E) displays more pronounced biphasic association, with a much larger rapid association than ⁇ -arrestinl-mYFP or ⁇ -arrestin2-mYFP at equivalent expression levels.
  • ⁇ -arrestinl-mYFP (Rl 69E) recruitment, most likely signifying a phosphorylation-dependent affinity.
  • the relative affinity of this mutant is similar to that of ⁇ -arrestin2- mYFP.
  • GRK activity was assessed by two methods: GRK overexpression and GRK silencing by siRNA.
  • Overexpression of GRK2 increased the rate of ⁇ - arrestin2-mYFP recruitment without altering recruitment amplitude (Fig. 19A). Similar results were found with each of the ubiquitous GRKs (GRK2, GRK3, GRK5, and GRK6) (data not shown). This suggests that any of these GRKs are capable, with overexpression, of phosphorylating the ⁇ 2 AR to induce ⁇ -arrestin recruitment. However, this does not address which GRKs are relevant at endogenous expression levels. To address this question, endogenous GRKs were silenced with siRNA as described earlier (Ren et al, Proc. Natl. Acad.
  • GRK6 has the most profound effect. It was verified that this is not an off-target effect of the particular GRK6 siRNA sequence by showing comparable results with a second, independent siRNA sequence for GRK6 (data not shown). Efficiency of GRK silencing was shown to be greater than 90% by immunoblot (Fig. 19C, bottom). Importantly, GRK silencing had no significant effect on the maximum FRET signal, consistent with an unchanged amount of ⁇ - arrestin2 recruited at equilibrium (Fig. 19D). However, the possibility that the nature of the receptor: ⁇ -arrestin interaction is altered by GRK silencing, altering FRET efficiency and masking differences in the amount of ⁇ -arrestin recruited,
  • GRK silencing had the same effects on rate and amplitude of ⁇ -arrestinl-mYFP recruitment as observed for ⁇ -arrestin2-YFP (data not shown).
  • the slowed rate of FRET increase corresponds to a slowed association of ⁇ -arrestin with ⁇ 2 AR.
  • FRET depends on fluorophore orientation as well as proximity
  • some of the FRET increase detected is caused by conformational changes of the ⁇ - arrestin: ⁇ 2 AR complex after the initial interaction. It was possible to discriminate between these two possibilities by measuring ⁇ -arrestin2-mYFP redistribution from cytosol to membrane by tracking directly excited YFP fluorescence in user- defined regions of cytosol and membrane. After isoproterenol stimulation, membrane YFP intensity increases while cytosolic YFP intensity decreases.
  • the ratio of these two signals serves as a direct measure of ⁇ -arrestin translocation, and is independent of FRET.
  • GRK siRNA slows translocation compared to a control siRNA, consistent with a GRK-controlled rate of ⁇ -arrestin association with ⁇ 2 AR (Fig. 24).
  • this finding is inconsistent with FRET changes induced by a GRK-controlled conformational change of the ⁇ - arrestin: ⁇ 2 AR complex.
  • ⁇ 2 AR phosphorylation was then assessed by 32 P incorporation to test if the GRKs that mediate bulk phosphorylation are the same as those required for endogenous ⁇ -arrestin recruitment. To simplify the interpretation of this experiment a mutant ⁇ 2 AR was used that lacks PKA
  • ⁇ 2 AR phosphorylation 85 phosphorylation sites but has unaltered phosphorylation by GRKs (Seibold et al, MoI. Pharmacol. 58:1162-1173 (2000)). Total phosphorylation after 5 minutes is most effectively reduced by GRK2 silencing (Fig. 20B). This indicates that not all ⁇ 2 AR phosphorylation is equivalent for recruiting ⁇ -arrestin, and that there is functional specificity for ⁇ 2 AR regulation by different GRKs. To further examine this possibility, ⁇ 2 AR phosphorylation was tested using an antibody that specifically recognizes phosphorylation of serines 355 and 356 on the ⁇ 2 AR (Tran et al, MoI. Pharmacol. 65:196-206 (2004)). In contrast to bulk ⁇ 2 AR phosphorylation, this site appears to be exclusively phosphorylated by GRK6 (Fig. 20C), confirming that GRKs target different sites on the ⁇ 2 AR.
  • GRK silencing does not appear to alter the amount of ⁇ -arrestin recruited, but only the rate at which recruitment occurs, it is apparent that either 1) the residual GRK expressed after silencing is sufficient, with impaired kinetics, to drive ⁇ -arrestin recruitment, or 2) the untargeted GRK isoforms are responsible for recruitment of ⁇ -arrestin2 after individual GRK silencing by a mechanism of compensatory phosphorylation. Since multiple GRKs (most prominently GRK2 and GRK6) contribute to ⁇ -arrestin recruitment in the HEK-293 cells used here, the latter hypothesis is favored.
  • ⁇ 2 AR phosphorylation sites responsible for ⁇ -arrestin recruitment are shared by GRKs, then ⁇ -arrestin recruitment in a cell type with a different GRK expression pattern should exhibit different sensitivity to GRK siRNA, corresponding to the relative expression of each GRK isoform.
  • a U2- OS osteosarcoma cell line stably expressing ⁇ 2 AR-mCFP and ⁇ -arrestin2-mYFP
  • GRK expression between these cells and the ⁇ 2 AR-mCFP/ ⁇ - arrestin2-mYFP HEK-293 cell line was compared by immunoblot (Fig. 21B).
  • U2-OS express relatively more GRK3 and GRK5, but little to no GRK6.
  • GRK silencing in the U2-OS line revealed that GRK2 and especially GRK3 are most efficacious at promoting ⁇ -arrestin2 recruitment, in correlation with the expression pattern of GRKs in the U2-OS line (Fig. 21C).
  • FRET has been used extensively as a non-destructive way of measuring protein:protein interactions, protein conformational changes, and physiochemical properties in living cells (Zhang et al, Nat. Rev. MoI. Cell. Biol. 3:906-918 (2002)). These approaches have been put to use in many fluorescent biosensors to report intracellular signals.
  • FRET between mCFP-tagged ⁇ 2 AR and mYFP-tagged ⁇ -arrestins is used as a measure of GRK activity.
  • Other groups have shown FRET or BRET, a related biophysical phenomenon, between receptors and ⁇ -arrestins as a method of reporting ⁇ -arrestin recruitment (Azzi et al, Proc. Natl. Acad. Sci.
  • FRET efficiency (the proportion of donor excitation emitted as FRET) depends upon the proximity ( ⁇ 10 nm) and orientation of the two interacting fluorophores (here, mCFP and mYFP). Changes in either proximity or orientation can alter FRET efficiency. Therefore, FRET efficiency does not necessarily directly reflect amount of interaction.
  • ⁇ - arrestin-mYFP can bind the ⁇ 2 AR-mCFP in different conformations, leading to different CFP:YFP orientation and thus different FRET efficiencies.
  • the kinetics and relative affinities of such interactions are independent of maximum FRET efficiency, so these parameters are more rigorous measures of ⁇ - arrestin-mYFP recruitment to ⁇ 2 AR-mCFP.
  • ⁇ 2 AR: ⁇ -arrestin interaction reported by FRET, was agonist-dependent, rapidly reversible, and localized to the plasma membrane (Fig. 15), as expected from previous work showing redistribution of GFP-tagged ⁇ -arrestin (Oakley et al, J. Biol. Chem.
  • GRKs are the rate-limiting step in ⁇ -arrestin recruitment
  • the rate of FRET increase is a functional reporter of GRK activity: any regulation or intervention that alters relevant GRK activity would be expected to alter the rate at which ⁇ - arrestin associates with receptor. Since ⁇ -arrestin binding desensitizes and internalizes the receptor, and results in a set of ⁇ -arrestin dependent signals
  • ⁇ 2 AR affinity for ⁇ -arrestin is regulated by two factors: the agonist-induced receptor conformation and receptor phosphorylation.
  • the agonist-induced conformational change is very rapid (Swaminath et al, J. Biol. Chem. 279:686-691 (2004)) but induces significant amounts of ⁇ -arrestin recruitment only when ⁇ -arrestin expression is very high (Fig. 16A).
  • GRK-mediated receptor phosphorylation leads to higher affinity binding, but at a rate set not by receptor occupancy directly, but rather by GRK activity.
  • Rl 69E mutant displays enhanced affinity for the unphosphorylated, agonist- induced receptor conformation.
  • this mutant is still sensitive to receptor phosphorylation, and thus is, in fact, only partially phosphorylation-independent.
  • this mutant is a useful probe for agonist-induced receptor conformation (mimicking very high expression of wild-type ⁇ -arrestin)
  • any endogenous ⁇ -arrestin associates with unphosphorylated ⁇ 2 AR.
  • non-phosphorylatable ⁇ 2 AR is deficient in desensitization and internalization suggests that these ⁇ -arrestin functions require receptor phosphorylation (Seibold et al, MoI. Pharmacol. 58:1162-1173 (2000)).
  • ⁇ -arrestin expression levels affect the amount of receptor: ⁇ -arrestin complex formed. This may have profound implications for the desensitization, surface expression, and ⁇ -arrestin signaling of
  • ANG Sigma-Aldrich, St. Louis, MO, USA
  • SII Cleveland Clinic core synthesis facility, Cleveland, OH, USA
  • PBS phosphate- buffered saline
  • PBS calcium and magnesium
  • Final concentrations in these assays were 10 ⁇ M for each drug.
  • Frozen stocks in either distilled water or PBS were maintained at 10 mM.
  • the protein kinase C (PKC) inhibitor Ro-3 1-8425 was dissolved in DMSO and stored at a concentration of 1 mM.
  • the angiotensin receptor blocker (ARB) valsartan was dissolved in 100% ethanol to a concentration of 20 mM, and used at a final concentration of 50 ⁇ M in calcium fluorimetry experiments.
  • ARB angiotensin receptor blocker
  • Neonatal rat atrial cardiomyocytes were isolated and cultured as described (Mohler et al, J. Biol. Chem. 277:10599-10607 (2002)). Cells were loaded with the dye Fura-2 (as per manufacturer's instructions, InvitrogenTM), and treated with either ANG (100 nM) or SII (10 ⁇ M), in the absence or presence of pre-treatment with 50 ⁇ M valsartan.
  • the instantaneous 340/3 80 nm excitation ratio (Violin et al, J. Cell Biol. 161 :899-909 (2003)) for Fura-2 was calculated and plotted as a function of time.
  • siRNA Small Interfering RNA
  • GRK2 (5'- AAGAAGUACGAGAAGCUGGAG-3')
  • GRK6 (5'- AACAGU AGGUUUGU AGUGAGC-3').
  • a non-silencing RNA duplex (5'- AAUUCUCCGAACGUGUCACGU-3') was used as a control for all siRNA experiments.
  • HEK293 cells were transfected with Gene Silencer (Gene Therapy
  • HEK293 cells stably transfected with ATi A R-mCFP and ⁇ -arrestin2- mYFP were used to measure GRK functional activity by quantifying the rate and extent of isoproterenol-stimulated ⁇ -arrestin association with the ATi A R as measured by FRET, as described elsewhere (Violin et al, J. Cell Biol. 161 :899- 909 (2003)).
  • FRET was calculated as %F, the percentage of CFP-excited fluorescence detected as YFP emission, corrected for both background signal and for spectral bleedthrough:
  • the FRET signal %F was modeled as an exponential function of time (GraphPad), with rate constant k o b s and equilibrium amplitude %F max , where k obs is a relative measure of GRK activity and %F max depends on expression of AT I A R- ⁇ CFP and ⁇ -arrestin2-
  • mice All animals used in these studies were adult male mice of 8 to 20 weeks of age. All mouse strains were back-crossed to the C57B1/6 background ⁇ 10 generations. Animals were handled according to approved protocols and animal welfare regulations of the Institutional Review Board at Duke University Medical Center. In addition to wild-type (WT) C57B1/6 mice, the following gene-targeted deficient [homozygous or heterozygous knockout (KO), as indicated] mouse strains were used, all of which have been previously described (Jaber et al, Proc. Natl. Acad. Sci. USA 93:12974-12979 (1996); Bohn et al, Science 286:2495-2498 (1999); Conner et al, Circ. Res.
  • 91 of single cells was assessed in real-time using video edge detection (i.e., cardiomyocyte length as a function of time).
  • % Fractional shortening an index of systolic function, was calculated directly from these measurements [(length at end-diastole - length at end-systole)/length at end-diastole].
  • Cardiomyocyte end velocities were derived from the measured length-time relationships, and their minima (maximum contraction; -dL/dt m a x ) and maxima (maximum relengthening; +dL/dt m a x ) are reported as indices of systolic and diastolic function, respectively. 10-15 cardiomyocytes per stimulation condition were assayed in each experiment; n denotes number of animals (i.e., independent experiments).
  • ⁇ -arrestins As noted above, accumulating biochemical evidence suggests that in many cases, one component of G protein-independent signaling via 7TMRs is mediated by ⁇ -arrestins.
  • cardiomyocytes from ⁇ -o arrestin2 KO mice were assayed for responses to ANG and SII, along with cardiomyocytes from a contemporaneous set of WT mice. While WT cardiomyocytes displayed similar increases in % fractional shortening in response to ANG and SII (Figs.
  • ⁇ -arrestin2 KO cardiomyocytes displayed minimal increases in % fractional shortening in response to SII despite preserveds responses to ANG [Fig. 29C - SII versus Basal (p > 0.05), ANG versus SII (p ⁇ 0.05); Fig. 29D - ANG versus SII (p ⁇ 0.05)]. Similar data were observed for -dL/dtmax and +dL/dt max (Fig. 33). These data demonstrate that AT IA R signaling via ⁇ -arrestin2 is required for SII to exert positive inotropic and lusitropic effects.
  • GRK5 KO cardiomyocytes exhibit equivalent increases in % fractional shortening in response to ANG or SII; similar data were observed for - dL/dt m a x and +dL/dt max (Fig. 34).
  • GRK6 KO cardiomyocytes like ⁇ -arrestin2 KO cardiomyocytes, display augmentations in % fractional shortening in response to ANG, but not to SII [Fig. 3OC - SII versus Basal (p > 0.05), ANG versus SII (p ⁇ 0.05); Fig. 3OD - ANG versus SII (p ⁇ 0.05)].
  • ANG may exert its inotropic effects primarily via activation of heterotrimeric G proteins.
  • approximately 30% of ANG-mediated cardiomyocyte functional responses were insensitive to
  • the first mechanism suggests Ca 2+ channel/pump proteins as possible ⁇ -arrestin-dependent effectors and/or interaction partners: (1) the L- type Ca 2+ channel, (2) the IP3 receptor, (3), the ryanodine receptor, and (4) SERCA.
  • myofilament proteins such as myosin, actin, troponin, and tropomyosin, may be effectors or interaction partners of ⁇ -arrestin. Studies examining ⁇ -arrestin interaction with such proteins, or examining the role of ⁇ - arrestin in regulating the functions of these various proteins, remain to be performed.
  • G protein-independent, ⁇ -arrestin/GRK-dependent signaling has been shown to be cytoprotective in several cellular systems, and thus might be beneficial to myocardial function, especially if sustained chronically.
  • cardiomyocytes display reduced cell death in comparison to cardiomyocytes from mice that over-express the WT AT )A R (Zhai et al, J. Clin. Invest. 115:3045-3056 (2005)).
  • Fig. 35A illustrates the effect of SII on mean arterial blood pressure as both percent and absolute changes from basal. SII significantly decreased mean arterial blood pressure in anesthetized wild-type mice (p ⁇ 0.01, Student's modified t-test).
  • Fig. 35A illustrates the effect of SII on mean arterial blood pressure as both percent and absolute changes from basal. SII significantly decreased mean arterial blood pressure in anesthetized wild-type mice (p ⁇ 0.01, Student's modified t-test).
  • 35B illustrates that SII pre-treatment (10 mg/kg) results in a leftward shift in the angiotensin II dose response (p ⁇ 0.05 control vs. post-SII two-way ANOVA w/ Bonferroni).
  • SII can lower blood pressure and acts as a competitive antagonist of angiotensin II in vivo.
  • Ketamine/xylazine induced halothane maintained wild-type c57B16 mice were vagotomized and mechanically ventilated. The mice then had a Millar conductance catheter introduced into the common carotid artery and passed retrograde into the LV cavity, for determining cyclic changes in ventricular pressure and volume, while simultaneously recording systemic arterial pressure via a left axillary arterial catheter. An internal jugular venous catheter was also be placed for the administration of drugs.
  • the effects of AngII or SII on dP/dtmax, dP/dtmin and heart rate are shown in Figs. 36-38, respectively. SII produced significant increases in dP/dtmax, dP/dtmin and heart rate. * /? ⁇ 0.05 response vs. basal, one-way ANOVA and Bonferroni correction for multiple comparisons.
  • Rabbit polyclonal ⁇ -arrestin antibody (AlCT) was generated as described by Attramadal et al (J. Biol. Chem. 267: 17882-17890 (1992)). Detection of pERK was with a rabbit polyclonal anti-phospho-p44/42 MAPK (Cell Signaling Technology, 1 :3,000 for Western blot). Total ERK1/2 was detected with anti-MAPK 1/2 (Upstate Technology Inc., 1 :6,000 for Western blot). Horseradish peroxidase-conjugated secondary antibodies were from Amersham Biosciences. M2 anti-FLAG affinity agarose beads, G418, mouse
  • Plasmids - FLAG- ⁇ 2AR/pcDNA3 (Shenoy et al, J. Biol. Chem. 281 :1261-1273 (2006)) and ⁇ -arrestin2-GFP (Shenoy et al, J. Biol. Chem. 281 :1261-1273 (2006)) were generated as described previously.
  • the ⁇ 2AR-V2R chimera receptor was a generous gift from Dr. Marc Caron (Duke University) and has been described previously (Oakley et al, J. Biol. Chem. 274:17201-17210 (2000)).
  • HEK-293 cells were obtained from ATCC and maintained in modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution (Sigma).
  • HEK-293 lines stably expressing 2pmoles/mg of ⁇ 2AR or ⁇ 2AR TYY were generated as described previously (Shenoy et al, J. Biol. Chem. 281 :1261-1273 (2006)).
  • HEK-293 cells stably expressing either ⁇ 2AR or ⁇ 2AR ⁇ were maintained in Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin solution, and 400 ⁇ g/ml G418 (Sigma).
  • ICUE2 cAMP Assay - Neocin-resistant HEK-293 cells stably overexpressing the human ⁇ 2AR (Shenoy et al, J. Biol. Chem. 281 :1261-1273 (2006)) were stably transfected with a zeocin-resistant plasmid encoding the cAMP biosensor ICUE2 (DiPilato et al, Proc. Natl. Acad. Sci USA 101 :16513- 16518 (2004)). These cells were clonally selected and validated as described (DiPilato et al, Proc. Natl. Acad. Sci USA 101 :16513-16518 (2004)).
  • Intracellular cAMP concentrations were measured as a FRET Ratio: the CFP channel intensity divided by the FRET channel intensity. These channels were detected by excitation through a 438/24 bandpass filter (24 nm bandpass centered at 438 nm) and emission through a 542/27 nm bandpass filter (for the FRET channel) and a 483/32 nm bandpass filter (for the CFP channel). All experiments were performed on a BMG Labtech NOVOstar plate reader using 96-well plates
  • Phospho-ERK and Phospho- ⁇ 2AR Assays were carried out as has previously been described (Shenoy et al, J. Biol. Chem. 281 :1261-1273 (2006)).
  • Immunoblotting was carried out as previously described (Ahn et al, J. Biol. Chem. 279:35518-35525 (2004)) except chemiluminescence detection was performed with horseradish peroxidase-coupled secondary antibody (Amersham Biosciences) and SuperSignal West Pico reagent (Pierce). Chemiluminescence was quantified by a charge-coupled device camera (Syngene ChemiGenius2) according to the manufacturer. Metabolic Labeling and ⁇ -arrestin Translocation Assays - Metabolic labeling and ⁇ -arrestin translocation assays were accomplished according to previously described protocols (Shenoy et al, J. Biol. Chem.
  • HEK-293 cells in 10 cm dishes were transiently transfected with the ⁇ 2V2R chimera (Oakley et al, J. Biol. Chem. 274:17201-17210 (2000)) (consisting of the first 341 aa of the ⁇ 2AR and the last 29 aa of the V2R) and ⁇ -arrestin-2-GFP using FuGENE (Roche Applied Science). Also, images of GFP fluorescence were taken starting 1 minute post-stimulation and subsequently at 1 -minute intervals up to 10 minutes.
  • cAMP accumulation monitored by ICUE2 Typically receptor ligands have been classified as either agonist (full or partial) or antagonist with respect to G protein coupling efficiency. Work over the last 20 years has expanded this classification to include the concept of inverse agonism and it has been observed that numerous classical neutral antagonists actually act as either partial agonists or inverse agonists (Kenakin, Trends Pharmacol. Sci. 25:186-192 (2004)).
  • a FRET based biosensor was used to monitor cAMP concentration in live cells in order to assess the extent to which known ⁇ AR antagonists can stimulate G 5 - dependent AC activation through the ⁇ 2AR (DiPilato et al, Proc. Natl. Acad. Sci USA 101 :16513-16518 (2004)).
  • Acebutolol, alprenolol, atenolol, labetalol, oxprenolol, pindolol and practolol displayed weak partial agonism for G 5 - dependent AC activation (Fig. 39A).
  • Ligands that did not stimulate significant cAMP generation were further analyzed for inverse agonism.
  • Betaxolol, bisoprolol, carvedilol, ICI 118,551, metoprolol, nadolol, propranolol, sotalol and timolol all functioned as inverse agonists decreasing constitutive cAMP accumulation (Fig 39B). It should be noted that, similar to previous reports (Kenakin, Trends Pharmacol. Sci. 25:186-192 (2004)), no neutral antagonists
  • ERK 1/2 activation Recent work has demonstrated that ⁇ -arrestins can serve as scaffolds for signaling networks including the MAPK, ERK 1/2 (DeFea et al, J. Cell. Biol. 148:1267-1281 (2000), Luttrell et al, Proc. Natl. Acad. Sci. USA 98:2449-2454 (2001)) and that this activation can be independent of G protein (Shenoy et al, J. Biol. Chem. 281 :1261-1273 (2006)).
  • HEK-293 cells stably expressing 2pmoles/mg ⁇ 2AR a wide range of phosphorylated ERK 1/2 (pERK) responses can be elicited by different ⁇ AR antagonists (Fig. 40A).
  • Acebutolol, atenolol, alprenolol, carvedilol, labetalol, oxprenolol, pindolol, practolol and propranolol all stimulate significant pERK to varying degrees.
  • a 30 minute treatment with carvedilol stimulated a 1.9 ⁇ 0.09 fold (p ⁇ 0.0001) increase in receptor phosphorylation at the known GRK sites, serine 355/356 (Fig. 41A) and a 1.9 ⁇ 0.1 (p ⁇ 0.0001) fold increaseo over basal in global receptor phosphorylation (Fig. 41B).
  • Carvedilol mediated receptor phosphorylation was blocked by propranolol pretreatment (data not shown).
  • ⁇ arrestin recruitment The ⁇ 2AR exhibits a transient, low affinity interaction with ⁇ -arrestins and undergoes rapid recycling to the plasma 5 membrane after internalization, a pattern known as "Class A" recruitment.
  • Class B receptors such as the vasopressin V2 receptor (V2R) have a more prolonged interaction with ⁇ -arrestin and are recycled to the plasma membrane more slowly than Class A receptors (Oakley et al, J. Biol. Chem. 275:17201-17210 (2000)).
  • V2R vasopressin V2 receptor
  • One previously devised method for increasing ⁇ -arrestin affinity, and therefore o assay sensitivity is to construct chimeric Class A receptors which possess a Class B receptor cytoplasmic tail (Oakley et al, J. Biol. Chem. 274:17201-17210 (2000)).
  • the ⁇ 2AR-V2R receptor chimera is one such example.
  • Carvedilol stimulated 5.5% ⁇ 1.7 (p ⁇ 0.05) receptor internalization, whereas the full agonist isoproterenol stimulated 38.7% ⁇ 3.5 (p ⁇ 0.0001) receptor internalization (Fig. 42B).
  • ⁇ -arrestin2 mediated ERK activation.
  • ERK 1/2 activation after cellular depletion of ⁇ -arrestin2 using ⁇ -arrestin2 specific siRNA in HEK-293 cells stably expressing ⁇ 2AR.
  • pERK was reduced by 42.3% ⁇ 2.2 at 5 minutes whereas for carvedilol, pERK was reduced by 71.0% ⁇ 4.5 (Fig. 43A). This correlated with the overall effect of ⁇ -arrestin2 siRNA to lower cellular levels of ⁇ -arrestin2 by -70% (data not shown).
  • pERK for isoproterenol was reduced by 38.5% ⁇ 5.2 at 5 minutes whereas for carvedilol, pERK was reduced by 70.1% ⁇ 4.4 after ⁇ - arrestin2 silencing (Fig. 43B). This also correlated with the overall efficiency of the ⁇ -arrestin2 siRNA to lower cellular levels of ⁇ -arrestin2 by ⁇ 70% (data not shown).
  • a second siRNA targeting ⁇ -arrestin2 was used to test
  • carvedilol is identified as a compound that possesses the unique signaling profile of negative efficacy for G s -dependent AC activation while simultaneously stimulating ⁇ -arrestin-dependent ERK 1/2 activation. Moreover, carvedilol stimulates phosphorylation of the ⁇ 2AR, ⁇ - arrestin translocation to the receptor, and receptor internalization, all of which are characteristic of ⁇ -arrestin-mediated cellular processes. Thus carvedilol acts as a biased ligand (Violin et al, Trends Pharmacol. Sci. 28(8):416-422 (2007)) signaling via ⁇ -arrestin-dependent ERK 1/2 activation in the absence of G protein activation. This bias may help explain carvedilol's unique clinical effectiveness in heart failure and other cardiovascular diseases.

Abstract

The present invention relates to G protein-coupled receptor ligands having a relative efficacy for stimulating β-arrestin/G protein-coupled receptor kinase (GRK) function (e.g., signaling) that is greater than their relative efficacy for stimulating G-protein signaling function. These 'β-arrestin/GRK biased ligands' can act as agonists or antagonists of G protein-mediated signaling and agonists of β-arrestin/GRK-mediated signal transduction. The invention further relates to methods of identifying such ligands and to methods of using same in a variety of therapeutic settings.

Description

BIASED LIGANDS AND METHODS OF IDENTIFYING SAME
This application claims priority from Provisional Application No. 60/838,474, filed August 18, 2006, the entire contents of which are incorporated herein by reference.
This invention was made with Government support under Grant Nos. RO1HL56689, 2RO1HL016037-33, 5RO1HL070631-04 and RO1HL-56687 awarded by the National Institutes of Health. The Government has certain rights in the invention.
TECHNICAL FIELD
The present invention relates to G protein-coupled receptor ligands having a relative efficacy for stimulating β-arrestin/G protein-coupled receptor kinase (GRK) function (e.g., signaling) that is greater than their relative efficacy for stimulating G-protein signaling function. These β-arrestin/GRK "biased ligands" can act as agonists or antagonists of G protein-mediated signaling and agonists of β-arrestin/GRK-mediated signal transduction. The invention further relates to methods of identifying such ligands and to methods of using same in a variety of therapeutic settings.
BACKGROUND
G protein-coupled receptors (GPCRs), also called seven transmembrane receptors (7TMRs), are the largest, most versatile and most ubiquitous of the several families of plasma membrane receptors. These receptors regulate virtually all known physiological processes in mammals. Moreover, they are the most common targets of currently used therapeutic drugs. Two classical examples of drugs that target these receptors are "β-blockers" and "angiotensin receptor blockers" (ARB 's), which block or antagonize the β-adrenergic receptor for adrenaline or the receptor for angiotensin, respectively. These two classes of drugs are among the most widely used medications for the treatment of a variety of cardiovascular illnesses, including hypertension, heart failure and coronary artery disease.
Drugs that target GPCRs have been developed based on a signaling paradigm in which stimulation of the receptor by an agonist (e.g., adrenaline) leads to activation of a heterotrimeric "G protein", which then leads to second messenger stimulated signaling (e.g., via cAMP) and changes in physiological function (e.g., heart rate). "Blockers" competitively antagonize these actions. The stimulatory effects of agonists like adrenaline and angiotensin II on their respective receptors are also rapidly attenuated by a physiological process called "desensitization" (Koch et al, Annu. Rev. Physiol. 62:237 (2000)). This occurs when stimulated receptors are modified by a G protein-coupled receptor kinase (GRK) or other kinase which phosphorylates activated receptors and facilitates the binding to the phosphorylated receptor by a second molecule, β-arrestin, which then sterically interdicts further coupling to G proteins (Lefkowitz, J. Biol. Chem. 273 : 18677 (1998)) and targets the receptor for internalization (Claing et al, Prog. Neurobiol. 66:61 (2002)). This interaction between β-arrestin and the receptor shuts off further G protein signaling and leads to desensitization.
The present invention results, at least in part, from the finding that the GRK and β-arrestin mechanism not only desensitizes G protein signaling but also leads to signaling in its own right. The signaling pathways that have been identified include certain important enzymatic cascades, such as the ERK MAP kinases, PI3 kinase, and Akt, as well as others (Maudsley et al, J. Biol. Chem. 275:9572 (2000); Luttrell et al, Proc. Natl. Acad. Sci. USA 98:2449 (2001); Pierce et al, Oncogene 20:1532 (2001); Wei et al, Proc. Natl. Acad. Sci. USA 100:10782 (2003); Kim et al, Proc. Natl. Acad. Sci. USA 102:1442 (2005)). These pathways have been shown to be "pro-survival" and generally anti- apoptotic, meaning that they prevent cell death under a variety of stressful conditions.
The invention provides compounds (β-arrestin/GRK biased ligands) with a unique profile. They can act as agonists or antagonists of G protein-mediated signaling and as agonists of G-protein independent β-arrestin/GRK-mediated signal transduction. Thus, while these compounds can act as "blockers" in the classical sense, they can also stimulate potentially cell protective pathways. The invention further provides methods of identifying compounds with this unique profile, that is, compounds that can promote β-arrestin-mediated signaling. The invention additionally provides treatment methods based on the use of these biased ligands (such treatment methods being applicable to human and non- human animals).
SUMMARY OF THE INVENTION
The present invention relates to β-arrestin/GRK biased ligands that can act as agonists or antagonists of G protein-mediated signaling and as agonists of β- arrestin/GRK-mediated signal transduction (agonists biased for β-arrestin signaling can be either G protein independent or can show greater β-arrestin than G protein signaling). The invention also relates to methods of identifying such biased ligands and to methods of using the biased ligands for the treatment of disease. Objects and advantages of the present invention will be clear from the description that follows. BRIEF DESCRIPTION OF THE DRAWINGS
Figures IA- ID. βiAR-mediated transactivation of EGFR requires GRK phosphorylation sites but not PKA phosphorylation sites. HEK 293 cells stably expressing WT β,AR (Fig. IA), PKA' β,AR (Fig. IB), GRK' β,AR (Fig. 1C) or PKA'GRK" β i AR (Fig. 1 D) and transfected with FLAG-EGFR are treated for 5 minutes with lμM dobutamine (Dob) and lOμM ICI 118,551 (ICI, β2AR antagonist) and compared with no stimulation (NS) or lOng/mL EGF stimulation. As indicated, WT βiAR (Fig. IA) and PKA" βiAR (Fig. IB) induce increases in phospho-EGFR and phospho-ERKl/2 in response to treatment with Dob, while GRK" βiAR (Fig. 1C) and PKA'GRK' βiAR (Fig. ID) lack this effect; * p<0.05 versus NS. EGFR internalization following Dob or EGF stimulation for 30 minutes is visualized using confocal microscopic analysis of HEK 293 cells stably expressing the above βiAR mutants and transfected with EGFR-GFP. In the absence of agonist, EGFR-GFP is visualized on the membrane in each stable cell line (Figs. 1A-1D; panel 1, arrows), while EGF stimulation results in redistribution of EGFR-GFP into cellular aggregates (Figs. 1 A-ID; panel 3). Treatment of either WT βiAR or PKA" βiAR cells with Dob results in a similar redistribution of EGFR into cellular aggregates (Figs. IA, IB; panel 2, arrowheads), an effect which is absent in GRK" βiAR and PKA" βi AR cells where EGFR-GFP remains on the membrane (Figs. 1C, ID; panel 2, arrows).
Figures 2A-2C. β-arrestin is required for βiAR-mediated EGFR transactivation. Fig. 2A. HEK 293 cells stably expressing WT βiAR, PKA' βiAR or GRK" βiAR are transfected with GFP-β-arrestin. In the absence of agonist, GFP-β-arrestin (green) has a cytosolic distribution (panels 1, 2, 3). Agonist stimulation (Dob) results in redistribution of GFP-β-arrestin to the membrane in cells expressing WT βiAR and PKA" βiAR (panels 4, 5, arrowheads), whereas no redistribution is observed in cells expressing GRK" βi AR (panel 6, arrows). Fig. 2B. HEK 293 cells stably expressing PKA" βiAR are transfected with FLAG- EGFR alone (MOCK) or with siRNAs targeting β-arrestinl (si-βarrl), β-arrestin2 (si-βarr2), β-arrestinl/2 (si-βarrl/2) or scrambled siRNA (si-Control). Reduced Dob-stimulated phospho-EGFR and -ERKl /2 are observed in cells transfected with siRNA targeting β-arrestin. Fig. 2C. HEK 293 cells stably expressing WT βiAR are transfected with EGFR-GFP and si-Control or si-βarrl/2 to knock down expression (side panel). In the absence of agonist, EGFR-GFP is located at the membrane (panels 1, 5, arrows), while EGF stimulation induces EGFR-GFP redistribution into aggregates (panels 4, 8). Treatment of si-Control transfected cells with Dob or isoproterenol (ISO) also results in redistribution of EGFR into aggregates (panels 2, 3, arrowheads), an effect which is diminished in si-βarrl/2 transfected cells where EGFR-GFP remains at the membrane (panels 6, 7 arrows).
Figures 3A-3F. GRK5 and 6 are required for β-arrestin-mediated EGFR transactivation. Fig. 3A. Stably expressing WT βi AR and GRK* βiAR cells transfected with constitutively active β-arrestin, rβarrR169E, both respond to Dob stimulation by increasing association of β-arrestin and AP2. Fig. 3B. GRK" βiAR cells are transfected with EGFR-GFP, localized to the plasma membrane (panel 1 , arrows), and YFP-rβarrR169E, localized in the cytosol (panel 2, arrowheads). EGF stimulation induces redistribution of EGFR-GFP from plasma membrane to aggregates (panel 7, arrows) with no effect on YFP-rβarrR169E (panel 8, arrowheads). Conversely, Dob stimulation results in redistribution of YFP- rβarrRl 69E from the cytosol to the plasma membrane (panel 5, arrowheads) with no change in EGFR-GFP distribution (panel 4, arrows). Fig. 3C. Transfection of GRK" β i AR cells with constitutively active rβarrRl 69E does not restore EGFR transactivation in response to Dob stimulation. Fig. 3D. HEK 293 cells transiently expressing PKA' βiAR are transfected with FLAG-EGFR alone (MOCK) or with siRNAs targeting ubiquitous GRKs (siGRK2, siGRK3, siGRK5 and siGRKό) or a scrambled siRNA sequence (si-Control). Figs. 3E, 3F. Summary of 6 independent experiments showing significant inhibition of EGFR transactivation (Fig. 3E) and ERK 1/2 activation (Fig. 3F) upon Dob stimulation in the cells transfected with siRNA targeting GRK5 or 6; *p<0.001 versus Dob- stimulated Mock, si-Control, si-GRK2 and siGRK3.
Figures 4 A-4C: In vivo βlAR-mediated EGFR transactivation requires
GRK5, GRK6 and β-arrestin2. β-arrestin2 knockout mice (βarr-2 KO, Fig. 4A), GRK5 KO mice (Fig. 4B), GRK6 KO mice (Fig. 4C) and their wild-type (WT) littermate controls are injected with Dob following ICI pretreatment or with EGF alone. Myocardial lysates are immunoblotted with anti-phospho- and anti-total- ERK 1/2 antibodies (lower panels). Accompanying histograms show summary data of 6 independent experiments depicting the fold increase ERK 1/2 phosphorylation following Dob treatment; * p<0.05 versus ICI. Dob-mediated ERK 1/2 phosphorylation is completely blocked in βarr-2 KO, GRK5 KO and GRK6 KO mice compared to their WT littermate controls.
Figures 5A-5E: Cardiac characteristics of transgenic mice overexpressing mouse WT β, AR, GRK" βiAR or PKA" βiAR. Fig. 5A. Myocardial expression levels of βAR are equivalent in WT β, AR TG (n=6), GRK' βi AR TG (n=5) and PKA' βiAR TG (n=7) mice and approximately 14-fold greater than in their non- transgenic (NTG) littermates (n=15); * p<0.05 versus NTG littermates. Fig. 5B. In vivo hemodynamics show βAR responsiveness as monitored by increase in left ventricular contractility (LV dP/dT max) in WT βiAR (closed circles; n=l 1), GRX' βiAR (closed squares; n=5), PKA" βiAR (open squares; n=7) TG mice and NTG littermates (open circles; n=18). Both GRK" βiAR and PKA" βiAR TG mice show enhanced contractile response; * p<0.05 versus NTG littermates. Fig. 5C. Conscious echocardiography in 5-6 and 12 month old mice indicates no significant differences in fractional shortening among NTG and TG mice at each age in sedentary condition. Figs. 5D, 5E. Immunoblotting of left ventricular lysates of NTG and the 3 lines of βi AR TG mice given intraperitoneal injections of Dob (lmg/kg, 10 min) or EGF (30μg/kg, 15 min) reveals increased ERK1/2 (Fig. 5D) and Akt (Fig. 5E) phosphorylation in NTG, WT βjAR TG and PKA" βiAR TG mice. TG overexpression of GRK" βi AR prevents Dob-mediated
ERK1/2 and Akt activation in the heart. Histograms depict the summaries of fold increase of phospho-ERKl/2 in response to Dob stimulation (n=4-8); * p<0.05 versus control.
Figures 6A-6G. Deterioration of cardiac function in GRK"βi AR-TG mice following chronic ISO treatment. Fig. 6A. WT βiAR TG and NTG mice show βAR downregulation in the LV membrane fraction following chronic ISO treatment (1 week) whereas GRK" βi AR TG and PKA" βiAR TG mice do not; f p<0.01 versus vehicle in corresponding group. Fig. 6B. Adenylyl cyclase activity following acute ISO-stimulation is enhanced in all vehicle-treated mice, but desensitized in all chronic ISO-treated NTG and the 3 lines of βi AR TG mice; *p<0.05 versus vehicle-treated NTG ISO, JpO.05 versus vehicle-treated WT βiAR TG and PKA" βiAR TG ISO, tp<0.01 versus vehicle-treated ISO in each corresponding group. Fig. 6C. Representative M-mode echocardiography pre- and post-chronic ISO in β IAR-TG mice and NTG littermates, with bar scales of 5 mm (vertical) and 1 sec (horizontal). Percent changes from pre-ISO treatment in LV end-diastolic dimension (Fig. 6D) and fractional shortening (FS, Fig. 6E) indicate significant LV dilatation and decreased FS in GRK' βiAR TG mice following chronic ISO treatment; J p<0.01 versus all TG groups, *p<0.05 versus NTG littermates. Fig. 6F. Representative HE, MT and TUNEL staining following chronic ISO treatment reveal increased interstitial fibrosis (blue stain, MT panels) and apoptosis (arrows, TUNEL panels) in GRK" βiAR TG mice. Fig. 6G. Percent TUNEL-positive nuclei following chronic ISO treatment in NTG and the 3 lines of βiAR TG mice (n=5 each); Jp<0.05 versus all groups.
Figures 7A-7E. Pharmacological inhibition of EGFR caused dilated cardiomyopathy following chronic ISO treatment. Fig. 7A. NTG mice are pretreated for 1 hour with Erlotinib (20mg/kg, EGFR antagonist) or DMSO (10%, Control) intraperitoneally, following which Dob (lmg/kg, 10 min) or EGF (30μg/kg, 15 min) are injected intraperitoneally. Immunoblotting of the cardiac lysates reveals increases in Dob- and EGF-stimulated ERK 1/2 phosphorylation which are blocked by Erlotinib pretreatment; * p<0.05 versus control (n=4-6 each). Figs. 7B, 7C. Serial echocardiography parameters (LV end diastolic dimension (Fig. 7B) and FS (Fig. 7C)) following chronic ISO treatment in conjunction with Erlotinib (20mg/kg/day) indicates Erlotinib treatment mimics the cardiac phenotype observed in chronic ISO-treated GRK* βiAR TG mice (Fig 6); * p<0.05 versus each group at each time point. Fig. 7D. Representative
TUNEL staining following chronic ISO with or without Erlotinib shows increased apoptosis (arrowheads) in LV sections from NTG mice undergoing chronic ISO with Erlotinib treatment, as described above. Fig. 7E. Percent TUNEL positive nuclei in LV sections from NTG mice undergoing the following chronic treatments: vehicle+DMSO (n=6), vehicle+Erlotinib (n=8), ISO+DMSO (n=7) and ISO+Erlotinib (n=9); * p<0.05 versus DMSO in same group. Figures 8A-8F. Dob-stimulated EGFR transactivation and ERKl/2 activation requires βiAR expression and is sensitive to EGFR inhibition and β- arrestin function. Fig. 8A. Endogenous βiAR-expressing U2S osteosarcoma cells induce EGFR and ERK 1/2 phosphorylation upon stimulated with Dob (lμM, 5 min) which is prevented by pretreatment AG1478 (1 μM, EGFR antagonist). Fig. 8B. HEK 293 cells lacking endogenous βiAR expression (top panel) do not elicit phosphorylation of ERK 1/2, whereas those transfected with WT βiAR elicit a Dob-stimulated ERKl/2 response that is sensitive to EGFR inhibition. Fig. 8C. HEK cells transfected with WT βiAR are unable to elicit a Dob-stimulated ERKl/2 response in the presence of siRNA targeting either β-arrestinl or 2.
Fig. 8D. Plot indicates the linear relationship between percentage knockdown of β-arrestins and the amount of EGFR transactivation following Dob stimulation of PKA'βlAR cells. Figs. 8E, 8F. Upper panels, HEK 293 cells stably expressing either WT β,AR (Fig. 8E) or PKA' βiAR (Fig. 8F) and transfected with FLAG- EGFR and βArrl V53D which mimics a constitutively phosphorylated, dominant negative state of β-arrestin. Lower panels, WT βiAR (Fig. 8E) or PKA' βiAR (Fig. 8F) stable cells are transfected with EGFR-GFP and βArrl V53D. EGFR- GFP is distributed at the plasma membrane in unstimulated cells (panel 1). βArrlV53D expression inhibits EGFR-GFP internalization following Dob stimulation (panel 2, arrows), whereas the absence of βArrl V53D results in EGFR-GFP internalization (panel 4, arrowheads). EGF stimulation induces EGFR-GFP internalization even in the presence of βArrl V53D (panel 3).
Figures 9A-9C. Transient βl AR expression is sufficient to enable β- arrestin- and GRK-dependent EGFR transactivation and ERKl/2 activation. Figs. 9 A. HEK 293 cells transiently expressing WT βiAR are transfected with FLAG-EGFR alone (MOCK) or with siRNAs targeting GRK 2, 3, 5 or 6. GRK siRNAs reduce Dob-stimulated phospho-ERKl/2 and phospho-EGFR and -ERK1/2, respectively. Fig. 9B. HEK 293 cells transiently expressing WT βiAR and FLAG-EGFR are treated with pertussis toxin (PTX, Gi protein inhibitor), H89 (PKA inhibitor) and siRNA targeting GRK5 or GRK6. Dob-mediated phospho-EGFR and -ERK 1/2 are only prevented by GRK5 or GRK6 siRNA knockdown. Fig. 9C. HEK 293 cells stably expressing WT β i AR or WT AT, AR with or without transient transfection of FLAG-EGFR are treated with Dob, EGF or angiotensin II (All, lμM, 5 min) with or without AG 1478. Dob-induced phospho-EGFR is detectable only with FLAG-EGFR transfection in WT βiAR cells, while ERK 1/2 phosphorylation occurs with either endogenous EGFR or transient FLAG-EGFR expression and is sensitive to AG 1478. All does not induce EGFR phosphorylation in WT AT1 AR cells and All-induced ERK 1/2 phosphorylation is insensitive to AGl 478.
Figure 10. In vivo transactivation of EGFR and activation of ERK1/2 requires GRK5 or GRK6 and β-arrestin2. As described in Figure 4, βarr-2 KO, GRK5 KO, GRK6 KO mice and their WT littermates undergo treatment with Dob. Myocardial lysates are immunoprecipitated with anti-EGFR antibody and blotted for phospho- and total-EGFR. Accompanying histograms show summary data of 6 independent experiments depicting the fold increase EGFR phosphorylation following Dob treatment; * p<0.05 versus ICI. Dob-mediated EGFR phosphorylation is blocked in βarr-2 KO, GRK5 and GRK6 KO mice compared to WT littermate controls.
Figures 1 IA and 1 IB. In vivo transactivation of EGFR is sensitive to
Erlotinib. Fig. HA. Schematic depicting the location of serine and threonine residues mutated in the 3rd intracellular loop and C-terminal tail of WT βiAR in order to generate PKA* βi AR and GRK" P1AR, respectively, for the generation of the 3 lines of βiAR TG mice. Fig. 1 IB. WT βiAR TG, PKA" βiAR TG and GRK" βiAR TG mice are pretreated with Erlotinib followed by intraperitoneal injections of Dob or EGF as described in Figure 7. GRK" βiAR TG mice do not elicit ERK 1/2 phosphorylation in response to Dob, while WT βiAR TG and PKA" βiAR TG do increase phospho-ERKl/2, a response that is completely blocked with pretreatment with Erlotinib.
Figure 12: β-arrestin translocation vs. G protein activation. This graph shows the relative properties of the noted compounds for biologic responses. The vertical axis shows recruitment of β-arrestin proteins, while the horizontal axis shows the generation of c AMP. Compounds with ethyl substitutions on the α- carbon such as cyclopentylbutanephrine, isoetharine, and ethylNE are relatively better for GRK activity compared to their cAMP production, and thus are above the line.
Figures 13A-13C. Application of the GRK activity assay to high- throughput 7TMR ligand studies.
Figures 14A and 14B. Measuring GRK function for biased ligands for the
AT1AR.
Figures 15A-15C. Recruitment of β-arrestin to the β2AR reported by FRET. Fig. 15 A. P2AR-InCFP co-expressed with β-arrestin2-mYFP undergoes FRET upon agonist-stimulated phosphorylation of the receptor. Fig. 15B.
FRETc, the FRET image corrected for spectral bleed-through at each pixel, is detected 15 minutes after addition of 1 μM isoproterenol, and corresponds to translocation of β-arrestin2-mYFP to membrane. To best illustrate the localization of β-arrestin-bound β2AR, %F is displayed as a pseudocolor spectrum, intensity-modulated to correspond to β2AR-mCFP fluorescence to differentiate free β2AR (no FRET, blue) and β-arrestin-β2AR complex (high FRET, red). Fig. 15C. FRET is quantified as a percentage of whole-cell total CFP-excited fluorescence (%F), and shows monophasic kinetics for both isoproterenol-stimulated recruitment and ensuing dissociation by propranolol. Data is representative of 3 independent experiments.
Figures 16A-16C. β2AR-mCFP has two binding affinities for β-arrestin2- mYFP. Fig. 16A. At low expression of β-arrestin2-m YFP (R.F.U.: relative fluorescent units of YFP) isoproterenol-stimulated association is monophasic with a half-time of 90 seconds; at higher expression levels, association is biphasic, with half times of 2 and 90 seconds. Data is from 5 individual cells representative of 25 from 6 separate experiments. Fig. 16B. A phosphorylation-deficient β2AR- mCFP exhibits a rapid association similar to wild-type β2AR-mCFP, but is markedly impaired in the slow association, consistent with a phosphorylation- independent rapid association and phosphorylation-dependent slow association. Data is average +/- s.e.m. of 3 separate experiments. Fig. 16C. The slow association is much higher affinity than the fast association, as described by the fit of a saturable binding curve for each phase. Data are from single cells from 6 independent experiments.
Figures 17A and 17B. A phosphorylation-independent β-arrestin mutant displays enhanced low-affinity binding. Fig. 17A. β-arrestin 1/2 recruitment is monophasic at moderate expression levels, but the recruitment of the phosphorylation-independent mutant β-arrestinl-mYFP R169E expressed at similar levels is biphasic, with an enhanced rapid recruitment. Fig. 17B. Comparing the maximum FRET of β-arrestin isoforms and phosphorylation- independent mutants across a range of expression levels reveals that β-arrestin2 binds the β2AR with significantly higher affinity than β-arrestin 1. The β-arrestin 1 R169E has significantly increased affinity than wild-type β-arrestinl. (* = PO.05). All data are average +/- s.e.m. of 3 independent experiments.
Figures 18A and 18B. The rate and magnitude of β-arrestin recruitment depend on agonist concentration. Fig. 18A. Cells stably expressing both β2AR- mCFP and β-arrestin2-mYFP were stimulated with varying concentrations of isoproterenol. Data is from one of 3 separate experiments. Fig. 18B. The rate (kobs) of β-arrestin-mYFP recruitment is less sensitive to isoproterenol than the final amount of recruitment (%Fmax). Data is average +/- s.e.m. of 3 independent experiments.
Figures 19A-19D. High-affinity β-arrestin2-mYFP binding is kinetically limited by GRKs. Fig. 19A. Overexpression of GRK2 increases the rate of β- arrestin2-mYFP recruitment compared to vector alone. Data is representative of 3 separate experiments. Fig. 19B. siRNA silencing of GRK6 reduces the rate of recruitment of β-arrestin 1-m YFP (wt siGRK6) compared to a scrambled siRNA (wt CTL), and reduces only the slow rate of phosphorylation independent β- arrestinl-mYFP (R169E siGRKό and R169E CTL) without affecting the rapid association. Data is average +/- s.e.m. of 3 separate experiments. Fig. 19C. siRNA silencing of GRK2, GRK3, GRK5, and GRK6 reveals that GRK2 and GRK6 have significant effects on rate (kObs) of β-arrestin2-mYFP recruitment (* = P<0.001). The effect of GRK6 siRNA is most profound, and was noted with two siRNA sequences (GRK6-1 and GRK6-2). A representative immunoblot (IB) for each GRK shows the effectiveness of siRNA silencing. Data is average +/- s.e.m. from three independent experiments performed in triplicate. Fig. 19D. The final amount of β-arrestin recruited (%Fmax) is not altered by GRK silencing, suggesting an enzymatically-limited recruitment of β-arrestin. Data is average +/- s.e.m. of the same experiments as in Fig. 19C.
Figures 20A-20C. β2AR immunoprecipitation reveals distinct GRK specificity for β-arrestin association, bulk phosphorylation, and phosphorylation of a single site in the absence overexpressed β-arrestins. Fig. 2OA. immunoprecipitation of transiently transfected flag-β2AR shows agonist-induced β-arrestin association with the β2AR after 5 minutes of stimulation with 10 μM isoproterenol. Silencing of GRK2 or GRK6 significantly impairs this association (* = PO.01, ** = PO.001). A representative experiment is shown, including immunoblots (IB) for immunoprecipitated β2AR and associated β-arrestin 1/2. Fig. 2OB. PKA-independent phosphorylation of the β2AR was assessed by 32P incorporation into a β2AR mutated at the PKA phosphorylation sites. Silencing of any GRK reveals a loss of agonist-stimulated receptor phosphorylation (* =
PO.001), but GRK2 silencing is significantly more potent than any other GRK (** = P< 0.01). A representative autoradiograph is shown. Fig. 2OC. immunoblotting with an antibody specific for phosphorylation at serine residues 355 and 356 reveals that GRK6 silencing dramatically inhibits phosphorylation at this site (* = PO.001). A representative experiment is shown, including immunoblots (IB) for phosphorylated and total immunoprecipitated receptor. All data is average +/- s.e.m. of 3 independent experiments.
Figures 21A-21D. U2-Osteosarcoma cells utilize a different set of GRKs for β-arrestin recruitment than HEK-293 cells as measured by FRET. Fig. 2 IA. Simultaneous silencing of GRK2 and GRK6 slows recruitment more than silencing either GRK alone (# = PO.01), consistent with a mechanism of compensatory phosphorylation. All treatments were significantly different from control (* = PO.001). An immunoblot is shown for each GRK to verify silencing. Fig. 2 IB. Immunoblotting reveals that U2-OS cells express relatively more GRK3 than HEK-293 cells and less GRK6. For each GRK, an immunoblot is shown of equal amounts of total protein from each cell line. Fig. 21C. siRNA silencing of GRKs reveals that GRK2 and GRK3 are most important for β- arrestin2-mYFP recruitment (each treatment compared to control: * = P<0.05, ** = PO.001). An immunoblot is shown for each detectable GRK to verify silencing (N.D. = not detected) Fig. 21D. Silencing GRK2, GRK3, or GRK2 and GRK3 together significantly slows the rate of β-arrestin recruitment (* = PO.001). Simultaneous silencing of GRK2 and GRK3 is more effective at reducing the rate of β-arrestin2-mYFP recruitment than either GRK alone (# = PO.05), consistent with compensatory phosphorylation between these GRKs. An immunoblot is shown for GRK2 and 3 to verify silencing. All data is average +/- s.e.m. of 3 independent experiments performed in triplicate. All immunoblots are representative of 3 independent experiments.
Figures 22A-22C. Immunoblot analysis of β-arrestin-mYFP overexpression. Equal amounts of protein were separated by SDS-PAGE, transferred to membrane, and immunoblotted with an anti-β-arrestinl/2 antibody. Fig. 22A. HEK-293 cells stably transfected with β2AR-mCFP and transiently transfected with either vector plasmid (pcDNA3.1) or β-arrestin2-mYFP reveal that transient transfection results in approximately 30-fold over overexpression of β-arrestin2-mYFP in comparison to endogenous β-arrestin. Fig. 22B. In the same HEK-β2AR-mCFP cells stably expressing β-arrestin2-mYFP, there is 8-fold more β-arrestin2-mYFP than endogenous β-arrestin. Fig. 22C. U2-0S cells stably expressing β2ARmCFP and β-arrestin2-mYFP express 13-fold more β- arrestin2-mYFP than endogenous β-arrestin in cells expressing only stably transfected β2AR-mCFP. Similar levels of overexpression were found for β- arrestinl-mYFP and β-arrestinl-mYFP (R169E). Relative expression was determined as the ratio of background-corrected intensity of chemiluminescense of the β-arrestin2-mYFP and endogenous β-arrestins.
Figures 23 A and 23B. High-resolution FRET shows β2AR-mCFP:β- arrestin-mYFP complex at the plasma membrane but not in vesicles containing internalized receptor. Fig. 23A. Prior to stimulation (top row), β2AR-mCFP (cyan) is found in plasma membrane, β-arrestin2-mYFP (yellow) is found in the cytosol, and there is almost no detectable FRETc (red, FRET image corrected for spectral overlap at each pixel). A %F pseudocolor scale showing no FRET as blue and high FRET as red, displayed with intensity corresponding to β2AR- mCFP intensity, shows no β-arrestin2-mYFP:β2AR-mCFP complex. In contrast, after 20 minutes of stimulation with 1 μM isproterenol (bottom row), β2AR- mCFP has visibly internalized into vesicles, β-arrestin2-mYFP has translocated to plasma membrane and punctate. FRETc is detected on the plasma membrane but not on vesicles containing internalized β2AR-mCFP. This is most evident in the %F pseudocolor scale, indicating that β2AR-mCFP:β-arrestin2-mYFP complex is only present on plasma membrane. Fig. 23B. A magnified section of the image from Fig. 23 A shows the plasmalemmal-limited FRET. The arrow points to a vesicle containing internalized β2ARmCFP but not β-arrestin2-mYFP, and lacking FRET. The circle-ended arrow points to a punctate structure containing both β2AR-mCFP and β-arrestin2-mYFP and high FRET. This is most likely an aggregation of β2ARmCFP:β-arrestin2-mYFP complex prior to internalization and dissociation of the complex. Figures 24 A and 24B. β-arrestin2-mYFP translocation. Fig. 24 A. β- arrestin2-mYFP translocation was assessed by defining translocation as the ratio of m YFP fluorescence at membrane regions over mYFP fluorescence at perinuclear cytosolic regions in U2-OS cells stably expressing both β2AR-mCFP and β-arrestin-mYFP. This measure of β-arrestin2-mYFP redistribution is independent of FRET and thus corresponds to β-arrestin-mYFP. This measure of β-arrestin2-mYFP redistribution is independent of FRET and thus corresponds to β-arrestin recruitment and not to β-arrestin or β2AR conformational changes. Re- analysis of GRK siRNA experiments show that β-arrestin recruitment is slowed after GRK2 and GRK2 silencing (2/3) compared to control siRNA (CTL), consistent with a GRK-controlled rate of β-arrestin: β2AR association. Fig. 24B. Analysis of the rates of β-arrestin-mYFP translocation show that GRK2 and GRK3 silencing slow recruitment and FRET (Fig. 21D) to similar extent. Similar re-analysis for each GRK in both U2-OS and HEK-293 cells show that slowed translocation correlates with slowed FRET increase (data not shown).
Figure 25. Effects of chronic SII administration on left ventricular systolic function in a genetic model of heart failure in ice [mice with myocardium-specific transgenic (Tg) over-expression of the calcium handling protein calsequestrin (CSQ); these mice develop "spontaneous" biventricular dysfunction and heart failure with a mean lifespan of 16 weeks].
Figure 26. Mobilization of calcium in response to ANG and SII. Neonatal rat atrial cardiomyocytes were loaded with the calcium-binding dye Fura-2, and stimulated either with ANG ( 100 nM), SII (IO μM), or ANG in the presence of pretreatment with the ATiR antagonist valsartan (50 μM). Calcium fluorimetric traces are shown, with the 340/3 80 nm excitation ratio (y-axis) plotted as a function of time (x-axis). Results displayed are mean ± standard error of the mean (SEM) of 3 independent experiments.
Figures 27A-27C. Contribution of specific GRK isoforms to agonist- induced recruitment of β-arrestin2 to the ATi A R- HEK293 cells stably expressing ATiAR-mCFP and β-arrestin2-mYFP were stimulated with ANG (100 nM) or SII (10 μM), in the setting of exposure to the indicated siRNA. Fig. 27 A. Modest effects of GRK2 or GRK6 deficiency on recruitment of β-arrestin2 to the ATi AR- Fig. 27B. Substantial effects of GRK6 deficiency, but not GRK2 deficiency, on recruitment of β-arrestin2 to the ATiAR. Fig. 27C. Representative immunoblot demonstrating efficacy of inhibition of GRK2 or GRK6 expression.
Figures 28A-28C. AT^R-dependent changes in systolic and diastolic cardiomyocyte function mediated by the natural agonist ANG and the biased agonist SII; effects of antagonism of PKC on changes in systolic and diastolic cardiomyocyte function in response to ANG and SII. Fig. 28A. Absence of effects of ANG and SII on fractional shortening of cardiomyocytes from ATi AR KO mice. Fractional shortening of cardiomyocytes from contemporaneous WT (n=4 animals; black bars) and ATiAR-defϊcient (KO) (n=7 animals; white bars) mice, under conditions of pacing alone (Basal), or additional exposure to 10 μM ANG or SII as indicated. In 4 experiments (i.e., 4 individual animals), KO cardiomyocytes were additionally stimulated with 1 μM isoproterenol (Iso; hatched bar). *p < 0.05 by one-way ANOVA with post hoc Bonferroni test relative to pertinent Basal; **p < 0.05 by one-way ANOVA with post hoc Bonferroni test relative to pertinent ATi AR KO (identical stimulation condition). Figs. 28B and 28C. Differential effects of PKC antagonism on inotropic responses to ANG and SII. Fractional shortening [Fig. 28B - absolute values for each variable under indicated stimulation conditions; Fig. 28C - percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment)] of cardiomyocytes from WT mice (n=4 animals), without (black bars) or with (white bars) pretreatment with the PKC inhibitor Ro- 31-8425 (1 μM), under conditions of pacing alone (Basal), or additional exposure to 10 μM ANG or SII as indicated. *p < 0.05 by one-way ANOVA with post hoc Bonferroni test relative to pertinent Basal (Fig. 28B) or ANG (Fig. 28C); **p < 0.05 by one-way ANOVA with post hoc Bonferroni test relative to identical stimulation condition (Fig. 28B only). Data displayed are mean ± SEM. In an individual experiment (i.e., a single animal), 10-15 cardiomyocytes were analyzed for each experimental condition. A mean result was calculated for these cells, and represents a single data point. This was performed for each experiment (i.e., individual animal), and the mean and SEM for a particular experimental condition were determined using n as the number of animals (i.e., independent experiments).
Figures 29A-29F. Effects of deficiency of β-arrestin2 on changes in systolic and diastolic cardiomyocyte function in response to ANG and SII. Figs. 29A and 29B. WT cardiomyocytes display positive inotropic responses to both ANG and SII. Fractional shortening of cardiomyocytes from a series of WT mice (n=12 animals) analyzed contemporaneously with the experiments in Fig. 29B and Fig. 30, under conditions of pacing alone (Basal), or additional exposure to 10 μM ANG or SII as indicated. Fig. 29A. Absolute values for each variable under indicated stimulation conditions. Fig. 29B. Percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment). Figs. 29C and 29D. β-arrestin2 KO cardiomyocytes display severely defective positive inotropic responses to SII, but unaffected responses to ANG. Fractional shortening of cardiomyocytes from β-arrestin2 KO mice (n=5 animals) under conditions of pacing alone (Basal), or additional exposure to 10 μM ANG or SII as indicated. Fig. 29C. Absolute values for each variable under indicated stimulation conditions. Fig. 29D. Percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment). Figs. 29E and 29F. In contrast, β-arrestinl KO cardiomyocytes exhibit equivalent positive inotropic responses to ANG and SII. Fractional shortening of cardiomyocytes from β-arrestinl KO mice (n=5 animals) under conditions of pacing alone (Basal), or additional exposure to 10 μM ANG or SII as indicated. Fig. 29E. Absolute values for each variable under indicated stimulation conditions. Fig. 29F. Percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment). *p < 0.05 by one-way ANOVA with post hoc Bonferroni test relative to Basal, and between ANG and SII when relevant (Figs. 29A/29C/29E; in Fig. 29C, the * for ANG thus represents significance relative to both Basal and SII, whereas in Fig. 29A, the * for either ANG or SII represents significance relative to Basal), and by Student's paired t-test between ANG and SII (Figs. 29B/29D/29F). Data displayed are mean ± SEM. In an individual experiment (i.e., a single animal), 10- 15 cardiomyocytes were analyzed for each experimental condition. A mean result was calculated for these cells, and represents a single data point. This was performed for each experiment (i.e., individual animal), and the mean and SEM for a particular experimental condition were determined using n as the number of animals (i.e., independent experiments).
Figures 30A-30F. Effects of deficiency of specific GRK isoforms on changes in systolic and diastolic cardiomyocyte function in response to ANG and SII. Fig. 30A and 3OB. GRK5 KO cardiomyocytes display positive inotropic responses to both ANG and SII. Fractional shortening of cardiomyocytes from GRK5 KO mice (n=5 animals) under conditions of pacing alone (Basal), or additional exposure to 10 μM ANG or SII as indicated. Fig. 3OA. Absolute values for each variable under indicated stimulation conditions. Fig. 3OB. Percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment). Figs. 30C and 30D. GRK6 KO cardiomyocytes display severely defective positive inotropic responses to SII, but unaffected responses to ANG. Fractional shortening of cardiomyocytes from GRK6 KO mice (n=5 animals) under conditions of pacing alone (Basal), or additional exposure to 10 μM ANG or SII as indicated. Fig. 3OC. Absolute values for each variable under indicated stimulation conditions. Fig. 30D. Percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment). Figs. 30E and 3OF. GRK2 heterozygous KO (+/-) cardiomyocytes display augmented positive inotropic responses to SII. Fractional shortening of cardiomyocytes from GRK2 +/- mice (n=5 animals) under conditions of pacing alone (Basal), or additional exposure to 10 μM ANG or SII as indicated. Fig. 3OE. Absolute values for each variable under indicated stimulation conditions. Fig. 3OF. Percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment). *p < 0.05 by one-way ANOVA with post hoc Bonferroni test relative to Basal, and between ANG and SII when relevant (Figs. 30A/30C/30E; in Fig. 30C, the * for ANG thus represents significance relative to both Basal and SII, whereas in Figs. 3OA and 30E, the * for either ANG or SII represents significance relative to Basal), and by Student's paired t-test between ANG and SII (Fig. 30B/30D/30F); **p < 0.05 by ANOVA with post hoc Bonferroni test for SII relative to both Basal and ANG (Fig. 3OE only). Data displayed are mean ± SEM. In an individual experiment (i.e., a single animal), 10-15 cardiomyocytes were analyzed for each experimental condition. A mean result was calculated for these cells, and represents a single data point. This was performed for each experiment (i.e., individual animal), and the mean and SEM for a particular experimental condition were determined using n as the number of animals (i.e., independent experiments).
Figures 31 and 3 IB. ATi AR-dependent changes in systolic and diastolic cardiomyocyte function mediated by the natural agonist ANG and the biased agonist SII. Fig. 3 IA. Absence of effects of ANG and SII on -dL/dtmax of cardiomyocytes from ATi AR KO mice. -dL/dtmax of cardiomyocytes from contemporaneous WT (n=4 animals; black bars) and ATi AR-deficient (KO) (n=7 animals; white bars) mice, under conditions of pacing alone (Basal), or additional exposure to 10 μM ANG or SII as indicated. Fig. 31B. Absence of effects of ANG and SII on +dL/dtm aχ of cardiomyocytes from ATi AR KO mice. +dL/dtmax of cardiomyocytes from contemporaneous WT (n=4 animals; black bars) and ATi AR-deficient (KO) (n=7 animals; white bars) mice, under conditions of pacing alone (Basal), or additional exposure to 10 μM ANG or SII as indicated. *p < 0.05 by one-way ANOVA withpost hoc Bonferroni test relative to pertinent Basal; **p < 0.05 by one-way ANOVA withpost hoc Bonferroni test relative to pertinent ATi AR KO (identical stimulation condition). Data displayed are mean ± SEM. In an individual experiment (i.e., a single animal), 10-15 cardiomyocytes were analyzed for each experimental condition. A mean result was calculated for these cells, and represents a single data point. This was < performed for each experiment (i.e., individual animal), and the mean and SEM for a particular experimental condition were determined using n as the number of animals (i.e., independent experiments).
Figures 32A and 32B. Differential effects of PKC antagonism on changes in systolic and diastolic cardiomyocyte function in response to ANG and SII. . dL/dtmax (Fig. 32A) and +dL/dtmax (Fig. 32B) of cardiomyocytes from WT mice (n=4), without (black bars) or with (white bars) pretreatment with the PKC inhibitor Ro-31-8425 (1 μM), under conditions of pacing alone (Basal), or additional exposure to 10 μM ANG or SII as indicated. Within Figs. 32A and 32B: (Left) absolute values for each variable under indicated stimulation conditions; (Right) percentage change in each variable in response to ANG or SII, 5 relative to ANG response (indexed as 100% in each experiment). *p < 0.05 by one-way ANOVA with post hoc Bonferroni test relative to pertinent Basal (Left) or ANG (Right); **p < 0.05 by one-way ANOVA with post hoc Bonferroni test relative to identical stimulation condition (Left only). Data displayed are mean ± SEM. In an individual experiment (i.e., a single animal), 10-15 cardiomyocytes o were analyzed for each experimental condition. A mean result was calculated for these cells, and represents a single data point. This was performed for each experiment (i.e., individual animal), and the mean and SEM for a particular experimental condition were determined using n as the number of animals (i.e., independent experiments). 5
Figures 33A-33C. Effects of deficiency of β-arrestin2 on changes in systolic and diastolic cardiomyocyte function in response to ANG and SII. Fig. 33 A. WT cardiomyocytes display positive inotropic and lusitropic responses to ANG and SII, as assessed by changes in -dL/dtmaχ and +dL/dtm aχ- -dL/dtmax o (Left) and +dL/dtmax (Right) of cardiomyocytes from a series of WT mice (n= 12 animals) analyzed contemporaneously with the experiments in Figs. 29 and 30 (and B of this Figure and Fig. 34), under conditions of pacing alone (Basal), or additional exposure to 10 μM ANG or SII as indicated. Within Fig. 33 A: (Above) absolute values for each variable under indicated stimulation conditions; (Below)5 percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment). Fig. 33B. β-arrestin2 KO cardiomyocytes display severely defective positive inotropic and lusitropic +dL/dtm a x. -dL/dtmax (Left) and +dL/dtmax (Right) of cardiomyocytes from β- arrestin2 KO mice (n=5 animals), under conditions of pacing alone (Basal), or additional exposure to 10 μM ANG or SII as indicated. Within Fig. 33B: (Above) absolute values for each variable under indicated stimulation conditions; (Below) percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment). Fig. 33C. β-arrestinl KO cardiomyocytes display equivalent positive inotropic and lusitropic responses to ANG and SII, as assessed by changes in -dL/dtmax and +dL/dtm ax . -dL/dtmax (Left) and +dL/dtmax (Right) of cardiomyocytes from β-arrestinl KO mice (n=5 animals), under conditions of pacing alone (Basal), or additional exposure to 10 μM ANG or SII as indicated. Within Fig. 33C: (Above) absolute values for each variable under indicated stimulation conditions; (Below) percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment). *p < 0.05 by one-way ANOVA with post hoc Bonferroni test relative to Basal, and between ANG and SII when relevant (Above in all panels; in Fig. 33B, the * for ANG thus represents significance relative to both Basal and SII, whereas in Figs. 33A and 33C, the * for either ANG or SII represents significance relative to Basal), and by Student's paired t-test between ANG and SII (Below in all panels). Data displayed are mean ± SEM. In an individual experiment (i.e., a single animal), 10-15 cardiomyocytes were analyzed for each experimental condition. A mean result was calculated for these cells, and represents a single data point. This was performed for each experiment (i.e., individual animal), and the mean and SEM for a particular experimental condition were determined using n as the number of animals (i.e., independent experiments).
Figures 34A-34C. Effects of deficiency of specific GRK isoforms on changes in cardiomyocytes display positive inotropic and lusitropic responses to ANG and SII, as assessed by changes in -dL/dtmax and +dL/dtm aχ . -dL/dtmax (Left) and +dL/dtmax (Right) of cardiomyocytes from GRK5 KO mice (n=5 animals), under conditions of pacing alone (Basal), or additional exposure to 10 μM ANG or SII as indicated. Within Fig. 34A: (Above) absolute values for each variable under indicated stimulation conditions; (Below) percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment). Fig. 34B. GRK6 KO cardiomyocytes display severely defective positive inotropic and lusitropic responses to SII, but unaffected responses to ANG, as assessed by changes in -dL/dtmaxand +dL/dtm aχ. -dL/dtmax (Left) and +dL/dtmax (Right) of cardiomyocytes from GRK6 KO mice (n=5 animals), under conditions of pacing alone (Basal), or additional exposure to 10 μM ANG or SII as indicated. Within Fig. 34B: (Above) absolute values for each variable under indicated stimulation conditions; (Below) percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment). Fig. 34C. GRK2 +/- cardiomyocytes display augmented positive inotropic responses to SII. -dL/dtmax (Left) and +dL/dtmax (Right) of cardiomyocytes from GRK2 +/- mice (n=5 animals) mice under conditions of pacing alone (Basal), or additional exposure to 10 μM ANG or SII as indicated. Within Fig. 34C: (Above) absolute values for each variable under indicated stimulation conditions; (Below) percentage change in each variable in response to ANG or SII, relative to ANG response (indexed as 100% in each experiment). *p < 0.05 by one-way ANOVA with post hoc Bonferroni test relative to Basal, and between ANG and SII when relevant (Above in all panels; in Fig. 34B, the * for ANG thus represents significance relative to both Basal and SII, whereas in Figs. 34A and 34C, the * for either ANG or SII represents significance relative to Basal), and by Student's paired t-test between ANG and SII (Below), **p < 0.05 by ANOVA v/ύ' hpost hoc Bonferroni test for SII relative to both Basal and ANG (Fig. 34C, Above only). Data displayed are mean ± SEM. In an individual experiment (i.e., a single animal), 10-15 cardiomyocytes were analyzed for each experimental condition. A mean result was calculated for these cells, and represents a single data point. This was performed for each experiment (i.e., individual animal), and the mean and SEM for a particular experimental condition were determined using n as the number of animals (i.e., independent experiments).
Figures 35A and 35B. Effects of intravenous bolus doses of SII on systemic arterial hemodynamics. (Fig. 35A) SII decreases basal (post-treatmeht) MAP. (Fig. 35B) SII is a functional competitive ARB.
Figure 36. Effects of SII on left ventricular (LV) systolic function - LV dP/dW.
Figure 37. Effects of SII on LV diastolic function - LV dP/dtmm.
Figure 38. Effects of SII on heart rate.
Figures 39A and 39B. cAMP responses monitored by ICUE2. HEK-293 cells stably expressing β2AR and the cAMP biosensor ICUE2 were treated for 2 minutes with a panel of ligands described as β2AR antagonists (Fig. 39A). cAMP agonism was measured as the rate of change of the ICUE2 FRET ratio corresponding to the rate of cAMP accumulation. Ligands that did not induce cAMP generation were tested for inverse agonism in the same cells (Fig. 39B). These cells exhibit constitutive β2AR activity that, while too weak to generate high basal cAMP, causes a rapid increase in cAMP when phosphodiesterases are inhibited with isoxybutylmethylxanthine (IBMX). This effect is receptor-specific, as there is no IBMX-induced cAMP increase in cells lacking overexpressed receptor (data not shown). Inverse agonism was measured by pretreating cells with ligand for 5 minutes and quantifying the rate of cAMP increase for 30 seconds after IBMX treatment. Inverse agonists are those ligands which do not stimulate cAMP accumulation on their own, and decrease the rate of IBMX- induced cAMP accumulation caused by constitutive receptor activity. Data represent mean ± S. E. from five independent experiments. **p < 0.001 vs. non- stimulated cells (NS).
Figures 4OA and 4OB. ERK activation in β2AR and β2ARτγγ stable cells. HEK-293 cells stably expressing β2AR (Fig. 40A) or β2ARτγγ (Fig. 40B) were stimulated with the panel of β2AR ligands used in Figure 39 at 10 μM for 5 minutes and cell lysates were analyzed for pERK and ERK by Western blot. pERK was normalized to total ERK protein. Data represent mean ± S. E. of at least three independent experiments done in duplicate. Quantification of pERK bands is as a percentage of maximal activity observed for isoproterenol. * p < 0.05 vs. NS, **p < 0.001 vs. NS.
Figures 41 A and 4 IB. β2AR phosphorylation stimulated by carvedilol. HEK-293 cells stably expressing β2AR were stimulated with 10 μM of ligand for 30 minutes and cell lysates were either analyzed for receptor phosphorylation by Western blot (Fig. 41A) or lysates were immunoprecipitated with anti-FLAG beads and analyzed by 32P metabolic labeling (Fig. 41B). Data represent mean ± S. E. of at least five independent experiments. *** p < 0.0001 vs. NS.
Figures 42 A and 42B. β-arrestin2-GFP translocation to the β2AR-V2R and receptor internalization stimulated by carvedilol. HEK-293 cells transiently expressing the β2AR-V2R chimera were stimulated for 2 minutes with either isoproterenol (Iso), carvedilol (Carv), or propranolol (Prop). β-arrestin2-GFP translocation to the β2AR-V2R was then monitored by confocal microscopy (Fig. 42A). Images are representative of six independent experiments. HEK-293 cells stably expressing β2AR were stimulated with 10 μM of ligand for 30 minutes and assayed for internalization by fluorescence-activated cell sorting (Fig. 42B). Data represent mean ± S. E. of 5 independent experiments done in duplicate. * p < 0.05 vs. NS, *** p< 0.0001 vs. NS.
Figures 43A and 43B. Carvedilol stimulated ERK 1/2 phosphorylation is abolished by siRNA targeting β-arrestin2. HEK-293 cells stably expressing β2AR (Fig. 43A) or β2ARτγγ (Fig. 43B) were stimulated with 10 μM of Iso or Carv in the presence of either control siRNA (CTL) or siRNA targeting β- arrestin2 (βarr2) for 5 minutes and cell lysates were analyzed for pERK, ERK and β-arrestin 1/2 by Western blot. Data represent mean ± S.E. of four independent experiments done in duplicate. Quantification of pERK bands is as a percentage of maximal activity observed for isoproterenol. **p < 0.01, ***p < 0.001.
Figures 44A and 44B. Carvedilol stimulated ERK 1/2 phosphorylation is not inhibited by pertussis toxin. HEK-293 cells stably expressing β2AR (Fig. 44A) or β2ARτγγ (Fig. 44B) were stimulated with 10 μM of Iso or Carv for 5 minutes after treatment of either DMSO or pertussis toxin (PTX) for 16 hours as indicated. Cell lysates were analyzed for pERK and ERK by Western blot. Data represent mean ± S.E. of five (Fig. 44A) and four (Fig. 44B) independent experiments done in duplicate. Quantification of pERK bands is as a percentage of maximal activity observed for isoproterenol. **p<0.01 vs. NS, ***p<0.0001 vs. NS.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to ligands for GPCR' s (7TMR' s) that have a relative efficacy for stimulating β-arrestin/GRK function (e.g., signaling) that is greater than their relative efficacy for stimulating G protein signaling function. Such β-arrestin/GRK "biased ligands" can act as agonists or antagonists for G protein-mediated signaling and agonists of β-arrestin/GRK-mediated signal transduction. The invention further relates to methods of identifying such biased ligands and to methods of using biased ligands to treat a variety of diseases/disorders including, but not limited to, pulmonary and cardiovascular disease, allergies/allergic diseases, immunological diseases, psychiatric disorders, psychological disorders, dermatological diseases, neurological diseases, autonomic diseases, inflammatory diseases, endocrine or metabolic diseases (e.g., diabetes and obesity), genitourinary disorders, and opthamological diseases (e.g. glaucoma). The relative efficacies of biased ligands of the instant invention can be readily appreciated from the graph below where the vertical axis is a measure of β-arrestin/GRK-dependent signaling (e.g., recruitment of β-arrestin to the receptor as assayed, for example, by FRET (see Examples below)) and the horizontal axis is a measure of G-protein dependent signaling (e.g., generation of cAMP). GPCR ligands that fall on the dashed (diagonal) line are "unbiased" in that, upon binding to the receptor, their relative effects on β-arrestin/GRK- dependent signaling and G-protein dependent signaling functions are essentially equivalent. The biased ligands of the instant invention, upon binding to the receptor, have a greater positive effect on β-arrestin/GRK-dependent function (e.g., ERK signaling, etc.) than on G-protein dependent signaling function and thus fall above and/or to the left of the dashed line and in the shaded portion of the graph (biased ligands of the invention include inverse agonists of G-protein dependent signaling, that is, ligands with negative efficacy for G-protein signaling falling to the left of the vertical axis and above the horizontal):
Figure imgf000031_0001
-1 The bias of a particular ligand can be expressed using the following equation (1):
tjjoς = r_-(G protein activity)-] r -(β-arrestin functionh where: e = base of the natural logarithm, G protein activity = 2nd messenger activity measurement, as a percentage of the activity of a reference agonist, and β-arrestin function = percentage of a reference agonist function.
In accordance with the invention, the reference agonist can be an endogenous ligand for the GPCR (wherein more than one endogenous ligand for the GPCR exists, the reference agonist is, preferably, the endogenous ligand of highest potency) or an exogenous ligand for the GPCR. For example, a reference agonist for the angiotensin II type 1 receptor is the endogenous ligand, angiotensin II, a reference agonist for the β2AR is the exogenous ligand, isoproterenol, a reference agonist for the αAR is the exogenous ligand, phenylephrine, and a reference agonist for the GPRl 09 A receptor is the ligand, nicotinic acid. In determining bias of a ligand (or candidate ligand (e.g., a test compound)), G protein activity mediated by a GPCR can be measured using any of a wide variety of assays, including those well known in the art. For example, G protein activity can be assayed by determining the level of calcium, cAMP, diacylglycerol, or inositol triphosphate in the presence and absence of the ligand (or candidate ligand). G protein activity can also be assayed, for example, by determining phosphatidylinositol turnover, GTP-γ-S loading, adenylate cyclase activity, GTP hydrolysis, etc. in the presence and absence of the ligand (or candidate ligand). (See, for example, Kostenis, Curr. Pharm. Res. 12(14): 1703- 1715 (2006).) Similarly, β-arrestin function mediated by a GPCR in response to a ligand
(or candidate ligand (e.g., a test compound)) can be measured using any of a variety of assays. For example, β-arrestin function recruitment to the GPCR or GPCR internalization can be assayed in the presence and absence of the ligand (or candidate ligand). Advantageously, the β-arrestin function in the presence and absence of a ligand (or candidate ligand) is measured using by resonance energy transfer, bimolecular fluorescence, enzyme complementation, visual translocation, co-immunoprecipitation, cell fractionation or interaction of β-arrestin with a naturally occurring binding partner. (See, for example, Violin et al, Trends Pharmacol. Sci. 28(8):416-427 (2007); Carter et al, J. Pharm. Exp. Ther. 2:839- 848 (2005).)
One skilled in the art will appreciate that GRK activity can be used as a surrogate for β-arrestin function, β-arrestin function mediated by a GPCR in response to a ligand (or candidate ligand) can thus be reflected by changes in GRX activity, as evidenced by changes in receptor internalization or phosphorylation.
While the relative efficacies for G protein activity and β-arrestin functions for a given ligand (or candidate ligand) acting on a GPCR are preferably determined by assays in eukaryotic cells (e.g., mammalian cells (e.g., human cells), insect cells, avian cells, or amphibian cells, advantageously, mammalian cells), one skilled in the art will appreciate from a reading of this disclosure that appropriate assays can also be performed in prokaryotic cells, reconstituted membranes, and using purified proteins in vitro. Examples of such assays include, but are not limited to, in vitro phosphorylation of purified receptor by GRXs, GTP-γ-S loading in purified membranes from cells or tissues, and in vitro binding of purified β-arrestins to purified receptors upon addition of ligand (or candidate ligand) (with or without GRXs present in the reaction). (See, for example, Pitcher et al, Science 257:1264-1267 (1992); Zamah et al, J. Biol.
Chem. 277:31249-31256 (2002); Benovic et al, Proc. Natl. Acad. Sci. 84:8879- 8882 (1987).)
The above equation (1) measures the distance from the dashed (diagonal) line in the above-presented graph, and expresses that distance, for compounds with G protein activity and β-arrestin function ranging between 0 and 1 , as a number between -0.63 and +0.63, where -0.63 is a perfectly G protein-biased ligand and +0.63 is a perfectly β-arrestin-biased ligand. This range will vary for "superagonists" with activity/function greater than 1 , and/or "inverse agonists" with activity/function less than 0. Full agonists, antagonists (also known as "blockers"), and partial agonists with equal efficacies for both pathways, have a value of zero (as discussed above (and further below), the number resulting from application of the equation above is relative to a reference agonist for a given GPCR). It will be appreciated that two ligands that differ significantly in their ability to stimulate each pathway (i.e., G protein activity and β arrestin function), yet he the same distance off the line, will have the same value in the above equation. As an example, using the Angiotensin II receptor: ligands = AngII (full agonist), SII (β-arrestin biased agonist), valsartan
(antagonist)
AngII Bias = [e^He^] = 0
Sn Bias = [e("0)]-[e(-])] = 0.63 Valsartan Bias = [e('0)]-[e("0)] = 0
The perfect G protein biased ligand for the AngII receptor would be: [e''1']-^'"11'] = -0.63.
By way of further example, if G protein activity mediated by a particular GPCR is determined for reference agonist "X" as being 200 units (as measured by assay "A") and the G protein activity mediated by that GPCR is determined for ligand (or candidate ligand) "Y" as being 50 units (also as measured by assay "A"), the G protein activity of ligand (or candidate ligand) "Y" relative to reference agonist "X" is 50/200 = 0.25. If β-arrestin function mediated by the GPCR for reference agonist "X" is 400 units (as measured by assay "B") and the same β-arrestin function mediated by that GPCR is determined for ligand (or candidate ligand) "Y" as being 300 units (also as measured by assay "B"), then the β-arrestin function of ligand (or candidate ligand) "Y" relative to reference agonist "X" is 300/400 = 0.75. Usingn equation (1), the bias of ligand (or candidate ligand) "Y" is thus: Bias = [e'° 25J - [e"° 75] = 0.78 - 0.47 = 0.31.
The value derived using the above equation (1) for preferred biased ligands of the invention is >0.05, >0.075, >0.1, >0.2, >0.3, >0.4 or >0.5. Biased ligands having a bias value in the range of 0.05-1, 0.075-1, 0.1-1, 0.2-1, 0.3-1,
0.4-1, or 0.5-1 are preferred.
Biased ligands of the instant invention can share with standard β-blockers or ARBs the ability to block the deleterious effects of over-stimulation of either β receptors or angiotensin receptors, which are mediated through G protein activation. This is the property that is responsible for the ability of classical β- blockers and ARBs to lower blood pressure, reduce exercise-induced angina and improve cardiac function in heart failure. However, the biased ligands can simultaneously activate cell protective or other therapeutically beneficial pathways through the β-arrestin dependent mechanism. Currently available and clinically used ARBs have been tested and none have been found to possess this activity (β-arrestin bias).
Biased ligands of the invention include, but are not limited to, catecholamine derivatives with α-carbon alkyl substituents. Such derivatives can be of formula I:
Figure imgf000035_0001
wherein Ri is an alkyl, preferably, a Ci to C4 alkyl, more preferably, ethyl,
R2 and R3 are the same or different and are -H, alkyl (preferably a Ci to C4 alkyl) or cycloalkyl (preferably, cyclobutyl, cyclopentyl or cyclohexyl); alternatively, R2 and R3 together with the nitrogen to which they are attached can form a ring (substituted or unsubstitued), preferably, a C3 to C6 saturated ring, more preferable a C5 saturated ring, and R4 is -OH or -H, or pharmaceutically acceptable salt thereof. Examples of specific biased ligands of formula I include cyclopentylbutanephrine and isoetharine. Compounds of formula I can be synthesized using standard protocols known in the art.
Biased ligands of the invention also include peptides, such as the mutant angiotensin II (Ang) peptide Sarl,Ile4,Ile8-Ang II (SII), as well as derivatives thereof and low molecular weight mimics thereof. SII is a biased agonist for β- arrestin-mediated signaling (ERK activation) and an antagonist for G protein dependent signaling (see Example 7). SII acts to antagonize the G protein- dependent pressor actions of Ang II while directly activating GRK6 and β- arrestin2 dependent signaling (the dependence of this signaling on a particular GRK or β-arrestin isoform is likely tissue specific). As shown in Example 7, this results in positive inotropic effects (augmented myocardial contractibility ex vivo and in vivo) (see also Example 6). SII is a known peptide and, while the invention includes new methods of using this peptide (for example, as an anti-hypertensive, and in the treatment of acute heart failure and other conditions requiring increased cardiac contractility), it does not include this peptide per se.
Biased ligands of the invention additionally include carvedilol and derivatives thereof. Such derivatives can be of formula II:
Figure imgf000036_0001
wherein R is a substituted or unsubstituted linear or branched alkyl, a substituted or unsubstituted cycloalkyl or a substituted or unsubstituted heterocyclic group. Preferably, R is a C1-C6 (advantageously, C1-C4) linear alkyl or a C3-C6 (advantageously, C3 or C4) branched alkyl or a C3-C7 cycloalkyl. More preferably, R is a Cl -C4 alkyl (e.g., -C(CH3)3 or -CH(CH3)2) or R is a cyclopentyl. Carvedilol and (+)-l — (carbazol-4-yloxy)-3- (isopropylamino)-2-propanol are both known compounds and, while the invention can include new methods of using these compounds, it does not include these compounds per se or method of using same in treating hypertension, heart failure or heart attack. Compounds of formula II can be synthesized using standard art- recognized protocols.
The invention further relates to compositions comprising at least one biased ligand formulated with an appropriate carrier. The composition can be in dosage unit form (e.g., a tablet or capsule). The composition can also be present, for example, as a solution (e.g., a sterile solution) or suspension, or as a gel, cream, ointment, aerosol or powder. Approaches suitable for delivering peptide and non-peptide biased ligands of the invention, including oral, transdermal, intrathecal, inhalation, IV, IP, IM, IN, delivery, are known in the art. (See, for example, Morishita et al, Drug Discovery Today 11:905-910 (2006), AIi et al, Letters in Peptide Science 8:289-294 (2002), and Hamman et al, Drug Target Insights 2:71-81 (2007), as well as the references cited in these reviews).
Optimum formulations and dosing regimens can be determined by one skilled in the art and can vary with the biased ligand, the patient and the effect sought.
The present invention also relates to methods of identifying β- arrestin/GRK biased ligands, that is, methods of identifying a biased ligand for a GPCR. Such methods can comprise: i) determining the effect of a test compound on GPCR-mediated G-protein activity, and ii) determining the effect of the test compound on GPCR-mediated β-arrestin function, wherein a test compound that has a greater positive effect on GPCR-mediated β-arrestin function than on GPCR-mediated G-protein activity, relative to a reference agonist for both GPCR- mediated G-protein activity and GPCR-mediated β-arrestin function, is a biased ligand. Such methods can be used to identify a candidate therapeutic that can be used to modulate (e.g., stimulate (enhance) or inhibit) a physiological process. For example, candidate therapeutics can be identified by: i) determining the effect of a test compound on G-protein activity mediated by a GPCR relevant to the physiological process, and ii) determining the effect of the test compound on β-arrestin function mediated by that GPCR, wherein a test compound that has a greater positive effect on β-arrestin function than on G-protein activity mediated by the GPCR, relative to a reference agonist for both the G-protein activity and β- arrestin function mediated by the GPCR, is such a candidate therapeutic. Included in this aspect of the invention are methods of identifying agents suitable for use in treating cardiovascular diseases/disorders (including hypertension, heart failure, coronary artery disease, pulmonary hypertension, peripheral vascular disease or arrhythmia), as well as pulmonary diseases/disorders (such as asthma, chronic obstructive airway disease and pulmonary fibrosis), ophthalmologic diseases/disorders (such as glaucoma), hematologic diseases/disorders (including thrombolytic disorders), endocrine or metabolic diseases/disorders (e.g., diabetes and obesity), neurological or psychiatric diseases/disorders (including
Parkinsonism or Alzheimer's), as well as other diseases/disorders including those referenced below.
One embodiment of this aspect of the invention comprises evaluating the relative efficacy of a test compound to stimulate G protein dependent pathways compared to its efficacy to stimulate β-arrestin/GRK function (e.g., association with the receptor or signaling), for example, to promote β-arrestin membrane translocation (the most proximal event in β-arrestin signaling). A test compound that has a relatively greater efficacy to stimulate β-arrestin/GRK function is a candidate compound for use in treating cardiovascular diseases/disorders, pulmonary diseases/disorders, glaucoma, hematologic diseases/disorders, etc. In a preferred approach, a fluorescence resonance energy transfer (FRET)-based assay is used to assess β-arrestin/GRK function stimulating efficacy. GRK/β- arrestin efficacy can be measured as the rate of β-arrestin recruitment to a receptor in response to ligand, where the receptor/β-arrestin interaction is measured by FRET or bioluminescent resonance energy transfer (BRET). For example, β2AR- mCFP and β-arrestin-mYFP undergo FRET after addition of agonists with a quantifiable rate. This rate of FRET increase is a measure of ligand-stimulated GRK activity, which regulates β-arrestin function, and thus quantifies a ligand's β-arrestin/GRK efficacy. Details of a particularly preferred assay are provided in Example 5. This method can be adapted for use with a fluorescence plate reader for high-throughput screening of agonists and antagonists, which can thereby provide a rapid screen for β-arrestin/GRK biased ligands. β-arrestin/GRK function can be measured for all manner of 7TMRs, e.g., the angiotensin II type 1 receptor.
As noted above, other assays that can be used to measure β-arrestin function include: receptor/β-arrestin co-immunoprecipitation, receptor/β-arrestin crosslinking, receptor/β-arrestin BRET, receptor/β-arrestin bimolecular fragmentation complementation, receptor/β-arrestin translocation imaging, receptor internalization, receptor phosphorylation, and β-arrestin associated phosphorylated ERK (Violin et al, Trends Pharmacol. Sci. 28(8):416-422 (2007)). As described above, approaches that can be used to measure G-protein mediated signaling function include assays for adenylate cyclase and/or cyclic AMP accumulation (ICUE (DiPilato et al, Proc. Natl. Acad. Sci. USA 101 :16513 (2004)), radioimmunoassays, ELISAs, GTPase assays, GTPgammaS loading assays, intracellular calcium accumulation assays, phosphotidyl inositol hydrolysis assays, diacyl glycerol production assays (e.g., liquid chromatography, FRET based DAGR assay (Violin et al, J. Biol. Chem. 161 :899 (2003)), receptor- G protein FRET assays, measures of receptor conformation change, receptor/G protein co-immunoprecipitation, ERK activation, phospholipase D activation, ion channel activation (including electrophysiologic methods), and cyclic GMP changes. (See, for example, Thomsen et al, Curr. Opin. Biotech. 16:655-665 (2005).)
The therapeutic efficacy of the biased ligands of the invention, including those identifiable using the methods described above, can be increased using modifications known in the art to improve pharmacodynamic profile (e.g., increased affinity, etc), to prevent degradation (for peptides this can include N-acetylation and C-amidation, etc), to increase absorption, to allow for different routes of administration and different dosing strategies (including the addition of polyethylene glycol (PEGylation), lipids and protective salting, etc) and to modulate excretion. (See, for example, Morashita et al, Drug Discovery Today 11(19/20):905-910 (2006); Hamman et al, Drug Target Insights 2:71-81 (2007); AIi et al, Letter in Peptide Science 8:289-294 (2002); Whitfield et al, J. Bone Mineral Res. 12(8): 1246 (1997).)
The biased ligands of the invention, which can be identified using, for example, methods described above, can be used in a variety of therapeutic settings, including those described above and below. For example, biased ligands that are βi AR agonists can be used as positive inotropes in the treatment of acute and/or severe systolic ventricular dysfunction/heart failure. Biased ligands that are βiAR antagonists can be used as antihypertensive agents both in acute (emergencies) and chronic settings (negative inotropic/chronotropic mechanism). Such ligands can also be used as cardioprotective/reverse remodeling agents in the treatment of chronic heart failure. Biased ligands that are β2AR agonists can be used as positive inotropes/afterload-reducing agents in the treatment of acute and/or severe systolic ventricular dysfunction/heart failure (e.g. as smooth muscle relaxants in obstetric setting as tocolytics).
Biased β2AR ligands that antagonize Gs-mediated signaling while stimulating β-arrestin mediated signaling (e.g., compounds of formula II) can be used in the treatment of cardiovascular disease and, in particular in heart failure (e.g., chronic heart failure) and post-acute myocardial infarction.
Biased ligands that are D] -dopaminergic receptor agonists can be used as antihypertensive agents with renoprotective/diuretic properties in the acute setting (e.g., emergencies) (the mechanism being arteriolar vasodilator, i.e., used as afterload-reducing agents in the acute setting with concomitant preload reduction.). Biased ligands that are antagonists of this receptor have antiemetic and digestive motility stimulatory effects. Such ligands can be used, for example, in treating gastrointestinal reflux while avoiding confounding neurological effects.
Biased ligands that act as AT i -Angiotensin II receptor antagonists can be used as antihypertensive agents in the chronic setting, promoting arteriolar vasodilation and protection from renal dysfunction (associated with diabetes). Additionally, such compounds can be used as afterload-reducing/reverse remodeling agents in chronic heart failure.
SII and other β-arrestin biased ligands for the ATI angiotensin receptor can be used for the treatment of acute heart failure. SII, for example, can bind the ATI angiotensin receptor and prevent the binding of endogenous angiotensin II, acting as a classical antagonist. Moreover, since SII and other β-arrestin biased ligands for the ATI angiotensin receptor block G protein mediated signaling, they can act as vasodilators to decrease total peripheral resistance (afterload), and inhibit aldosterone secretion preventing sodium and fluid retention. SII stimulates β-arrestin mediated signaling, which has been shown to increase contractility in isolated cardiac myocytes, which would lead to an increase in inotropy of the whole heart. Moreover, stimulation of β-arrestin mediated signaling has been shown to be anti-apoptotic. Patients presenting with late stage acute heart failure are typically given inotropic agents to increase cardiac performance. The βAR agonist dobutamine is a commonly utilized inotropic agent. However, the continuous infusion of dobutamine over periods as short as 24-72 hours is associated with the development of significant tachyphylaxis and, more importantly, an increase in cardiomyocyte apoptosis. Increased cardiomyocyte death can have a negative impact on long-term cardiac function and overall survival. In accordance with the invention, β-arrestin biased ligands for the ATI angiotensin receptor, for example, SII, can be used in combination with or in place of existing inotrope therapies such as dobutamine due to their unique properties described above. Additionally, β-arrestin biased βAR ligands (which are also conventional agonists or antagonists for G protein stimulation) can be used for the treatment of acute heart failure with similar benefits over conventional non-biased agonists as described above for the ATI angiotensin receptor biased ligands.
Biased ligands that act as ETA/ETβ-Endothelin receptor antagonists can be used as anti -pulmonary hypertensive agents both in acute (pulmonary hypertensive crisis) and chronic settings (arteriolar vasodilator mechanism) such ligands can be used, for example, as right ventricular (RV) afterload-reducing agents in the acute and chronic (also reverse remodeling) setting.
Biased ligands that act as Vi -vasopressin receptor agonists can be used as vasoconstrictors in the treatment of acute and refractory distributive shock. Such ligands may have pulmonary vasodilator effects. Biased ligands that act as V]- vasopressin receptor antagonists have investigational use as afterload-reducing agents in heart failure.
Biased ligands of the invention that act as A2A-adenosine receptor agonists can be used diagnostically as coronary arterial/arteriolar vasodilators in myocardial function/perfusion pharmacologic stress studies and therapeutically in the treatment of supraventricular arrhythmias. Biased ligands of the invention that act as prostaglandin Ei receptor agonists can be used to maintain patency of the ductus arteriosus and those that act as prostaglandin Ei receptor antagonists can be used to promote closure of the ductus arteriosus. Biased ligands of the invention that act as prostaglandin I2 receptor agonists can be used as anti-pulmonary hypertensive agents both in the acute (pulmonary hypertensive crisis) and chronic setting, that is, they can be used as RV afterload-reducing agents in the acute and chronic (also reverse remodeling) setting. In the pulmonary area, biased ligands of the invention have uses that include the following .
Biased ligands that act as β2-adrenergic receptor agonists can be used as bronchodilators (airways resistance-reducing agents) in obstructive airways/lung disease both in the acute and chronic setting. Biased ligands that act as β2- adrenergic receptor antagonists can be used investigational^ (Collaerts-Vegh et al, Proc. Natl. Acad. Sci. USA 101 :4948-4953 (2004)) in the long-term management of asthma. Biased ligands that act as Mi -acetylcholine receptor antagonists can be used as bronchodilators (airways resistance-reducing agents) and mucous-reducing agents in obstructive airways/lung disease both in the acute and chronic setting. Biased ligands that act as leukotriene D4 receptor antagonists can be used as anti-inflammatory agents in the management of asthma in the chronic setting.
Specific examples of βarrestin biased ligands (agonists and antagonists) of β2AR that can be used in the treatment of asthma and chronic obstructive pulmonary disease (COPD), as well as other respiratory and pulmonary disorders, include compounds of formula I above (e.g., cyclopentylbutanephrine) (isoetharine and ethylnorepinephrine have been previously used to treat asthma and thus methods of treating asthma using these specific compounds are not within the scope of the present invention). These compounds are all partial agonists for G protein signaling on β2AR and are thus expected to function as classic bronchodialators but with the additional benefits of augmented β-arrestin mediated signaling. The benefits of β-arrestin mediated signaling could also potentially be obtained through β-arrestin biased ligands for β2AR that are devoid of G protein signaling.
In the hematologic area, biased ligands of the invention that act as P2Y12 purinergic receptor antagonists can be used as antiplatelet agents in a variety of settings, most commonly post-percutaneous vascular intervention with stent placement (coronary being most common). They can also be used for thrombotic stroke. Biased ligands that act as adenosine receptor agonists can be used as antiplatelet agents in a variety of thrombotic disorders, most commonly stroke. Biased ligands that act as thromboxane receptor antagonists can be used as antiplatelet agents in a variety of thrombotic disorders, most commonly acute coronary syndrome and its prophylaxis. Biased ligands that act as thrombin receptor agonists can be used topically intraoperatively in the treatment of local bleeding while those that act as antagonists can be used as anticoagulant agents in the setting of contraindication to (or failure of, or complications from) conventional anticoagulation with heparin.
In the allergy area, biased ligands of the invention that act as Hj/H2 - histamine receptor antagonists can be used in the acute setting in the treatment of anaphylactic distributive shock and in the chronic setting as an "anti- inflammatory" in atopic conditions. In the anesthesia area, biased ligands of the invention that act as opioid receptor agonists can be used for pain management and those that act as opioid receptor antagonists can be used for reversal of opioid effects (e.g. drug overdose). Biased ligands that act as Gamma Amino Butyric Acid (GABA) Type B receptor agonists can be used for muscle relaxation.
In the development/oncology areas, biased ligands of the invention that act as smoothened receptor antagonists can be used investigational^ in malignancies 5 that reactivate developmental pathways (e.g., pancreatic and other GI, prostate).
In the gastrointestinal area, biased ligands that act as gastrin receptor agonists can be used in determining stomach acid production. Biased ligands that act as secretin receptor agonists can be used intraoperatively for pancreatic duct identification. Biased ligands that act as serotonin receptor (5HT) Type 3 i o receptor antagonists can be used for management of nausea and vomiting, particularly in patients receiving chemotherapy. Biased ligands that act as cannabinoid receptor agonists can be used to stimulate appetite particularly in patients with cancer while those acting as antagonists can be used in development for appetite suppression. Biased ligands that act as cholecystokinin receptor is agonists can be used to assess biliary dyskinesia prior to cholecystectomy.
In the endocrine/metabolic areas, biased ligands of the invention that act as Parathyroid Hormone (PTH)/Parathyroid Hormone related Peptide (PTHrP) receptor agonists can be used in the treatment of osteoporosis. Biased ligands that act as Thyroid Stimulating Hormone (TSH) receptor agonists can be used in
20 screening for thyroid cancer recurrence. Biased ligands that act as Luteinizing Hormone (LH)/Follicle Stimulating Hormone (FSH)/Gonadotropin Releasing Hormone (GnRH) receptor agonists can be used in the treatment of infertility, endometriosis, and central precocious puberty. Biased ligands that act as Adrenocorticotropic Hormone (ACTH) receptor agonists can be used in various
25 provocative endocrine tests (specifically to test adrenal function). Biased ligands that act as calcitonin receptor agonists can be used in the treatment of osteoporosis, pain following fracture, and management of hypercalcemia. Biased ligands that act as Calcium Sensing Receptor (CaSR) receptor agonists/modulators can be used in the management of hypercalcemia associated with renal failure and for patients with inoperable parathyroid carcinoma. Biased ligands that act as glucagon receptor agonists can be used for the treatment of symptomatic hypoglycemia. Biased ligands that act as growth hormone receptor agonists can be used in patients with pituitary insufficiency and for children with reduced growth. Biased ligands that act as dopamine receptor agonists can be used used in the management of prolactin secreting adenomas and for the treatment of Parkinsonism. Biased ligands that act as somatostatin receptor agonists can be used in the management of diarrhea, neuroendocrine GI malignancies, and growth hormone excess. Biased ligands that act as melanocortin receptor antagonists can be used for the management of weight. Biased ligands that act as ghrelin receptor agonists can be used for management of gastric motility. Biased ligands that act as oxytocin receptor agonists can be used for induction of labor. Biased ligands that act as vasopressin receptor agonists can be used in the management of diabetes insipidus.
In the areas of neurology/psychiatry, biased ligands of the invention that act as dopamine receptor agonists can be used in the treatment of Parkinsonism and those that act as antagonists can be used as anti-emetics and to increase bowel motility. Biased ligands that act as serotonin receptor agonists can be used in the treatment of migraine headaches and those that act as antagonists can be used as antipsychotics. Biased ligands that act as metabotropic glutamate receptor agonists can be used as anticonvulsants and those that act as antagonists can be used in the management of chronic pain. Biased ligands that act as acetylcholine receptor agonists can be used for the treatment of cognitive decline associated with Alzheimer's disease and those that act as antagonists can be used as a sedative prior to anesthesia and as an antispasmodic in disorders characterized by restlessness or agitation (e.g. delirium tremens, Parkinsonism). Biased ligands that act as Neuropeptide Y (NPY) receptor antagonists can be used for reducing caloric intake. Biased ligands that act as orexin antagonists can be used for the treatment of addiction.
In the area of ophthalmology, biased ligands of the invention that act as β- adrenergic receptor antagonists can be used for the management of glaucoma. In addition to the above, biased ligands of the invention that act as melatonin receptor agonists can be used for induction and maintenance of sleep. Biased ligands that act as trace amine receptor agonists (such as tyramine, phenyl ethylamine, tryptamine and octopamine) have cardiovascular effects similar to that observed with β-adrenergic receptors. Further, biased ligands of the invention that act as agonists or antagonists of the ghrelin, orexin, amylin, NPY, cannabanoid, obestatin, and/or melanin concentrating hormone receptors can be used to treat endocrine or metabolic diseases or disorders, such as diabetes and obesity.
Numerous studies have shown that βAR agonists can promote skeletal muscle growth and combat skeletal muscle atrophy associated with aging. The ability of such agonists compounds to affect skeletal muscle cell function, growth and survival has been attributed primarily to the activation of β2ARs, and a number of downstream signaling processes involving G-α-s, G-β-γ and ERK. The actual role of these processes, however, has not been strictly demonstrated, and it is well known that the β2AR can signal through additional pathways, such as those involving the β-arrestins. Moreover, β-arrestin signaling has been shown to be anabolic and anti-apoptotic, processes one might associate with promoting skeletal muscle growth and combating skeletal muscle atrophy associated with aging. In accordance with the present invention, β-arrestin biased ligands for the β2AR, such as cyclopentylbutanephrine, isoetharine and ethyl-norepinephrine, are used for the treatment of skeletal muscle atrophy, sarcopenia, age related frailty and improved healing after skeletal muscle injury. (See, for example, Sumukadas et al, Gerontology 52:237-42 (2006), Emery et al, Biosci. Rep 4:83-91 (1984), Shi et al, Am. J. Physiol. Cell Physiol. (12/6/06).doi:10.1152/ajpcell.00466.2006, Mersmann, J. Anim. Sci. 76:160-172 (1998), Doherty, J. Appln. Physiol. 95:1717-1727 (2003), Marzetti et al, Exp. Geron. 41 :1234-1238 (2006), Yimlamai et al, J. Appl. Physiol. 99:71-80 (2005), Libera et al, J. MoI. Cell. Card. 38:803- 807 (2005), Bodine, Med. Sci. Sports Exer. 1950-1957 (2006), Roubenoff, J. Geron. 58 A: 1012-1017 (2003), Glass, Nat. Cell Biol. 5:87 (2003), Kline et al, J. Appl. Physiol. 102:740-747 (2007), Solomon et al, J. Endocin. 191 :349-360 (2006), Gregorevic et al, Am. J. Physiol. Heart Circ. Physiol. 289:H344-H349 (2005); Beitzel et al, J. Appl. Physiol. 96:1385-1392 (2004), Ryall et al, Am. J. Physiol. Regul. Integr. Comp. Physiol. 283:R1386-R1394 (2002), Ryall et al, Brit. J. Pharmacol. 147:587-595 (2006), Ryall et al, J. Physiol. 555.1 :175-188 (2003).) In addition to the above, β-arrestin biased ligands of the invention can also be used in the treatment of diseases/disorders associated with physiological processes mediated by one or more of the following receptors: the α-adrenergic, receptor, ADP receptor, apelin (APJ) receptor, prostaglandin receptor, neurotensin receptor, neuromedin U & B receptor, cholecystokinin receptor, chemokine receptor, endothelin receptor, orexin receptor, bradykinin receptor, GP30 receptor, mas receptor, ghrelin receptor, amylin receptor, GPRl 09a receptor, calcitonin receptor, calcitonin gene related peptide receptor, adrenomedulin receptor, rhodopsin receptor, cone opsin receptor, frizzled recpeptor, bombesin receptor, C5a anaphylatoxin receptor, FMLP receptor, interleukin receptor, prolactin releasing peptide receptor, neurotensin receptor, tachykinin receptor, conopressin receptor, galanin receptor, thrombin receptor, protease activated receptor, neuropeptide FF receptor, orexigenic neuropeptide QRFP receptor, urotensin receptor, GPR37 receptor, neuromedin U receptor, melanin concentrating hormone receptor, prokineticin receptor, leutropin- choriogonadatropic receptor, thyrotrophin receptor, gonadatropin receptor, olfactory receptor, taste receptor, purinoceptor, platelet activating receptor, metatonin receptor, viral receptor, EDG receptor, RDCl receptor, EBV induced receptor, orphan receptor, LGR-like receptor, GPR receptor, GPR45-like receptor, cysteinyl leukotriene receptor, free fatty acid receptor, corticotrophin releasing factor receptor, gastric inhibitory peptide receptor, PACAP receptor, VIP receptor, diuretic hormone receptor, EMRl receptor, latrophilin receptor, ETL receptor, brain-specific angiogenesis inhibitor receptor, cadsherin EGF LAG receptor, very large G-protein coupled receptor, metabotropic glutamine receptor, ocular albinism protein receptor, and vomerolnasal receptor. Certain aspects of the invention can be described in greater detail in the non-limiting Example that follows. (See also Wei et al, Proc. Natl. Acad. Sci. USA 100:10782-10787 (2003); Wei et al, J. Biol. Chem. 279:48255-48261 (2004); Barnes et al, J. Biol. Chem. 280:8041-8050 (2005); Ren et al, Proc. Natl. Acad. Sci. USA 102:1448-1453 (2005); Kim et al, Proc. Natl. Acad. Sci. USA 102:1442-1447 (2005); Ahn et al, J. Biol. Chem. 297:7808-7811 (2004); Ahn et al, Proc. Natl. Acad. Sci. USA 100:1740-1744 (2003); Ahn et al, J. Biol. Chem. 279:35518-35525 (2004); Gesty-Palmer et al, J. Biol. Chem. 281 :10856-10864 (2006); Hunton et al, MoI. Pharm. 67:1229-1236 (2005); Rajagopal et al, Circulation 112(17):U237-951 Suppl. 5 (2005); Rajagopal et al, J. Clin. Invest. 115(11):2971 (2005)). (The entirety of U.S. Provisional Application
Nos. 60/902,353 and 60/907,439 are also incorporated herein by reference.)
EXAMPLE 1
Experimental Details Cell Culture HEK 293 cells were maintained as previously described (Naga Prasad et al, J.
Cell. Biol. 158:563 (2002)). The cells were transfected with cDNA encoding 2 μg FLAG-EGFR, EGFR-GFP, β-arrestin-GFP, β-arrestinl V53D or rβarrR169E- YFP with lipofectamine reagent (Invitrogen, CA). Transfected cells were incubated overnight in serum-free medium supplemented with 0.1% bovine serum albumin (BSA), 1OmM HEPES (pH 7.4), and 1% penicillin prior to stimulation. Under serum starvation condition, cells were preincubated with ICI-118, 551 for 30 min and then stimulated with Dob or EGF for 4-5 min. Cell lines stably expressing WTp1AR, PKA' βiAR, GRK' βiAR and PKA7GRK' βiAR have been previously described (Rapacciuolo et al, J. Biol. Chem. 278:35403 (2003)). U2S sarcoma cell line was maintained in minimal essential medium supplemented with 10 % FBS and antibiotics.
Immunoprecipitation, immunoblotting and detection
Following stimulation, cells were washed once with PBS, solubilized in ImI of lysis buffer (5mM Hepes, 25OmM NaCl, 10% glycerol, 0.5% Nonidet P- 40, 2mM EDTA and protease inhibitors) as previuosly described (Naga Prasad et al, Nat. Cell. Biol. 7:785 (2005)). Prior to immunoprecipitation, 25 μl of whole cell lysates was aliquoted into separate tube for protein estimation and analyzing total cellular phospho-ERKl/2. Immunoprecipitation of the FLAG epitope was carried out as previously described (Rapacciuolo et al, J. Biol. Chem. 278:35403 (2003)). Immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose membrane or PVDF (BioRad) for immunoblotting. Anti- phosphotyrosine (PY20) (BD Transduction Laboratories, Lexington, KY) was used to detect tyrosine phosphorylation of EGFR at 1 :3000 and phospho-ERKl/2 (Cell Signaling) was also used at 1 :3000. Immunoblotting for total EGFR (Upstate) and total ERK (Santa Cruz) was carried out at 1 :2000. EGFR from the myocardial lysates was immunoprecipitated using anti-EGFR antibody (Upstate) and immunoblotted for phospho-EGFR using anti-phospho-EGFR (Tyr845) (Cell Signaling) at 1 :1000. Detection was carried out using enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech). Densitometric analysis was performed using BioRad Fluoro-S Multi-image software, β-arrestin immunoblotting was carried out using AlCT rabbit polyclonal antibody at a 5 dilution of 1 :3,000 as previously described (Wei et al, Proc. Natl. Acad. Sci. USA 100: 10782 (2003)). Anti-GRK specific antibodies (Santa Cruz, CA) were used to detect GRK 2, 3, 5 and 6 as described previously (Ren et al, Proc. Natl. Acad. Sci. USA 102:1448 (2005)). β-actin and FLAG immunoblotting were carried out using monoclonal antibodies at dilutions of 1 :3000 each (Sigma). i o Immunoblotting for myocardial pERK and pAkt was performed as previously described (Perrino et al, J. Clin. Invest. 116:1547-1560 (2006)). Hearts were homogenized with NP -40 lysis buffer containing 137 mmol/1 NaCl, 20 mmol/1 Tris pH 7.4, 1% NP-40, 20% glycerol, 10 mmol/1 PMSF, 1 mmol/1 Na3VO4, 10 mmol/1 NaF, 2.5 μg/ml aprotinin, and 2.5 μg/ml leupeptin,
15 phosphatase inhibitor cocktail I and II (Sigma).
Confocal Laser Microscopy
Confocal microscopy was performed as previously described (Naga Prasad et al, Nat. Cell. Biol. 7:785 (2005)). Briefly, HEK 293 cells stably
20 expressing FLAG tagged WTβiAR and βiAR mutants were transfected with cDNA encoding fluorescently labeled EGFR (EGFR-GFP) (2 μg). Following transfection, cells were trypsinized and plated onto 35 mm glass-bottomed culture dishes (MatTek Corporation). Following stimulation, cells were washed once with PBS and fixed in 4% paraformaldehyde for 30 minutes. EGFR receptor
25 internalization following Dob, ISO or EGF stimulation was visualized by green fluorescence using single sequential line of excitation filters. EGFR-GFP internalization and βarrR169E-YFP recruitment was visualized using a combination of excitation 488 and emission filters between 499-520 nm. YFP visualization was carried out with LSM meta (520-552 nm).
siRNA experiments targeting β-arrestins and GRKs siRNA targeting β-arrestin 1 , 2 or both were generated by BLAST search algorithim for a unique 21 nt sequence for β-arrestins from National Center for Biotechnology Information. The sequence of the 21 nt siRNA's has been previous described (Ahn et al, J. Biol. Chem. 279:35518 (2004), Ahn et al, J. Biol. Chem. 279:7808 (2004)). The sequence of siRNA's targeting GRK 2, 3, 5, or 6 have been previously described (Ren et al, Proc. Natl. Acad. Sci. USA 102: 1448 (2005), Kim et al, Proc. Natl. Acad. Sci. USA 102:1442 (2005)). 30-40% confluent HEK 293 cells stably expressing either PKA' βiAR or WT βiAR in 6 well dishes were transfected with 0.2 μg of FLAG-EGFR and 3.5 μg of siRNA using the GeneSilencer Transfection reagent (Gene therapy systems) as previously described (Ren et al, Proc. Natl. Acad. Sci. USA 102: 1448 (2005)).
For confocal microscopy experiments with β-arrestin knockdown, WT βl AR cells underwent transfection with 1 μg EGFR-GFP and 3.5 or 7 μg of siRNA targeting both β-arrestins and control siRNA, and were replated onto 35mm glass-plated culture dishes after 60 hours. For GRK knockdown experiments, HEK 293 cells were transfected with 0.2 μg of FLAG-EGFR, 4 μg of siRNA targeting either GRK 2, 3, 5 or 6 and an appropriate amount of plasmid cDNA encoding either WT βiAR or PKA" βiAR. The expression of the receptors in transient transfection was 475-600 fmol/mg protein. All assays were performed 60-72 hours after siRNA transfection. Cells were serum starved for 12 hr before stimulation. Treatment protocol for mice
C57BL/6 mice or β-arrestin-2 knockout, GRK5 knockout or GRK6 knockout mice were administered with ICI 118,551 (5 mg) intra-peritoneal. After 5 min of ICI pre-treatment, mice were administered with saline, βiAR specific agonist Dob (10 mg/kg) as a single intra-peritoneal dose or epidermal growth factor (EGF) (300 μg/kg) as a single intra-venous bolus. Following a wait of 5 min after Dob or EGF administration, the experiment was terminated. The heart was excised and flash frozen in liquid N2 for biochemical assays.
Histological analysis
Heart specimens were fixed with 10% neutral buffered formalin, embedded in paraffin, and sectioned at 5-μm thickness. Sections are stained with Hematoxylin-Eosin (HE) and Masson Trichrome (MT). DNA fragmentation was detected in situ using TUNEL (Perrino et al, J. Clin. Invest. 116:1547-60 (2006)). In brief, deparaffinized sections were incubated with proteinase K, and DNA fragments were labeled with fluorescein-conjugated dUTP using TdT (Roche Diagnostics Corp.). The total number of nuclei was determined by manual counting of DAPI-stained nuclei in 6 fields of each section using the *200 magnification, and the number of TUNEL-positive nuclei was counted in the entire section.
Membrane Fractionaton, βAR Radioligand Binding and Adenylyl cyclase activity Plasma membrane and cytosolic fractions from left ventricles flash frozen in liquid N2 were separated by centrifugation at 37,00Og as previously described (Esposito et al, Circulation 105:85 (2002); Perrino et al, J. Clin. Invest. 116: 1547- 60 (2006)). Receptor binding with 20μg of protein from plasma membrane was performed using [125I] cyanopindolol (35OpM) as described previously (Esposito et al, Circulation 105:85 (2002)). Receptor density (fmol) was normalized to milligrams (mg) of membrane protein. Adenylyl cyclase assays were performed as described previously (Esposito et al, Circulation 105:85 (2002)) using 20μg of membrane fraction. Generated cyclic adenosine monophosphate (cAMP) was quantified using a liquid scintillation counter (MINAXI-4000, Packard Instrument Co., PerkinElmer, Boston, Massachusetts, USA)
Generation of Transgenic mice
The FLAG-tagged mouse wild type βl -adrenergic receptor (WTβlAR) and two different mutants lacking either the putative GRK phosphorylation sites (GRK'βlAR) or the putative PKA phosphorylation sites (PKA'βlAR) were directionally subcloned into a vector downstream of α-myosin heavy chain gene promoter and upstream of the SV40 polyadenylation site (Nienaber et al, J. Clin. Invest. 112:1067 (2003)). The βiAR mutants were generated by mutating the following serine/threonine amino acids to alanine at residues 412, 417, 426, 427, 437, 443, 444, 445, 448, 450, 451, 461, 462, 464 for the GRK'βlAR mutant and the four serine amino acids to alanine at residues 295, 296, 301, and 401 in PKA" βlAR (Fig. 10). Transgenic founders were identified by Southern blot analysis of tail DNA using the SV40 poly (A) as a probe. Transgenic founder mice were backcrossed into a C57BL/6 background for at least 4 generations before being used in experiments to investigate the phenotype. Mice were screened by PCR with sense primer, 5'-CATGGGTGTGTTCACGCTC, located in mouse βlAR coding sequence, and an antisense primer, 5'-CCTCTACAGATGTGATATGGC, located in SV40 poly (A) sites. βAR radioligand binding assay was carried out on multiple generations to analyze and confirm the receptor expression. Animals were handled according to the approved protocols and animal welfare regulations of the Institutional Review Board at Duke University Medical Center. Echocardiography
Echocardiography was performed on conscious mice with either HDI5000 echocardiograph (Philips) or Veno770 (Visualsonics, Inc, Toronto, Canada) as described previously (Esposito et al, Circulation 105:85 (2002); Perrino et al, J. Clin. Invest. 116:1547-60 (2006)).
Mini-osmotic pump implantation
Mini-osmotic pumps were implanted as described previously (Nienaber et al, J. Clin. Invest. 112:1067 (2003)). Isoproterenol (ISO) was dissolved in 0.002% ascorbic acid, and pumps (Alzet model 1007D; DURECT Corp., Cupertino, California, USA) are filled to deliver at rate of 3mg/kg/day over a period of 7 days. In control mice, vehicle (0.002% ascorbic acid) was used. In erlotinib treatment, mice were given erlotinib (20mg/kg/day) intraperitoneally for 3 weeks. Erlotinib were dissolved into dimethyl sulfoxide (DMSO), and diluted to 10%DMSO with 0.9% physiological saline. The control mice in erlotinib treatment were given an equal volume of 10%DMSO. Echocardiography was performed pre and post ISO treatment.
Statistical Analysis
Data are expressed as mean±SE. Statistical significance was determined using one-way ANOVA (with Bonferroni correction for multiple comparisons). p< 0.05 was considered significant.
Results
Stimulation of βiARs induces EGFR transactivation
A determination was made as to whether βiAR stimulation could mediate EGFR transactivation and induce activation of downstream signaling pathways. Cells stably expressing WT βiARs were transfected with FLAG-EGFR and stimulated with either the βiAR specific agonist Dobutamine (Dob) or EGF following pretreatment with the β2AR specific antagonist ICI-118,551 (ICI), to block endogenous β2ARs. Stimulation with Dob resulted in a significant increase in phosphorylation of the EGFR along with activation of ERK, which was somewhat less marked than direct stimulation with EGF ligand (Fig. IA upper panel). To test whether activation of βiARs leads to internalization of EGFRs cells, stably expressing WT βjARs were transfected with green fluorescent protein tagged-EGFR (GFP-EGFR) and stimulated with Dob or EGF. EGFR internalization was visualized by confocal microscopy. In the absence of agonist, EGFR has a uniform membrane distribution (Fig. IA, lower panel 1, arrows). In contrast, Dob stimulation results in internalization of EGFRs as visualized by marked redistribution into cellular aggregates (Fig. IA, lower panel 2, arrowheads). Dob-induced EGFR internalization was qualitatively similar to that evoked by EGF treatment itself (Fig. IA, lower panel 3).
Phosphorylation ofβjAR by GRK is necessary for EGFR transactivation
To test whether GRK mediated phosphorylation of the βiAR following agonist stimulation is a required step in EGFR transactivation, the mouse wild type βiAR (WT) and 3 phosphorylation site deficient mutants were used that had been previously cloned and characterized (Rapacciuolo et al, J. Biol. Chem.
278:35403 (2003)). The 3 phosphorylation defective mutants contain alanine for serine or threonine substitutions at: 1) the 4 putative PKA phosphorylation sites in the third intracellular loop and proximal carboxy tail (PKA" βi AR), 2) the 14 putative GRK phosphorylation sites within the carboxy tail (GRK' βiAR), and 3) both sets of phosphorylation site substitutions (PKAVGRK' βi AR) (Rapacciuolo et al, J. Biol. Chem. 278:35403 (2003)). To investigate whether GRK phosphorylation of the βi AR is required for EGFR transactivation, HEK 293 cells stably expressing PKA" βiAR (receptors with only GRK phosphorylation sites) were stimulated with agonist and EGFR activation measured. Dob stimulation resulted in robust phosphorylation of the EGFR and ERK activation (Fig. IB), albeit somewhat less than direct EGF stimulation. Agonist stimulation of PKA' βiARs also triggered robust EGFR internalization as shown by the coalescence of GFP fluorescence into bright patches within the cell (Fig. IB, lower panel 2, arrowheads), which again was comparable to direct EGF stimulation (Fig. IB, lower panel 3).
To test whether GRK phosphorylation sites are essential for EGFR transactivation, GRK" βiAR (receptors with only PKA phosphorylation sites) expressing cells were transfected with FLAG-EGFR. Dob stimulation of GRK" βiAR showed no significant EGFR transactivation, minimal ERK activation (Fig. 1C) and absence of EGFR internalization (Fig. 1C, lower panel 2). As expected, β,ARs lacking both PKA and GRK phosphorylation sites also failed to trigger EGFR transactivation (Fig. 1 D). Additionally, using U2 S sarcoma cells expressing endogenous βiARs, and HEK 293 cells transfected with βiARs, strong EGFR phosphorylation and ERK activation were shown upon treatment with Dob, that was blocked by pretreatment with the specific EGFR antagonist AGl 478 (Fig. 8A, 8B). Taken together, these results demonstrate that, in response to βiAR agonist stimulation in several cell types, the EGFR is transactivated and the mechanism for transactivation requires phosphorylation of the βiAR on consensus GRK phosphorylation sites, a process known to promote β-arrestin recruitment and activation (Lefkowitz and Shenoy, Science 308:512 (2005)).
Both β-arrestin-1 and -2 are required for βiAR transactivation of EGFR Previous studies have shown that recruitment of activated c-Src to the β2AR complex by β-arrestin is needed for ERK activation (Luttrell et al, Proc. Natl. Acad. Sci USA 98:2449 (2001)), Luttrell et al, Science 283:655 (1999)). To test the possible role of β-arrestin in EGFR transactivation, HEK 293 cells stably expressing either WT βiAR or βiAR mutants were transfected with GFP-β- arrestin and stimulated with Dob. Robust membrane translocation of GFP-β- arrestin was seen in cells expressing WT βiARs and PKA" βiARs (Fig. 2 A, panels, 4 and 5, arrowheads), but was completely absent in cells expressing GRK" βiARs (Fig. 2A, panel 6, arrows).
To directly determine the requirement of β-arrestin- 1 or -2 for βi AR- mediated EGFR transactivation, siRNA was used that specifically targets either β- arrestin-1 or -2 or both (Ahn et al, J. Biol. Chem. 279:7807 (2004)). Dob stimulation of PKA" βiARs resulted in intense phosphorylation of the EGFR associated with significant ERK activation in mock and scrambled siRNA transfected cells (Fig. 2B). In contrast, transactivation of the EGFR and associated ERK activation were significantly blocked in the presence of siRNA targeting either β-arrestin- 1 or -2 or both (Fig. 2B). Immunoblotting for β-arrestin shows selective and effective depletion of specific β-arrestins in the presence of their respective siRNA's consistent with previous studies (Fig. 2B, lower panels) (Ahn et al, J. Biol. Chem. 279:7807 (2004)). Similar data was obtained when using the WT βiARs (Fig. 8C). Moreover, siRNA targeting of β-arrestin 1/2 blocked EGFR internalization following either Dob or isoproterenol stimulation, but not with EGF stimulation (Fig. 2C, panels 6 and 7, arrows). Transfection with siRNA targeting β-arrestinl/2 blocked Dob-mediated EGFR internalization in a dose dependent fashion, consistent with data showing a concentration-dependent relationship between the level of β-arrestin knockdown and βiAR-mediated EGFR transactivation (Fig. 8D). Lastly, consistent with the siRNA experiments, transactivation of the EGFR was also completely blocked in WT βiAR or PKA' βiAR expressing cells in the presence of dominant negative β-arrestin (βArrV43D) (Fig. 8E, 8F).
Inability of a constitutively activated β-arrestin to rescue GRK βiAR-mediated EGFR transactivation
In the cytosol, β-arrestins are constitutively phosphorylated proteins and become dephosphorylated at the plasma membrane upon binding of activated 7TMRs. β-arrestin dephosphorylation promotes the recruitment of adapter proteins such as clathrin and AP-2, which are required for receptor endocytosis (Lin et al, J. Biol. Chem. 272:31051 (1997), Laporte et al, Proc. Natl. Acad. Sci., USA 96:3712 (1999), Naga Prasad et al, J. Cell. Biol. 158:563 (2002)). To test whether targeting of β-arrestin to activated 7TMRs in the plasma membrane can rescue EGFR transactivation by the GRK" βiAR, a "phosphorylation independent" β-arrestin mutant (βarrR169E-YFP tagged) that mimics dephosphorylated β-arrestin was used. βarrR169E binds with higher affinity to activated receptors and scaffold proteins (Kovoor et al, J. Biol. Chem. 274:6831 (1999), Violin et al, J. Biol. Chem. 281:20577-20588 (2006)). HEK 293 cells stably expressing GRK" βiAR were transfected with βarrR169E-YFP along with FLAG-EGFR. Dob stimulation of GRK" βiARs resulted in significant recruitment of βarrR 169E- YFP to GRK" β i ARs as assessed by immunoblotting for β-arrestin co-immunoprecipitating with the receptor (Fig. 3A, upper panel). Moreover, translocation of βarrR 169E- YFP to agonist-stimulated GRK" βiARs was associated with robust AP-2 recruitment to the receptor complex, confirming that this β-arrestin mutant functions as a constitutive active β-arrestin for GRX" βiARs in the plasma membrane (Fig. 3A, lower panel).
Confocal microscopy experiments were carried out to test whether the recruitment of constitutively active β-arrestin to the agonist stimulated GRK' βiAR would support transactivation. HEK 293 cells stably expressing GRK" βiAR were co-transfected with βarrR169E-YFP and EGFR-GFP and stimulated with Dob. EGFR was visualized in green and β-arrestin- YFP was visualized using a red filter to distinguish the YFP fluorescence of βarrR169E from the GFP fluorescence of EGFR. In the absence of Dob, the majority of βarrRl 69E- YFPwas localized in the cytosol (Fig. 3B, panel 2, arrowheads), with EGFR on the membrane (Fig. 3B, panel 1, arrows). However, Dob stimulation of GRK" βiAR was unable to induce EGFR internalization (Fig. 3B, panel 4, arrows) or EGFR phosphorylation (Fig. 3C), despite the prominent membrane translocation of β-arrestin (Fig. 3B, panel 5, arrowheads). In contrast, EGF stimulation caused EGFR internalization (Fig. 3B, panel 7, arrows) with no discernable membrane recruitment of β-arrestin (Fig. 3B, panel 8, arrowheads). These data suggest that receptor phosphorylation by GRK is a critical component in the βiAR-mediated EGFR transactivation.
Selective role for GRK5/6 in promoting βiAR mediated EGFR transactivation
Since EGFR transactivation was not rescued by targeting β-arrestin to the GRK" βiAR, a determination was made as to the role for the various GRKs in the transactivation process. GRKs, based on their sequence similarity and distribution, have been divided into three subfamilies. GRKs 1 and 7 are exclusively expressed in the retina, GRKs 2 and 3 interact with Gpv subunits through their pleckstrin homology domain and GRKs 4, 5 and 6 are a sub- family that are membrane associated (Pitcher et al, Annu. Rev. Biochem. 67:653 (1998), Willets et al, Trends Pharmaceol. Sci. 24:626 (2003)). Since GRKs 2, 3, 5 and 6 are expressed ubiquitously in mammalian tissues, their role in transactivation was investigated. HEK 293 cells were transfected with PKA" βiAR alone or along with siRNAs targeting GRK 2, 3, 5 or 6. Dob stimulation resulted in robust phosphorylation of EGFR associated with significant ERK activation in the presence of siRNA knocking down expression of GRK 2 and 3 (Fig. 3D, 3E, 3F). In contrast, βi AR-mediated EGFR transactivation and downstream ERK activation were nearly completely blocked in the presence of siRNA targeting GRK 5 or 6 (Fig. 3D, 3E, 3F). Similarly, GRK 5 or 6 knock down blocked WT βi AR-mediated EGFR phosphorylation and ERK activation, which was insensitive to Gi inhibition by pertussis toxin or PKA inhibition by H89 (Fig. 9A, 9B). Lastly, in contrast to βiAR-mediated EGFR transactivation, angiotensin II stimulation (All, l μM) in cells expressing ATiRs did not induce EGFR phosphorylation and All-induced ERK 1/2 phosphorylation was insensitive to AG 1478 (Fig. 9C).
Transactivation ofβjARs in vivo requires both β-arrestin and GRK5/6
To test whether this newly defined β-arrestin and GRK5/6 dependent signaling pathway can be recapitulated in the heart in vivo, mice lacking the gene encoding β-arrestin2, GRK 5 or GRK 6 were used. Mice were pretreated with ICI and then challenged with Dob or EGF. Myocardial lysates were immunoblotted for pERK. Consistent with the cell culture experiments, Dob-mediated ERK activation was completely blocked in the β-arrestin-2 knock out mice compared to their wild type littermate controls (Fig. 4A). Moreover, ERK activation was also completely blocked in GRK 5 and 6 knockout mice (Fig. 4B, 4C). In the same hearts following ICI and Dob treatment, EGFR was immunoprecipitated from the myocardial lysates and immunoblotted for phosphotyrosine. Although detection of phosphorylated EGFR from myocardial lysates was less consistent than from cell culture lysates, Dob-mediated EGFR phosphorylation appears to be blocked in hearts from β-arrestin-2, GRK5 and GRK6 knockout mice (Fig. 10). These data demonstrate that, in vivo, the mechanism for βiAR mediated EGFR transactivation requires both GRK 5, GRK 6, and β-arrestin recruitment to agonist-stimulated βiARs.
Cardioprotective role of β ιAR-mediated EGFR transactivation in vivo
While the epidermal growth factor receptor family appears to play a critical role in normal heart development (Chen et al, Nat. Genet. 24:296 (2000), Crone et al, Nat. Med. 8:459 (2002), Gassmann et al, Nature 378:390 (1995), Lee et al, Nature 378:394 (1995)), emerging evidence indicates that transactivation of the EGFR by HB-EGF may also be an important molecular mechanism for normal heart function (Iwamoto et al, Proc. Natl. Acad. Sci. USA 100:3221 (2003)) and that such transactivation may be perturbed during the development of pathological cardiac hypertrophy (Yoshioka et al, Proc. Natl. Acad. Sci. USA 102: 10622 (2005)). Since stimulation of β-arrestin-mediated signaling has been shown to activate a number of cellular signaling pathways (Lefkowitz and Shenoy, Science 308:512 (2005)), a test was made as to whether β-arrestin- dependent βiAR-mediated transactivation in the heart plays an important homeostatic role under normal and stress conditions. To address this question, transgenic (TG) mice were generated with cardiac-specific overexpression of the mouse WT βiAR (WT β,AR TG) and the two mutant βiARs lacking either GRK phosphorylation sites (GRK" βiAR TG) or PKA phosphorylation sites (PKA" βiAR TG) used in the cell culture experiments (Fig. 1 IA). βAR expression evaluated by 125I-cyanopindolol radioligand binding on cardiac membranes from left ventricles revealed -14 fold increase in receptor levels in all TG lines compared to non transgenic mice (NTG) (Fig. 5A). In vivo hemodynamic studies were next performed to determine the level of agonist-stimulated βAR responsiveness (i.e., in vivo G protein coupling) in the 3 lines of transgenic mice. As expected, isoproterenol (ISO)-stimulated cardiac contractility (as measured by LV dP/dtmax) was significantly enhanced in the GRK" or PKA' βi AR TG mice compared to NTG or WT βiAR TG mice (Fig. 5B) indicating a state of diminished βAR desensitization. In vivo, diminished βAR desensitization in the heart is observed in genetically engineered mice containing a 50% reduction in GRK2 levels (Rockman et al, J. Biol. Chem. 273:18180 (1998)), and in mice overexpressing a inhibitor peptide for GRK2 (Koch et al, Science 268:1350 (1995)). Cardiac function was preserved in all 3 lines of transgenic mice at 5-6 months and 12 months of age (Fig. 5C, Table 1). These data are consistent with the concept that homologous and heterologous desensitization of the receptor requires phosphorylation of specific GRK residues in the c-terminal tail and PKA residues in the 3r intracellular loop. Interestingly, overexpression of mouse βiARs has little untoward effects on cardiac function under normal conditions, a finding that is in contrast to TG overexpression of human βiARs in mice (Engelhardt et al, Proc. Natl. Acad. Sci. USA 96:7059 (1999)).
lablβ 1. Echocardiographlc mesuremenls in NTG, WT and mutant β1AR TG mice
5-6 months 12 months p IAR TG P1AR 1 G
NI G WT GRK- PKA" NTG WT GRIC PKA-
(n=2 l) ("=13) (n=14) ("=11) ("=2D (n=13) (n=14) (n=7)
LVEDD.mni 3.1±0.1 2.8±0.1 3.2±0.1 3.1±0.1 3.5±0. t 3.310.1 3.6±0.2 3.3±0.1
LVESD.inm U±O.O 1.0±0.1 1.ϋ±0.1 1.1+.0.1 1.6+.0.1 1.5±0.1 1.7±0.2 1.3±0.1
IVS.min ϋ.9±0.0 O.δ±O.O O.β±O.O O.β±O.O 0.9±0.0 O.β±O.O O.Θ±O.O 0.9±0.0
U)
LVPW1IHIn OJ±O.O OJ±O.O OJ±O.O 0.8±0.1 0.8+0.0 OJ±O.O OJ±O.O 0.9±0.1
FS,% 62.9±1.4 66.5±1.7 68.8±2.5 63.7±2.4 54.5±1.5 54.9±1.5 54.3±3.2 61.4±2.5
Vclc,circ/s 4.1±0.1 4.7±0.1 4.5±0.2 4.6±0.2 3.6±0.1 3.8+0.1 3.7±0.2 3.9±0.2
Head Rate.bpm 658±12 646±16 663±12 623±22 673±9 670±11 677±9 648±30
LeIt venlilcular end diastolic demenslon(LVEDD), ten ventricular systolic demension (LVESD). intraventricular septum (IVS),
IeIt ventiicular posterior wall (LVPW), fractional shortening (FS), heart rate corrected mean velocity of circumferential fiber shortening (Vcfc).
Statistical analysis was peiformed using l-test with Bonferronl coriection for multiple compailson. No statistical dirierences was found for any of the β1AR versus NI G mice at 5-6 months or 12 months.
Note same serial echocaidiographic measurements for NTG, WT and GRICβiAR TG mice except PKA'βiAR TG mice.
6B). Taken together, these data suggest that while either GRK- or PKA-mediated phosphorylation of the βiAR is sufficient to impair G protein coupling, both are required for agonist-dependent receptor downregulation.
Since heart failure is a condition that is associated with excess sympathetic stimulation, and β-arrestin-mediated signaling has been linked to the activation of protective signaling pathways (Lefkowitz and Shenoy, Science 308:512 (2005)), a test was made to determine whether βiAR-mediated EGFR transactivation would confer cardioprotection under conditions of chronic catecholamine stimulation. Remarkably, GRK" βi AR TG mice showed significant deterioration in cardiac function with marked left ventricular dilatation and reduced fractional shortening after 7 days of the βAR agonist isoproterenol (ISO) (Figs. 6C, 6D, 6E, Table 2).
64
In contrast, ISO had no deleterious effects on the NTG, WT βiAR TG or PKA" βiAR TG mice (Figs. 6C, 6D, 6E, Table 2), despite the fact that both WT β,AR TG and PKA" βi AR TG mice show enhanced agonist-stimulated second messenger signaling at baseline (Fig. 6B). Histological analysis of hearts from GRK" βiAR TG mice following chronic ISO treatment showed increased interstitial fibrosis and apoptotic nuclei compared to the WT βiAR and PKA" βiAR overexpressing mice (Figs. 6F, 6G). Taken together, these findings suggest that phosphorylation of βiAR by GRK may induce activation of cytoprotective signaling pathways, which are independent of classic G protein dependent signaling. Indeed, while all 3 TG lines show increased G protein signaling in the heart (as measured by enhanced adenylyl cyclase activity), only the hearts containing GRK" βi ARs that are unable to stimulate EGFR transactivation respond to catecholamines with a deterioration in cardiac function and greater myocyte apoptosis. Table 2 serial echocardiography in chronic ISO treatment
NTG (n=21) piARTG
WT (n=9) GRIC (n=14) PKA" (n=9) pre post pre post pie post pre post
Vehicle tiealmenl
Seiial echocardiography
LVEDD.mm 3.1±0.1 3.010.1 3.1 ±0.1 3.210.1 3.310.1 3.110.1 3.010.1 3.210.1
LVESD.mm 1.1±0.1 1.1±0.1 1.3±0.1 1.210.1 1.310.2 1.210.1 1.010.1 1.010.1
IVS.mm 0.8+0.0* 0.8+0.0* 0.710.0 0.610.0 0.810.0* 0.910.0* 0.810.0* 0.810.0*
LVPW.mm O.βiϋ.O* 0.7±0.0 0.6±0.0 0.6±0.0 0.810.1* 0.710.0 0.710.1 0.710.0
FS.% 65.6±1.7 64.5±1.7 60.2±2.0 62.712.3 61.112.6 61.911.7 67.012.2* 67.812.3
Vclc,ciιc/s 5.010.2 4 B±0.3 4.2±0.2 4.5±0.3 4.7±0.3 4.510.2 4.710.4 4.110.3
Head Rale.bpm 624H5 678±9 64 I±21 682±18 605±26 68419 622±37 673±11
Morpliometry
LV/BW, mg/g 3.5±0.1* 2.8±0.1 3.410.1* 3.210.2
NTG I [ιι=21) P1AR TG
Ch
WT (n=9) GRIC (n=13) PKA' (n=9) pie post pre post pre post pre post
ISO lieatment
Seiial echocardiography
LVEDD.mm 3.1±0.1 3.1 ±0.1 3.2±0.1 3.310.1 3.110.1 3.610.1 **-t 3.210.1 3.4±0.1
LVESD.mm 1.2±0.1 1.0±0.1 1.110.1 1.210.1 1.310.1 1.8±0.1"'t 1.310.1 1.310.1
IVS.mπi 0.8+0.0 0 9±0.0 0.7±0.0 0.910.0 0.910.0* 1.010.0 0.8+0.1 0.910.1
LVPW.mm O.β±O.O O.β±O.O 0.710.1 0.910.0 0.810.1 O.β±O.O 0.710.1 0.810.1
FS.% 61.9±1.1 67.6H.8* 64.1H.9 62.411.8 60.2+2.2 50.411. B"-t 60.811.7 61.5±1.7
Vcfc,circ/s 4.7±0.2 4.910.2 4.410.2 4.410.2 4.710.2 3.5±0.2*'t 3.810.2* 3.910.1
Heart Rate.bpm 637±15 697±13 643±22 725113 619122 697±13 558141* 671129
Morphometry
LV/BW. mg/g 3.910.1 3.910.1 4.510.1* 4.210.2
LeIt ventricular end diastolic demeπsioπ(LVEDD), left ventricular systolic deinension (LVESD), intraventricular septum (IVS)1IeH ventricular posterior wall (LVPW), fractional shortening (FS), heart rale corrected mean velocity of circumferential fiber shortening (Vcfc), left ventricular weighl/body weight (LV/BW) iepeated 2 way ANOVA with Newman-Keuls lest for echocardiography, multiple comparison with Bonfeironi conection for LV/BW. ', p<0.05 versus WTI G, "; p<001 versus Nl G, Wl"! G and PKATG, t; p<0 001 post versus pre
To explore whether the mechanism for the deleterious effect of catecholamines in the GRK" βi AR TG mice is an inability to activate the EGFR in response to βAR stimulation, normal mice were treated with the pharmacological EGFR inhibitor, erlotinib. Acutely, erlotinib treatment in vivo blocked myocardial ERK signaling by both βiAR mediated transactivation and direct EGF stimulation in WT mice (Fig. 7A), and in hearts of the WT βiAR TG and PKA' βiAR TG mice (Fig. HB). Cardiac function in normal C57BL/6 mice following a 2 week treatment with both erlotinib (20mg/kg, ip daily) and ISO was then measured. Cardiac function significantly deteriorated in mice treated with both ISO and erlotinib as shown by the significant increase in LV diastolic dimensions and reduced fractional shortening (Figs. 7B, 7C). Moreover, erlotinib-induced cardiac dysfunction was associated with a ~3 fold increase in apoptosis compared to ISO treated mice without the EGFR inhibitor (Figs. 7D, 7E). These data support the the hypothesis that β-arrestin mediated transactivation of the EGFR confers cardioprotection under conditions of catecholamine excess.
Summarizing, in the above study, a new signaling pathway for the βiAR is identified that uses β-arrestin and the EGF receptor to activate mitogenic and cell survival signaling pathways. The essential role of β-arrestin and GRK5/6 in this process is shown. Moreover, it is demonstrated in vivo that βiAR transactivation of cardiac EGFRs has a cardioprotective role against chronic catecholamine stimulation.
Until recently, negative regulation of G-protein signaling, i.e., desensitization, was the only known role for β-arrestins in 7TMR function. While β-arrestins desensitize 7TMR signaling by sterically inhibiting G protein coupling to agonist activated receptors, they also function to bring activated receptors to clathrin-coated pits for endocytosis (Lefkowitz and Shenoy, Science 308:512 (2005)). It is now appreciated that, in the context of the endocytic process, β- arrestins serve as ligand-activated scaffolds to initiate 7TMR signaling networks,
67 such as MAPK and Akt-cell survival pathways (Lefkowitz and Shenoy, Science 308:512 (2005)).
An ever expanding diversity is now being uncovered as to how 7TMRs activate MAPK signaling. For example, one pathway by which agonist- stimulated 7TMRs activate ERK signaling is to switch their G-protein coupling from Gs to Gi leading to the activation of the small GTPase protein RAS (Daaka et al, Nature 390:88 (1997), Martin et al, Cell Signal 16:1397 (2004)). A second recognized pathway for the activation of ERK is via the ability of β-arrestin to recruit the non-receptor tyrosine kinase c-Src following agonist stimulation (Luttrell et al, Science 283:655 (1999)). A recently discovered third mechanism is one where β-arrestins act to scaffold and assemble specific components of a MAPK module to enhance the activation and targeting of ERK (Luttrell et al, Proc. Natl. Acad. Sci. USA 98:2449 (2001), DeFea et al, J. Cell. Biol. 148:1267 (2000)) or JNK pathways (McDonald et al, Science 290:1574 (2000)) to specific subcellular locations. Of importance is that the latter 2 mechanisms depend on the recruitment of β-arrestin to activated 7TMRs but are independent of G protein activation. To date, the concept that βAR stimulation links the translocation and activation of β-arrestin to transactivation of the EGFR has not been appreciated. In addition to identifying a novel and direct functional role for β-arrestin in EGFR transactivation, the above study shows the in vivo relevance for this signaling pathway in the heart under conditions of catecholamine excess.
One of the more intriguing findings regarding β-arrestin signaling is the issue of isoform specificity. Accumulating evidence suggests considerable complexity in the specificity of the β-arrestin isoform that regulates ERK signaling following ligand-activation of a 7TMR. In one instance, termed reciprocal regulation, a particular receptor relies upon only one β-arrestin isoform, and the other β-arrestin isoform functions to inhibit signaling. This is seen for the ATIAR (Ahn et al, J. Biol. Chem. 279:7807 (2004)), the vasopressin V2R (Ren et
68 al, Proc. Natl. Acad. Sci. USA 102:1448 (2005)) and the protease-activated receptor- 1 (Kuo et al, Cell Signal (2006)). Interestingly in the case of the lysophosphatidic acid receptor, β-arrestin2 functions to attenuate EGF receptor transactivation, while promoting a second wave of ERK-mediated signaling (Gesty-Palmer et al, J. Biol. Chem. 280:32157 (2005)). In contrast to reciprocal regulation, for receptors such as the β2AR (Shenoy et al, J. Biol. Chem. 281 :1261 (2006)) and the parathyroid hormone receptor (Gesty-Palmer et al, J. Biol. Chem. 281 :10856 (2006)), there is co-dependent regulation since β-arrestin mediated signaling relies on both β-arrestin isoforms. In co-dependent regulation, the selective elimination of either isoform eliminates β-arrestin dependent ERK activation. In the study of βi AR mediated ERK activation, not only was the EGFR found to be essential for ERK signaling but also the transactivation process was found to require both β-arrestins -1 and -2, i.e., co-dependent regulation. While the mechanism for differences in the requirement of β-arrestins for ERK activation is not known, a possible explanation may be preferential recruitment of β-arrestin homo- or hetero-oligomers (Storez et al, J. Biol. Chem. 280:40210 (2005)). Whether the activated β-arrestins that form signaling complexes upon binding 7TMRs are in fact homo- or hetero-dimers or even monomers will require further study. The importance of GRK 5 or 6 for transactivation is highlighted by both the in vitro and in vivo studies on hearts of GRK 5 and 6 knockout mice. One possible mechanism for this finding is via phosphorylation of the βiAR at preferred residues on the c-terminal tail, which enables the receptor to interact with and activate β-arrestin in a conformationally specific fashion. The requirement for precise receptor-β-arrestin interaction suggests that a GRK5/6 dependent receptor conformation allows for docking of β-arrestin in a 3- dimensional structure that permits activation of downstream target proteins including possibly activation of the matrix metalloproteases. This concept is
69 supported by the data showing that 1) the non-GRK phosphorylatable receptor, GRK" βiAR, is unable to recruit β-arrestin and support EGFR transactivation, and 2) the simple targeting of "constitutively active" β-arrestin to βiARs lacking GRK phosphorylation sites, while adequate to recruit AP2, is insufficient to induce βAR-mediated EGFR transactivation. The data also suggest that there are distinct receptor-β-arrestin interactions that favor desensitization, e.g., following GRK2 phosphorylation, while other receptor-β-arrestin interactions favor G protein- independent signaling, e.g., following GRK 5/6 phosphorylation. These data are also consistent with the recent studies for the β2AR (Shenoy et al, J. Biol., Chem. 281 :1261 (2006)), AT,AR (Kim et al, Proc. Natl. Acad. Sci USA 102:1442
(2005)) and the V2 vasopressin receptor (Ren et al, Proc. Natl. Acad. Sci. USA 102:1448 (2005)).
It has been shown previously that overexpression of a GRK2 inhibitor peptide preserves βAR responsiveness and improves survival in a number of experimental models of heart failure (Rockman et al, Nature 415:206 (2002)). At first glance these results may seem surprising since enhancing catecholamine sensitivity should result in enhanced toxicity. However, the present data shows that reduction of GRK2 levels by siRNA in vitro enhances βjAR-mediated EGFR transactivation, which would be expected to enhance cell survival pathways in vivo, and may explain the salutary effects seen with GRK2 inhibition in the heart.
The importance of β-arrestin-mediated EGFR signaling in the heart is highlighted by the transgenic mouse studies, which demonstrate how the loss of βiAR mediated EGFR transactivation results in enhanced apoptosis and cardiac deterioration. Apoptotic heart cell death has been implicated in the overall process of myocardial remodeling and the transition from cardiac hypertrophy to chronic heart failure (Kang and Izumo, Circ. Res. 86:1107 (2000), Olivetti et al, N. Engl. J. Med. 336:1131 (1997)), which is due in part to chronic β,AR stimulation (Communal et al, Circulation 100:2210 (1999), Zaugg et al,
70 Circulation 102:344 (2000), Zhu et al, J. Clin. Invest. 1 11 :617 (2003)). It is shown here that βiAR mediated ERX and Akt activation in the heart, via βiAR- mediated EGFR transactivation, are important mechanisms counteracting catecholamine induced cardiomyocyte toxicity. Understanding the molecular mechanism underlying 7TMR-mediated
EGFR transactivation is important because many of the downstream signaling pathways stimulated by receptor tyrosine kinases have been linked with pathologic processes. For example, while there is evidence showing that HB- EGF ligands can contribute to the hypertrophic signaling in the heart (Yoshioka et al, Proc. Natl. Acad. Sci. USA 102:10622 (2005), Asakura et al, Nat. Med.
8:35(2002)), it is also known that the EGFR family plays a critical role in normal heart development and prevention of dilated cardiomyopathy (Chen et al, Nat. Genet. 24:296 (2000), Crone et al, Nat. Med. 8:459 (2002), Lee et al, Nature 378:394 (1995)). Additional investigation into the precise molecular mechanisms by which the EGFR signals in the cardiomyocyte is needed to better understand the physiological importance of these processes.
In conclusion, a new signaling mechanism has been identified that involves the recruitment of β-arrestin to activated βiARs in a GRK5/6 dependent manner that leads to EGFR transactivation. Activation of this βiAR- transactivation pathway provides cardioprotection in vivo under conditions of catecholamine toxicity. The concept that a receptor conformation, which allows binding and activation of β-arrestin, can activate a cardiomyocyte protective signaling pathway independent of G protein activation has considerable therapeutic implications.
71 EXAMPLE 2
β-AR: Three catecholamine derivatives with α-carbon alkyl substitutions have been identified that appear to have a bias for β-arrestin-mediated signaling (Fig. 12). Specifically, these compounds promote greater GRK/β-arrestin function (as assessed by recruitment of β-arrestin to the receptor) than would be predicted based on the ability of these compounds to stimulate G protein mediated cAMP accumulation (see Fig. 12)."
Bias was determined by comparing efficacy for stimulating G protein signals to efficacy for stimulating β-arrestin/GRK functions. Specifically, for a panel of β2AR ligands, ligand-stimulated cAMP generation was quantified to measure G protein efficacy, and the ligand-stimulated rate of β-arrestin recruitment was measured by FRET between β2AR-mCFP and β-arrestin2-mYFP to measure β-arrestin/GRK efficacy. For most ligands, relative G protein and β- arrestin/GRK efficacies correlated extremely well. However, for a subset of ligands, all of which were catecholamines with α-ethyl substituents, β- arrestin/GRK efficacy was substantially greater than G protein efficacy, indicating that these are β-arrestin-biased ligands.
The introduction of substitutions at the α-carbon position of β-adrenergic receptor agonists and antagonists can lead to ligands that differentially recruit cytosolic β-arrestin to the ligand-bound receptor. Such receptors can signal in a biased manner, i.e., selectively via β-arrestin-dependent pathways more than via classical G protein dependent pathways. Cellular outputs for this differential recruitment and signaling that can be assayed include, but are not limited to: 1) assays for β-arrestin recruitment following ligand addition; 2) assays for GRK activity/recruitment; 3) assays of G protein dependent cellular activity such as cAMP; 4) assays of MAPK activation such as pERK generation and 5) β-arrestin- dependent, βi-AR mediated epidermal growth factor receptor transactivation.
72 EXAMPLE 3
When cells expressing appropriately tagged 7TMR and β-arrestin are assessed for FRET in a fluorescent plate reader, several ligand properties can be discerned. Fig. 13A shows a dose-response curve for the classical agonist isoproterenol in cells expressing β2AR-mCFP and β-arrestin2-mYFP in a 96-well plate. The assay was run in equilibrium mode, allowing the FRET signal to stabilize for 10-20 minutes before acquiring data. Fig. 13B shows a dose- response curve for the classical antagonist propranolol reversing the isoproterenol-stimulated FRET signal. This assay was also run at equilibrium in a 96-well plate. In contrast, this assay can be run in kinetic mode to measure GRK function, as described above. Fig. 13C shows the kinetics of the FRET change in response to either agonist or buffer. In this case, the β-arrestinl mutant R169E was used to measure both GRK-independent FRET (fast response from receptor conformational changes) and GRK-dependent FRET (slow response), as described above.
EXAMPLE 4
It has been shown that the GRK activity assay functions for multiple receptors. In addition to the β2-adrenergic receptor (Fig. 12), GRK function for the Angiotensin II type 1 receptor (ATlR) has also been measured. Fig. 14A shows that siRNA-mediated silencing of GRKs slows the rate of β-arrestin recruitment to the ATiAR in response to 10OnM angiotensin. GRK2 silencing is more effective at slowing β-arrestin recruitment than GRK6 silencing. In constrast, Fig. 14B shows that for the biased agonist SII (Sar1 He4 He8- Angiotensin II), GRK2 silencing has no effect on β-arrestin recruitment, and GRK6 is very effective at slowing β-arrestin recruitment. This demonstarates that
73 biased ligands can select from subsets of GRK functions to determine the signaling outcome of receptor stimulation.
EXAMPLE 5
Experimental Details Materials
Isoproterenol was obtained from Sigma. [125I]-Iodocyanopindolol was obtained from Perkin Elmer. H-89 was obtained from EMD Biosciences. Anti- phospho-β2AR (p355/p356) was from Santa Cruz Biotechnology. Anti-beta- arrestin (AlCT) is described elsewhere (Attramadal et al, J. Biol. Chem. 267:17882-17890 (1992)). Anti-FLAG beads were from Sigma. All other reagents were from Sigma.
Plasmids
Rat β-arrestinl and rat β-arrestin2 were amplified by PCR to encode Hind III and Sal I restriction sites at the 5' and 3' ends, respectively, with the terminator codon replaced with a sequence encoding a diglycine linker. These products were cut, purified, and ligated into a pcDNA3.1-mYFP vector (Violin et al, J. Cell. Biol. 161 :899-909 (2003)) to generate β-arrestinl-mYFP and β- arrestin2-mYFP. The β-arrestin-mYFP inserts were then transferred to a pcDNA3.1-zeo vector providing zeocin resistance. The R169E mutation in β- arrestinl-mYFP was generated by the QuickChange protocol (Stratagene). Rat β2AR was amplified by PCR to encode a Hind III restriction site, flag epitope, and signal peptide sequence at the 5' end (Guan et al, J. Biol. Chem. 267:21995- 21998 (1992)), and an Xho I restriction site at the 3' end with the terminating codon replaced with a sequence encoding a diglycine linker. This product was cut, purified, and ligated into a pcDNA3.1-mCFP vector (Violin et al, J. Cell. Biol. 161 :899-909 (2003)) to generate β2AR-mCFP. All plasmids were amplified
74 in bacteria, kit purified (Qiagen) and validated by capillary electrophoresis sequencing.
Small interfering RNA (siRNA) silencing of gene expression Chemically synthesized double-stranded siRNA duplexes (with 3' dTdT overhangs) were purchased from Dharmacon for the following targets, as described and validated elsewhere (Ren et al, Proc. Natl. Acad. Sci. USA 102:1448-1453 (2005), Kim et al, Proc. Natl. Acad. Sci. USA 102:1442-1447 (2005)): GRK2 (5'-AAGAAGUACGAGAAGCUGGAG-S') GRK3 (5'- AAGCAAGCUGUAGAACACGUA-3') GRK5 (5'-AAGCCGUGCAAAGAACUCUUU-S') GRK6
(5'-AACAGUAGGUUUGUAGUGAGCO'). A non-silencing RNA duplex (5'-AAUUCUCCG AACGUGUCACGU-3') was used as a control for all siRNA experiments. A second GRK6 sequence (5'-AAGUGAACAGUAGGUUUGUAG-S ') was used to demonstrate that the siRNA effect was target-specific. HEK-293 cells were transfected with Gene Silencer (Gene Therapy Systems), and U2-OS cells were transfected with Lipofectamine 2000 (Invitrogen), according to manufacturers' instructions. Silencing was quantified by immunoblotting. Only experiments with verified silencing were used; average silencing for these experiments was as follows, for HEK-293 cells: GRK2 (91%), GRK3 (97%), GRK5 (85%), and GRK6 (96%). For U2-OS cells silencing was GRK2 (94%), GRK3 (99%), and GRK5 (97%). GRK6 was not detected in U2-OS cells.
Cell Culture
HEK-293 cells and U2-OS cells were maintained in modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution (Sigma). HEK-293 cells were transfected with FuGeneό (Roche); U2-OS cells were transfected with Lipofectamine 2000
75 (Invitrogen). AU transfections used 3.0 μg of plasmid in a 10 cm tissue culture plate. Cells expressing β2AR-mCFP were selected with either 400 ng/ml G418 (Sigma) and colonies of stable transfectants were isolated. A single line of stably transfected cells was chosen as representative based on membrane targeting and isoproterenol-induced internalization of the β2AR-mCFP. Surface expression of β2AR was measured by l25I-cyanopindolol binding as described (Shenoy et al, J. Biol. Chem. 280:15315-15324 (2005)), and determined to be 1.0 pmol receptor/mg protein for HEK-293 and 3.2 pmol/mg protein for U2-OS. Doubly- stable cells were generated by transfecting β-arrestin2-mYFP-Zeo selecting single colonies with 150 μg/ml zeocin (Invitrogen). β-arrestin-mYFP overexpression was assessed by immunoblot and compared to endogenous β-arrestin (Fig. 22)
Imaging
Cells were washed once and placed in imaging buffer (125 mM NaCl, 5 mM KCl, 1.5mM MgCl2, 1.5 mM CaCl2, 1OmM glucose, 0.2% BSA, 10 mM HEPES, pH 7.4) and imaged in the dark on a stage heated to 37 0C. Images were acquired on a Zeiss Axiovert 200M microscope (Carl Zeiss Microimaging, Inc.) with a Roper Micromax cooled charge-coupled device camera (Photometries) controlled by SlideBook 4.0 (Intelligent Imaging Innovations). CFP and FRET images were obtained through a 436/20 excitation filter (20 nm bandpass centered at 436 nm), a 455DCLP (dichroic longpass mirror), and separate emission filters (480/30 for CFP and 535/30 for FRET). YFP intensity was imaged through a 500/20 excitation filter, 515LP dichroic mirror, and 535/30 emission filter. All optical filters were obtained from Chroma Technologies. Excitation and emission filters were switched in filter wheels (Lambda 10-2; Sutter). Integration times were varied between 100 and 300 ms to optimize signal and minimize photobleaching. Spectral bleedthrough was determined by acquiring CFP, FRET, and YFP intensity images of samples expressing CFP only and YFP only, and was
76 linear with respect to fluorophore expression. This imaging system exhibits 43% CFP/FRET bleedthrough and 24% YFP/FRET bleedthrough. CFP/YFP bleedthrough was undetectable. FRET intensity corrected for bleedthrough (FPvETc) was defined as:
FRETc = FRET - 0.43 • CFP - 0.24 • YFP
FPvETc for all FRET images was calculated on a pixel-by-pixel basis for localization of FRET. In contrast, all graphs display calculations based on intensity from whole cells or sets of cells.
Immunoblotting
Cells were lysed in SDS sample buffer, and adjusted to equal protein concentration by protein assay of a parallel set of cells, as described (Ahn et al, J. Biol. Chem. 279:35518-35525 (2004)). Equal amounts of protein were separated on 10% Tris-glycine polyacrylamide gels (Invitrogen), and transferred to polyvinylidine fluoride membranes for immunoblotting. GRKs were detected with isoform-specific antibodies from Santa Cruz Biotechnologies for GRK2 (sc- 562), GRK3 (sc-563), GRK5 (sc-565), and GRK6 (sc-566), according to the manufacturer's instructions. Chemiluminescent detection was performed with horseradish peroxidase-coupled secondary antibody (Amersham Pharmacia) and SuperSignal West Pico reagent (Pierce). Chemiluminescence was quantified by a charge-coupled device camera (Syngene ChemiGenius2); representative images are shown as inverted greyscale.
Receptor Phosphorylation Three days after transfection, HEK 293 cells plated in 100-mm dishes were incubated at 370C for 60 min in phosphate-free MEM containing [32P]Pj
77 (100 μCi/ml; 1 Ci = 37 GBq). The amount of [32P]Pj in the medium was increased to 200 μCi/ml in the GRK6 siRNA-transfected cells to normalize for a 50% decrease in the uptake found in these cells. After agonist stimulation, the lysate protein concentrations were normalized according to the relative receptor expression. Immunoprecipitation was carried out to determine phosphorylation as described (Ren et al, Proc. Natl. Acad. Sci. USA 102:1448-1453 (2005)).
Coimmunoprecipitation
3 days after transfection with a FLAG-β2AR plasmid and the different siRNAs, 100-mm plates were incubated in 4.0 ml of Dulbecco's phosphate buffered saline with 10 mM Hepes for 1 h at 370C and then stimulated. Cells were subjected to cross-linking by dithiobis(succinimidylpropionate) from Pierce (DeFea et al, J. Cell. Biol. 148:1267-1281 (2000)). Immunoprecipitates were analyzed by immunoblotting with the AlCT polyclonal antibody (1 :3,000 dilution), which detects β-arrestin 1 and 2 (Attramadal et al, J. Biol. Chem. 267:17882-17890 (1992)).
Statistics
All statistics were performed with GraphPad Prism 3.02 software. For determining association kinetics, both one-phase and two-phase associations were compared to determine best fit. For comparison of experimental treatments, one- way ANOVA was used, with a Bonferroni multiple comparison test as appropriate.
Results
In an attempt to elucidate β2AR signal modulation by the arrestin/GRK axis, the HEK-293 cell line was chosen as a model system. These cells express the 4 ubiquitously expressed non-visual GRKs (2, 3, 5, and 6) and are readily
78 transfected with either DNA plasmids or siRNA (Ren et al, Proc. Natl. Acad. Sci. USA 102:1448-1453 (2005), Kim et al, Proc. Natl. Acad. Sci. USA 102:1442- 1447 (2005)). The choice was made to assay β-arrestin recruitment to the receptor by FRET to quantify both the kinetics and relative amount of β-arrestin recruitment. FRET has been used previously to assay β2AR interaction with β- arrestin2 (Krasel et al, J. Biol. Chem. 280:9528-9535 (2005)), as was the similar bioluminescence resonance energy transfer (BRET) assay (Azzi et al, Proc. Natl. Acad. Sci. USA 100:11406-11411 (2003)). Whereas BRET is more simply adapted to plate-reader systems than FRET, only FRET is compatible with single- cell and imaging assays. FRET constructs were generated for an imaging assay, allowing evaluation of both the subcellular location and single-cell kinetics of β- arrestin recruitment. Briefly, the monomelic cyan and yellow variants of the Green Fluorescent Protein (mCFP and mYFP) were fused to the human β2AR and various β-arrestin constructs to generate β2AR-mCFP and β-arrestin-mYFP for co-expression. These constructs interact after addition of the agonist isoproterenol, and a portion of excited-state CFP is non-radiatively transferred to YFP, resulting in yellow rather than cyan emission (Fig. 15A). The proportion of energy thus transferred (FRETc, corrected for spectral overlap (Gordon et al, Biophys. J. 74:2702-2713 (1998))) is quantifiable as a percentage of total fluorescence emitted %F, analogous to fractional receptor occupancy:
FRFTr
FRET (%F) = \00 '
CFP + FRETc
%F differs from absolute FRET efficiency (%E) only in the absence of a term correcting for hardware-specific differences in the quantum yield of CFP and FRET emission (Gordon et al, Biophys. J. 74:2702-2713 (1998)). In addition,
79 FRET can be imaged after correcting for spectral overlap, allowing localization of the β2AR:β-arrestin interaction (Fig. 15B). At higher resolution, this method of FRET imaging discriminates between β2AR-mCFP bound to β-arrestin-mYFP at the plasma membrane and internalized vesicular β2AR-mCFP which has lost the association with β-arrestin-mYFP. (Fig. 23). When FRET is measured for a field of cells, the kinetics of this interaction are monoexponential, and rapidly reversed with addition of the antagonist propranolol (Fig. 15C).
The kinetics of recruitment was tested in cells stably transfected with β2AR-mCFP and transiently transfected with β-arrestin2-mYFP. These cells express constant amounts of β2AR-mCFP (1 pmol/mg protein, data not shown) and varying amounts of β-arrestin2-mYFP. Transiently transfected cells, for which single-cell overexpression is highly variable, were used to assess the effects of β-arrestin expression on the β-arrestin:β2AR interaction. It was found that in cells with low to moderate expression of β-arrestin-mYFP, as measured by single- cell YFP fluorescence, isoproterenol-induced recruitment is monoexponential (Fig. 16A). Furthermore, higher β-arrestin2-mYFP expression yielded increased recruitment, as measured by maximum fractional FRET. In contrast, in cells with high expression of β-arrestin2-mYFP, recruitment is biphasic, exhibiting a rapid association (less than 5 second half-time) as well as the slow association seen with low expression. At all levels of β-arrestin2-mYFP expression, the slow rate was the same (Ua = 2.5 +/- 0.2 minutes, n = 15), with no correlation between β- arrestin2-mYFP expression and t\n (R2 = 0.18). This is consistent with a bimolecular interaction dependent on a single rate-limiting activity. Presumably this limit is set by the rate of receptor phosphorylation by GRKs. To test this, β- arrestin2-mYFP was transiently expressed with either wild-type β2AR-mCFP (WT) or with a non-phosphorylatable mutant β2AR-mCFP (GRK-/PKA-) (Seibold et al, MoI. Pharmacol. 58:1 162-1173 (2000)). As expected, the non- phosphorylatable receptor exhibits very low agonist-induced FRET (Fig. 16B).
80 For this experiment, β-arrestin was highly expressed, and WT and GRK-/PKA- receptor recruited similar amounts of rapidly-associating β-arrestin. The defect in GRK-/PKA- receptor is thus only in the slow phase of β-arrestin recruitment. This is consistent with conformation-dependent rapid association and phosphorylation- dependent slow association of β-arrestin2 with the β2AR. Indeed, as shown below, GRK activity influences only the slow phase of β-arrestin recruitment.
The relative affinities (ICF) of these two states (agonist-induced conformation and the phosphorylated state) for β-arrestin2-mYFP were next established by fitting the amplitude of slow and fast FRET changes with varying amounts of β-arrestin2-mYFP expression to a saturable binding model (Fig. 16C):
%F • YFP
FRET (%F) = =
YFP+ KFRET
It was not possible to accurately measure the relative affinity of the rapid, phosphorylation-independent component since the extremely high expression levels of β-arrestin2-mYFP necessary to saturate this component lead to YFP aggregation and altered receptor trafficking (data not shown). However, it is apparent from the data that the affinity of β-arrestin2-mYFP for the agonist- occupied phosphorylated receptor is at least two orders of magnitude higher than for the agonist occupied receptor prior to phosphorylation.
These results suggest that β-arrestin2 recruitment kinetics can be used to assess both agonist-induced receptor conformation and agonist-induced receptor phosphorylation, β-arrestin 1 is also reported to be less effective than β-arrestin2 for β2AR internalization (Kohout et al, Proc. Natl. Acad. Sci. USA 98:1601-1606 (2001)), so the kinetics and relative binding affinity of β-arrestin 1-m YFP and β- arrestin2-mYFP were compared. In addition, the "phosphorylation-independent" mutant β-arrestin 1-m YFP R169E was tested (Kovoor et al, J. Biol. Chem. 274:6831-6834 (1999)). This mutant exhibits a disrupted "polar core" which consists of closely aligned amino acid side chains of opposite charge. The R169E
81 mutation mimics the disruption of this region that is thought to occur upon β- arrestin binding phosphorylated receptor, leading to a conformational change with higher affinity for receptor and several β-arrestin-scaffolded proteins (Gurevich and Gurevich, Trends Pharmacol. Sci. 25:105-111 (2004)). It was found that β- arrestinl-mYFP is recruited with a slow rate similar to β-arrestin2-mYFP but a lower affinity (Fig. 17). Phosphorylation-independent β-arrestinl-mYFP (Rl 69E) displays more pronounced biphasic association, with a much larger rapid association than β-arrestinl-mYFP or β-arrestin2-mYFP at equivalent expression levels. However, there is also a large slow component of β-arrestinl-mYFP (Rl 69E) recruitment, most likely signifying a phosphorylation-dependent affinity. Interestingly, the relative affinity of this mutant is similar to that of β-arrestin2- mYFP. Together, these results suggest that β-arrestin2 binds the β2AR with higher affinity than β-arrestinl, but with similar kinetics, limited by the rate of receptor phosphorylation. "Phosphorylation-independent" β-arrestinl-mYFP (Rl 69E) appears in this assay to be partially phosphorylation independent, with an enhanced affinity contributed by the agonist-induced, unphosphorylated receptor.
The roles of individual GRKs in regulating β-arrestin recruitment were next addressed. To minimize variability in the β-arrestin recruitment assay, cell lines stably expressing either β-arrestinl-mYFP, β-arrestin2-mYFP or β-arrestinl- mYFP (Rl 69E) in addition to β2AR-mCFP were generated. Based on the findings above, these cell lines allow testing the GRK requirements for both slow, phosphorylation-dependent and rapid, phosphorylation-independent β-arrestin recruitment. These cell lines each express 8-fold to 30-fold β-arrestin-mYFP over endogenous β-arrestin as shown by immunoblot (Fig. 22); however, since the phosphorylation-dependent phase of the β-arrestin:β2AR interaction is independent of β-arrestin expression (Fig. 16A), results from these cell lines can be extrapolated to GRK function for endogenous β-arrestins. The dose-response
82 relationship of the kinetics and amplitude of β-arrestin2-mYFP recruitment to the β2AR-mCFP was tested first. Increasing concentrations of isoproterenol caused increasing rate and amount of recruitment (Fig. 18A). The rate and amplitude of recruitment after 1 μM isoproterenol was similar for the double-stable transfectants and transiently transfected cells, indicating that the double-stable transfectants are representative of the parent cell lines (data not shown). Quantification of the dose-response relationship reveals that the amount of β- arrestin recruited (%Fmax) fits a saturable one-site binding model (Fig. 18B), and is indistinguishable from predicted receptor occupancy (Benovic et al, Biochemistry 23:4510-4518 (1984)). In contrast, the rate of β-arrestin recruitment is less sensitive to isoproterenol and exhibits a "shallow slope" with a Hill coefficient of 0.67. This most likely reflects the kinetics of the receptoπligand interaction and receptor conformational change. The decision was made to continue the studies using high agonist concentration (1 μM) to maximize both amount and rate of β-arrestin recruitment, using both of these parameters to test GRK activities.
GRK activity was assessed by two methods: GRK overexpression and GRK silencing by siRNA. Overexpression of GRK2 increased the rate of β- arrestin2-mYFP recruitment without altering recruitment amplitude (Fig. 19A). Similar results were found with each of the ubiquitous GRKs (GRK2, GRK3, GRK5, and GRK6) (data not shown). This suggests that any of these GRKs are capable, with overexpression, of phosphorylating the β2AR to induce β-arrestin recruitment. However, this does not address which GRKs are relevant at endogenous expression levels. To address this question, endogenous GRKs were silenced with siRNA as described earlier (Ren et al, Proc. Natl. Acad. Sci. USA 102:1448-1453 (2005), Kim et al, Proc. Natl. Acad. Sci. USA 102:1442-1447 (2005)). The effects of GRK6 silencing on stably expressed β-arrestin 1-m YFP and β-arrestin 1-m YFP (Rl 69E) were tested first; both YFP constructs were
83 expressed to similar levels and P2AR-InCFP expression and distribution were comparable (data not shown). Cells transfected with a scrambled control siRNA displayed monophasic and biphasic recruitment for β-arrestinl and β-arrestinl (Rl 69E), respectively, just as in untransfected cells (Fig. 19B). Transfection of siRNA to silence GRK6 resulted in a reduced rate of slow-phase recruitment, without altering the rapid recruitment of β-arrestinl -mYFP (Rl 69E). Similar results, with a less pronounced effect, were noted with GRK2 silencing (data not shown). This is consistent with a GRK-independent rapid recruitment and GRK- mediated phosphorylation-dependent slow recruitment. Importantly, the final amplitude of recruitment is unaffected by GRK siRNA, suggesting that the primary defect noted with GRK silencing is kinetic. Protein kinase A, which also phosphorylates and desensitizes the β2AR, does not appear to affects β-arrestin recruitment, as 20 μM H-89 does not alter either rate or amplitude of isoproterenol-induced FRET (data not shown). Additionally, 20 μM H-89 does not detectably alter the appearance of isoproterenol-stimulated vesicles containing β2AR-mCFP (data not shown). This is consistent with the inability of PKA to mediate phosphorylation relevant for β-arrestin recruitment to the β2AR.
Each of the ubiquitous non- visual GRKs were next tested: siRNA silencing of GRK2 and GRK6 significantly slow β-arrestin2-mYFP recruitment (Fig. 19C) but GRK6 has the most profound effect. It was verified that this is not an off-target effect of the particular GRK6 siRNA sequence by showing comparable results with a second, independent siRNA sequence for GRK6 (data not shown). Efficiency of GRK silencing was shown to be greater than 90% by immunoblot (Fig. 19C, bottom). Importantly, GRK silencing had no significant effect on the maximum FRET signal, consistent with an unchanged amount of β- arrestin2 recruited at equilibrium (Fig. 19D). However, the possibility that the nature of the receptor: β-arrestin interaction is altered by GRK silencing, altering FRET efficiency and masking differences in the amount of β-arrestin recruited,
84 cannot be excluded. Notably, GRK silencing had the same effects on rate and amplitude of β-arrestinl-mYFP recruitment as observed for β-arrestin2-YFP (data not shown).
It is most likely that the slowed rate of FRET increase corresponds to a slowed association of β-arrestin with β2AR. However, since FRET depends on fluorophore orientation as well as proximity, it is also possible that some of the FRET increase detected is caused by conformational changes of the β- arrestin:β2AR complex after the initial interaction. It was possible to discriminate between these two possibilities by measuring β-arrestin2-mYFP redistribution from cytosol to membrane by tracking directly excited YFP fluorescence in user- defined regions of cytosol and membrane. After isoproterenol stimulation, membrane YFP intensity increases while cytosolic YFP intensity decreases. The ratio of these two signals serves as a direct measure of β-arrestin translocation, and is independent of FRET. As expected, GRK siRNA slows translocation compared to a control siRNA, consistent with a GRK-controlled rate of β-arrestin association with β2AR (Fig. 24). Furthermore, this finding is inconsistent with FRET changes induced by a GRK-controlled conformational change of the β- arrestin:β2AR complex.
The effect of GRK silencing on β-arrestin recruitment was confirmed using co-immunoprecipitation of endogenous β-arrestins. Immunoprecipitation of a flag-tagged β2AR showed increased β-arrestin association following 5 minutes of 10 μM isoproterenol (Fig. 20A). This effect was inhibited by silencing either GRK2 or, even more effectively, GRK6. These data confirm the FRET assay results (Fig. 19C), and suggest that the β2AR exhibits some specificity for GRK- mediated β-arrestin recruitment. β2AR phosphorylation was then assessed by 32P incorporation to test if the GRKs that mediate bulk phosphorylation are the same as those required for endogenous β-arrestin recruitment. To simplify the interpretation of this experiment a mutant β2AR was used that lacks PKA
85 phosphorylation sites but has unaltered phosphorylation by GRKs (Seibold et al, MoI. Pharmacol. 58:1162-1173 (2000)). Total phosphorylation after 5 minutes is most effectively reduced by GRK2 silencing (Fig. 20B). This indicates that not all β2AR phosphorylation is equivalent for recruiting β-arrestin, and that there is functional specificity for β2AR regulation by different GRKs. To further examine this possibility, β2AR phosphorylation was tested using an antibody that specifically recognizes phosphorylation of serines 355 and 356 on the β2AR (Tran et al, MoI. Pharmacol. 65:196-206 (2004)). In contrast to bulk β2AR phosphorylation, this site appears to be exclusively phosphorylated by GRK6 (Fig. 20C), confirming that GRKs target different sites on the β2AR.
Since GRK silencing does not appear to alter the amount of β-arrestin recruited, but only the rate at which recruitment occurs, it is apparent that either 1) the residual GRK expressed after silencing is sufficient, with impaired kinetics, to drive β-arrestin recruitment, or 2) the untargeted GRK isoforms are responsible for recruitment of β-arrestin2 after individual GRK silencing by a mechanism of compensatory phosphorylation. Since multiple GRKs (most prominently GRK2 and GRK6) contribute to β-arrestin recruitment in the HEK-293 cells used here, the latter hypothesis is favored. Indeed, simultaneous silencing of GRK2 and GRK6 reduced the rate of β-arrestin2-mYFP recruitment, as measured by FRET, more than any single GRK siRNA alone (Fig. 21A). Thus GRK2 and GRK6 can compensate for each other for β-arrestin recruitment. The most likely mechanism of this GRK2 and GRK6 interplay is either shared substrate residues on the β2AR or different substrate sites with equal affinity for β-arrestin2.
If β2AR phosphorylation sites responsible for β-arrestin recruitment are shared by GRKs, then β-arrestin recruitment in a cell type with a different GRK expression pattern should exhibit different sensitivity to GRK siRNA, corresponding to the relative expression of each GRK isoform. To test this, a U2- OS osteosarcoma cell line stably expressing β2AR-mCFP and β-arrestin2-mYFP
86 was generated. GRK expression between these cells and the β2AR-mCFP/β- arrestin2-mYFP HEK-293 cell line was compared by immunoblot (Fig. 21B). In comparison to HEK-293 cells, U2-OS express relatively more GRK3 and GRK5, but little to no GRK6. GRK silencing in the U2-OS line revealed that GRK2 and especially GRK3 are most efficacious at promoting β-arrestin2 recruitment, in correlation with the expression pattern of GRKs in the U2-OS line (Fig. 21C). As in HEK-293 cells, an additive effect of simultaneous silencing the two GRKs that individually contribute most to β-arrestin recruitment, in this case GRK2 and GRK3, was found (Fig. 21D). Thus, it is concluded that β2AR regulation by GRK-mediated β-arrestin recruitment strongly depends on a cell's complement of GRKs.
Summarizing, FRET has been used extensively as a non-destructive way of measuring protein:protein interactions, protein conformational changes, and physiochemical properties in living cells (Zhang et al, Nat. Rev. MoI. Cell. Biol. 3:906-918 (2002)). These approaches have been put to use in many fluorescent biosensors to report intracellular signals. Here, FRET between mCFP-tagged β2AR and mYFP-tagged β-arrestins is used as a measure of GRK activity. Other groups have shown FRET or BRET, a related biophysical phenomenon, between receptors and β-arrestins as a method of reporting β-arrestin recruitment (Azzi et al, Proc. Natl. Acad. Sci. USA 100:11406-11411 (2003)). The choice was made to develop a FRET assay because, unlike BRET, FRET can be used as an imaging technique, allowing single-cell and subcellular measurements of receptor: β- arrestin interaction. One previous report shows FRET between β2AR and β- arrestins which required overexpressed GRK2 (Krasel et al, J. Biol. Chem. 280:9528-9535 (2005)); the present assay differs in that the receptor: β-arrestin interaction driven by endogenous GRKs is measured. This permitted several novel observations pertaining to GRK regulation of β-arrestin function for the β2AR: 1 ) the rate of β-arrestin recruitment is a specific reporter of endogenous or
87 exogenous GRK activity, and 2) the β2AR exhibits cell-type dependent GRK specificity for its regulation by β-arrestin. Also shown, for the first time, is the rapid agonist-induced conformational change of the β2AR in live cells, as detected by the low-affinity interaction of β2AR-mCFP and β-arrestin-mYFP. FRET efficiency (the proportion of donor excitation emitted as FRET) depends upon the proximity (<10 nm) and orientation of the two interacting fluorophores (here, mCFP and mYFP). Changes in either proximity or orientation can alter FRET efficiency. Therefore, FRET efficiency does not necessarily directly reflect amount of interaction. For instance, it is conceivable that β- arrestin-mYFP can bind the β2AR-mCFP in different conformations, leading to different CFP:YFP orientation and thus different FRET efficiencies. However, the kinetics and relative affinities of such interactions are independent of maximum FRET efficiency, so these parameters are more rigorous measures of β- arrestin-mYFP recruitment to β2AR-mCFP. β2 AR: β-arrestin interaction, reported by FRET, was agonist-dependent, rapidly reversible, and localized to the plasma membrane (Fig. 15), as expected from previous work showing redistribution of GFP-tagged β-arrestin (Oakley et al, J. Biol. Chem. 276:19452-19460 (2001)). This association displayed both phosphorylation-dependent and -independent affinities. These affinities were assessed by measuring single-cell FRET over time in cells with constant levels of β2AR-mCFP but varying amounts of β-arrestin-mYFP. These studies revealed a fast, low-affinity phosphorylation-independent association, and a slower, GRK phosphorylation-dependent high-affinity interaction (Figs. 16A, 16C). The phosphorylation-independent affinity is insensitive to phosphoacceptor site mutation (Fig. 16B) and GRK silencing (Fig. 19B), consistent with a low affinity of β-arrestin for the agonist-induced "active" receptor conformation. In contrast, the much higher affinity slow component is both sensitive to phosphoacceptor site mutation (Fig. 16B) and GRK overexpression or siRNA (Fig. 19A, 19B). The
88 rate of this slow association is independent of β-arrestin-mYFP expression (Figure 2A), suggesting that the GRK-sensitivity of this assay can be extrapolated to endogenous β-arrestin. Importantly, only the phosphorylation-independent association can be detected with very high β-arrestin expression, so it is unclear if this low-affinity state has any physiological relevance. Nonetheless, this rapid, low affinity binding is a direct readout of receptor conformational change, and comports with other assays of receptor conformation, both with purified proteins (Swaminath et al, J. Biol. Chem. 279:19452-19460 (2001)) and in live cells (Liapakis et al, MoI. Pharmacol. 65:1181-1190 (2004), Hoffmann et al, Nat. Methods 2:171-176 (2005)).
Interestingly, the major effect of altered GRK expression is kinetic. The rate of the slow, high affinity β-arrestin interaction is enhanced by GRK overexpression (Fig. 19A) and reduced by GRK silencing (Figs. 19B, 19C). In contrast, neither overexpression nor silencing of GRKs appreciably alters the final maximum amount of FRET. As noted, FRET efficiency depends on both proximity and orientation of the interacting fluorophores. This indicates that the amount of β-arrestin recruited at equilibrium is the same in all conditions, but the possibility cannot be excluded that differences in β-arrestin recruitment are masked by parallel differences in β-arrestin or receptor conformation that inversely alter FRET to result in an unchanged FRET signal. Regardless, since GRKs are the rate-limiting step in β-arrestin recruitment, the rate of FRET increase is a functional reporter of GRK activity: any regulation or intervention that alters relevant GRK activity would be expected to alter the rate at which β- arrestin associates with receptor. Since β-arrestin binding desensitizes and internalizes the receptor, and results in a set of β-arrestin dependent signals
(Shenoy et al, J. Biol. Chem. 280:15315-15324 (2005)), such rate changes may have distinct signaling effects. As the understanding of heptahelical receptor
89 signaling is refined, it will be important to address both the amount and rate of β- arrestin recruitment as important parameters of receptor regulation.
It is clear from this work that, as previously shown in vitro (Gurevich et al, J. Biol. Chem. 270:720-731 (1995)), β2AR affinity for β-arrestin is regulated by two factors: the agonist-induced receptor conformation and receptor phosphorylation. The agonist-induced conformational change is very rapid (Swaminath et al, J. Biol. Chem. 279:686-691 (2004)) but induces significant amounts of β-arrestin recruitment only when β-arrestin expression is very high (Fig. 16A). GRK-mediated receptor phosphorylation leads to higher affinity binding, but at a rate set not by receptor occupancy directly, but rather by GRK activity. Presumably, the relative contributions of receptor conformation and phosphorylation vary for different receptors; for example, the LTB4 receptor appears to recruit β-arrestin completely independently of phosphorylation (Jala et al, J. Biol. Chem. 280:4880-4887 (2005)). Interestingly, it seems that the "phosphorylation-independent" β-arrestin 1
Rl 69E mutant displays enhanced affinity for the unphosphorylated, agonist- induced receptor conformation. However, this mutant is still sensitive to receptor phosphorylation, and thus is, in fact, only partially phosphorylation-independent. Although this mutant is a useful probe for agonist-induced receptor conformation (mimicking very high expression of wild-type β-arrestin), it is unclear if, under physiological conditions, any endogenous β-arrestin associates with unphosphorylated β2AR. Certainly, the fact that non-phosphorylatable β2AR is deficient in desensitization and internalization suggests that these β-arrestin functions require receptor phosphorylation (Seibold et al, MoI. Pharmacol. 58:1162-1173 (2000)).
One implication of the results is that β-arrestin expression levels affect the amount of receptor: β-arrestin complex formed. This may have profound implications for the desensitization, surface expression, and β-arrestin signaling of
90 receptors in cells with different β-arrestin expression levels. For assay systems that rely on exogenous β-arrestin, such as FRET and BRET, it is important to consider the effect of β-arrestin overexpression on the assay readout. To manage this concern, cell lines that stably express both β2AR-mCFP and β-arrestin-mYFP were generated. These cells make it possible to monitor β-arrestin recruitment even when measuring relatively few cells. Both the rate and amount of β-arrestin recruitment depend on agonist concentration (Fig. 17). Interestingly, the relative amount of β-arrestin recruited correlates well with receptor occupancy whereas the rate of β-arrestin recruitment is less sensitive. This likely reflects the kinetics of receptor :ligand interaction and receptor conformational change, and may allow the receptor to discriminate agonist concentrations across a wider range than would be predicted by receptor occupancy at equilibrium.
Since receptor phosphorylation is rate-limiting for β-arrestin recruitment and function, the effect of individual GRKs was measured by siRNA-mediated silencing. Results from two cell types lead to the striking conclusions that 1) β- arrestin recruitment to the β2AR is not specific to a single GRK, and 2) different cell types can use different GRKs to accomplish β-arrestin recruitment to the same receptor. This is deduced from the fact that GRK silencing does not appear to reduce the amount of β-arrestin:receptor complex. Instead, several GRKs contribute to β-arrestin recruitment in a complementary manner, as GRK silencing reduces the rate of β-arrestin recruitment. In the absence of any particular GRK, other GRKs still result in complete β-arrestin recruitment but with slowed kinetics. This is consistent with two possible mechanisms: compensatory GRK activity, and compensatory β-arrestin binding. Compensatory kinase activity would occur if GRKs shared the same phosphoacceptor sites on the receptor; the silencing of one GRK would result in the same phosphorylated residues but with delayed kinetics as the remaining GRKs take longer to complete the process. In this scheme, total GRK activity is
91 the relevant parameter for β-arrestin recruitment, and silencing of one GRK is akin to partial inhibition of total GRK activity. Compensatory β-arrestin binding, on the other hand, could result if GRKs phosphorylate different residues, but β- arrestin cannot distinguish between them. Such a mechanism would be consistent with β-arrestin having no set sequence specificity for binding but, instead, requiring some net charge introduced by phosphorylation (Gurevich and Gurevich, Trends Pharmacol. Sci. 25:105-111 (2004)). This would also be consistent with a kinetic role for individual GRKs: in the absence of a given GRK, it may take longer for the required number of phosphorylations to accumulate on the amino acids targeted by the remaining GRKs. Either of these mechanisms is consistent with these findings. The most promising method of distinguishing these hypotheses is a proteomic characterization of β2AR phosphorylation sites for each GRK. Indeed, preliminary work suggests this is a feasible approach (Trester-Zedlitz et al, Biochemistry 44:6133-6143 (2005)). It may be possible, by combining phosphorylation site identification with GRK silencing, to determine what specificity exists for GRK phosphorylation of the β2 adrenergic and other receptors. The current work, however, suggests that such putative specificity plays a role only in the kinetics of β-arrestin recruitment.
Recent results suggest that GRKs can have specific effects on β-arrestin signaling distinct from their effects on β-arrestin recruitment (Ren et al, Proc. Natl. Acad. Sci. USA 102:1448-1453 (2005), Kim et al, Proc. Natl. Acad. Sci. USA 102:1442-1447 (2005)). If these discrepancies are also found for the β2AR, it would suggest that not all β-arrestin recruitment is equivalent. For example, the rate and order of receptor phosphorylations might not affect the amount of β- arrestin recruited, but could place the β-arrestin in either distinct conformations or orientations on the cytoplasmic receptor surface, leading to different regulatory effects on the receptor signal output. Such differences in β-arrestin conformation/orientation were not evident by FRET in the present system.
92 (prior to mini-pump implantation), and after 2 weeks of exposure to either treatment. Left ventricular fractional shortening, an index of left ventricular systolic function, was assessed at both timepoints (Fig. 25). The difference in fractional shortening after 2 weeks of treatment is plotted on the y-axis, for each animal subgroup (n=6); as can be observed, the nature history of the CSQ+ mice is to develop deteriorating left ventricular systolic function as a function of time. Black bars denote data from CSQ' mice, while white bars denote data from CSQ+ mice. Data are presented as mean + SEM. *p < 0.05 by ANOVA with post-hoc Bonferroni test.
EXAMPLE 7
Experimental Details
Reagents
ANG (Sigma-Aldrich, St. Louis, MO, USA) and SII (Cleveland Clinic core synthesis facility, Cleveland, OH, USA) were dissolved in phosphate- buffered saline (PBS) without calcium and magnesium (Sigma-Aldrich, St. Louis, MO, USA) to concentrations of 1 mM prior to use in cardiomyocyte functional assays. Final concentrations in these assays were 10 μM for each drug. Frozen stocks in either distilled water or PBS were maintained at 10 mM. The protein kinase C (PKC) inhibitor Ro-3 1-8425 (Calbiochem, San Diego, USA) was dissolved in DMSO and stored at a concentration of 1 mM. Final concentration in the assays was 1 μM. The angiotensin receptor blocker (ARB) valsartan was dissolved in 100% ethanol to a concentration of 20 mM, and used at a final concentration of 50 μM in calcium fluorimetry experiments.
94 Calcium Fluorimetry
Neonatal rat atrial cardiomyocytes were isolated and cultured as described (Mohler et al, J. Biol. Chem. 277:10599-10607 (2002)). Cells were loaded with the dye Fura-2 (as per manufacturer's instructions, Invitrogen™), and treated with either ANG (100 nM) or SII (10 μM), in the absence or presence of pre-treatment with 50 μM valsartan. The instantaneous 340/3 80 nm excitation ratio (Violin et al, J. Cell Biol. 161 :899-909 (2003)) for Fura-2 was calculated and plotted as a function of time.
Fluorescent Resonance Energy Transfer CFRET) Assays Plasmids β-arrestin2-mYFP is described elsewhere (Violin et al, J. Biol. Chem. 281 :20577-20588 (2006), May 10 E.Pub (2006)). Rat AT,ARwas amplified by PCR to encode Hind III and Xho I restriction sites at the 5' and 3' ends, respectively, with the termination codon replaced to encode a diglycine linker. The PCR product was cut, purified, and ligated into a pcDNA3.1 -mCFP vector (Violin et al, J. Cell Biol. 161:899-909 (2003)) to generate AT, AR-mCFP.
Small Interfering RNA (siRNA) Silencing of Gene Expression Chemically synthesized siRNA duplexes with 3' dTdT overhangs were obtained from Dharmacon for GRK2 and GRK6, and have been described and validated elsewhere (Ren et al, Proc. Natl. Acad. Sci. USA 102:1448-1453
(2005); Kim et al, Proc. Natl. Acad. Sci. USA 102:1442-1447 (2005)): GRK2 (5'- AAGAAGUACGAGAAGCUGGAG-3') GRK6 (5'- AACAGU AGGUUUGU AGUGAGC-3'). A non-silencing RNA duplex (5'- AAUUCUCCGAACGUGUCACGU-3') was used as a control for all siRNA experiments. HEK293 cells were transfected with Gene Silencer (Gene Therapy
95 Systems) according to manufacturer's instructions. Silencing was quantified by immunoblotting, and only experiments with verified silencing were used. Imaging
Cells were washed once and placed in an isotonic imaging buffer (125 mM NaCl, 5 mM KCl, 1.5mM MgCl2, 1.5 mM CaCl2, 1 OmM glucose, 0.2% BSA, 10 mM HEPES, pH 7.4) and imaged in the dark on a stage heated to 37°C. Images were acquired on a Zeiss Axiovert 200M microscope (Carl Zeiss Microimaging, Inc.) with a Roper Micromax cooled charge-coupled device camera (Photometries) controlled by SlideBook 4.1 software (Intelligent Imaging Innovations). Details of imaging protocols have been described elsewhere (Violin et al, J. Cell Biol. 161 :899-909 (2003)). Real-Time GRK Activity Assay
HEK293 cells stably transfected with ATiAR-mCFP and β-arrestin2- mYFP were used to measure GRK functional activity by quantifying the rate and extent of isoproterenol-stimulated β-arrestin association with the ATi AR as measured by FRET, as described elsewhere (Violin et al, J. Cell Biol. 161 :899- 909 (2003)). FRET was calculated as %F, the percentage of CFP-excited fluorescence detected as YFP emission, corrected for both background signal and for spectral bleedthrough:
FRET (%F) =100 • FRETc
CFP + FRETc FRETc = FRET-0A3 • CFP-0.24 • YFP
Assuming pseudo-first-order receptor-β-arrestin association kinetics, the FRET signal %F was modeled as an exponential function of time (GraphPad), with rate constant kobs and equilibrium amplitude %Fmax, where kobs is a relative measure of GRK activity and %Fmax depends on expression of ATI AR-ΓΠCFP and β-arrestin2-
96 mYFP, the affinity of the interaction between the two molecules, and the average distance and orientation between CFP and YFP in the interacting state: %F= %Fmax ' (\-e koi»'1) Immunoblotting Efficacy of GRK silencing was validated by immunoblotting cell lysates for specific GRX isoforms. This was performed as detailed in (Violin et al, J. Biol. Chem. 281:20577-20588 (2006)).
Animals
All animals used in these studies were adult male mice of 8 to 20 weeks of age. All mouse strains were back-crossed to the C57B1/6 background <10 generations. Animals were handled according to approved protocols and animal welfare regulations of the Institutional Review Board at Duke University Medical Center. In addition to wild-type (WT) C57B1/6 mice, the following gene-targeted deficient [homozygous or heterozygous knockout (KO), as indicated] mouse strains were used, all of which have been previously described (Jaber et al, Proc. Natl. Acad. Sci. USA 93:12974-12979 (1996); Bohn et al, Science 286:2495-2498 (1999); Conner et al, Circ. Res. 81 :1021-1026 (1997); Fong et al, Proc. Natl. Acad. Sci. USA 99:7478-7483 (2002); Grainetdinov et al, Neuron 24:1029-1036 (1999); Ito et al, Proc. Natl. Acad. Sci. USA 92:3521-3525 (1995)): AT,AR KO, βarrestin2 KO, GRK2 heterozygous KO (+/-), GRK5 KO, and GRK6 KO.
Cardiomyocvte Isolation and Functional Assays
Adult mouse cardiomyocytes from the aforementioned strains were isolated as described previously (Barki-Harrington et al, Circulation 108:1611- 1618 (2003)). Purified cardiomyocytes were treated with relevant drugs, and were then subjected to electrical pacing at 1 Hz under visualization with an inverted microscope (Nikon Eclipse TE300). Cyclic shortening and relengthening
91 of single cells was assessed in real-time using video edge detection (i.e., cardiomyocyte length as a function of time). % Fractional shortening, an index of systolic function, was calculated directly from these measurements [(length at end-diastole - length at end-systole)/length at end-diastole]. Cardiomyocyte end velocities were derived from the measured length-time relationships, and their minima (maximum contraction; -dL/dtm a x) and maxima (maximum relengthening; +dL/dtm a x) are reported as indices of systolic and diastolic function, respectively. 10-15 cardiomyocytes per stimulation condition were assayed in each experiment; n denotes number of animals (i.e., independent experiments).
Statistical Analyses
Statistical analyses performed were one-way analyses of variance (ANOVA) withpost hoc Bonferroni tests, or Student's paired t tests. A p value of < 0.05 was considered statistically significant.
Results
Intracellular Calcium Mobilization in Cardiomyocytes Stimulated with
ANG or SII
It has been previously shown, in cells heterologously over-expressing the ATi AR, that SII fails to elicit activation of Gαq and phosphatidylinositol turnover, events proximal to intracellular calcium mobilization (Wei et al, Proc. Natl. Acad. Sci. USA 100:10782-10787 (2003)). However, Sll-mediated activation of Gαq- dependent signals has not been assessed in cells that endogenously express the ATJAR at physiologically normal levels. To determine whether SII activates Gαq- dependent pathways under these conditions, calcium mobilization in neonatal rat atrial cardiomyocytes (these cells can be obtained at high yield and maintained in culture (Mohler et al, J. Biol. Chem. 277:10599-10607 (2002)) in response to
98 exposure to ANG or SII was assessed. As shown in Fig. 26, cells treated with ANG displayed robust calcium mobilization, whereas cells treated with SII did not. Furthermore, ANG-induced calcium mobilization was dependent upon activation of the ATi AR, since it was abolished by pre-treatment with the ARB valsartan. These findings demonstrate that SII does not mediate calcium mobilization in cardiomyocytes expressing endogenous levels of the ATi AR-
Control of β-Arrestin Recruitment to the ATIAR by Specific GRKs
It has been previously shown that SII stimulates β-arrestin2 -dependent ERK activation through the AT]AR (Wei et al, Proc. Natl. Acad. Sci. USA 100:10782-10787 (2003)), and that the activities of GRK5 and GRK6 are required for this PKC-independent ERK activation (Kim et al, Proc. Natl. Acad. Sci. USA 102:1442-1447 (2005)). Prior to carrying out studies of the functional effects of β-arrestin-mediated signaling in cardiomyocytes, further characterization of β- arrestin2 recruitment to the AT^R by ANG and SII and its control by specific GRK isoforms was carried out. Using FRET, it was found that both ANG and SII led to β-arrestin2 recruitment to the ATIAR with comparable kinetics and magnitude (Fig. 27). Recruitment in response to ANG was slightly diminished in the presence of siRNA targeted against GRK6, and to a lesser extent, GRK2 (Fig. 27A). However, in the case of SII-induced β-arrestin2 recruitment, siRNA against GRK6 led to substantial impairment (Fig. 27B). No effects of GRK5 siRNA on β- arrestin2 recruitment were observed for either ANG or SII (data not shown). These findings suggest that SII-induced signaling via β-arrestin2 is dependent upon the upstream activity of GRK6.
99 Effects of SII on Cardiomyocyte Function Via Signaling Through the AT1AR
Having demonstrated that SII fails to activate Gαq-dependent signals in cardiomyocytes, a determination was made as to whether, like ANG (see Fig. 26), SII has effects on cardiomyocyte function. As shown in Fig. 28A, WT cardiomyocytes displayed augmented % fractional shortening in response to treatment with either ANG or SII in comparison to conditions of pacing alone, i.e., both drugs exerted essentially identical positive inotropic effects. Furthermore, both ANG- and SII-induced positive inotropic responses were absent in cardiomyocytes from ATIAR KO mice (Fig. 28A) which, nevertheless, responded robustly to the β-AR agonist isoproterenol. This demonstrates that the effects of both ATJAR ligands are mediated by signaling through the ATJ AR. Similar data were observed for -dL/dtmax and +dL/dtmax (Fig. 31). Thus, G protein-independent signal transduction via the ATI AR ΪS sufficient to mediate receptor-dependent positive inotropic and lusitropic responses.
Dependence of ANG Versus SII Effects on Activation of PKC
Next, having shown that both ANG and SII induce positive inotropic and lusitropic responses, the dependence of these effects on PKC activity was tested. ANG-induced positive inotropic effects in cardiomyocytes from other species (e.g., cat) have been shown to require PKC activity (Booz and Baber, Heart
Failure Reviews 3:125-130 (1998)); however, since SII does not activate Gαq, it was hypothesized that its effects would not be PKC-dependent. As shown in Figs. 28B and 28C, while ANG-induced positive inotropic responses were inhibited substantially (by 71%) by the PKC inhibitor Ro-31-8425, responses to SII were unaffected. Similar findings for -dL/dtmax and +dL/dtmax were observed (Fig. 32). Thus, the functional effects of SII are independent of PKC activation,
100 consistent with selective transmission of Gαq-independent signals. In contrast, in this experimental system, the functional effects of ANG are predominantly dependent upon PKC activation, and are presumably dependent upon upstream activation of Gαq.
5 Dependence of ANG Versus SII Effects on β-Arrestin2
As noted above, accumulating biochemical evidence suggests that in many cases, one component of G protein-independent signaling via 7TMRs is mediated by β-arrestins. In order to test whether the positive inotropic responses of cardiomyocytes to SII are, in fact, β-arrestin-dependent, cardiomyocytes from β-o arrestin2 KO mice were assayed for responses to ANG and SII, along with cardiomyocytes from a contemporaneous set of WT mice. While WT cardiomyocytes displayed similar increases in % fractional shortening in response to ANG and SII (Figs. 29A and 29B), β-arrestin2 KO cardiomyocytes displayed minimal increases in % fractional shortening in response to SII despite preserveds responses to ANG [Fig. 29C - SII versus Basal (p > 0.05), ANG versus SII (p < 0.05); Fig. 29D - ANG versus SII (p < 0.05)]. Similar data were observed for -dL/dtmax and +dL/dtmax (Fig. 33). These data demonstrate that ATIAR signaling via β-arrestin2 is required for SII to exert positive inotropic and lusitropic effects. It has been previously reported that β-arrestinl negatively regulates SII- o mediated ERK activation by the ATi AR in HEK293 cells (Ahn et al, J. Biol. Chem. 279:7807-7811 (2004)). Additionally, β-arrestinl KO mice have previously been shown to exhibit augmented cardiovascular system responses to β-AR stimulation with isoproterenol in vivo (Conner et al, Circ. Res. 81 :1021- 1026 (1997)). Thus, an examination was made of responses to ANG and SII in5 cardiomyocytes isolated from β-arrestinl KO mice. ANG and SII were found to elicit equivalent increases in % fractional shortening of β-arrestinl KO cardiomyocytes (Figs. 29E and 29F). Similar data were observed for -dL/dtmax
101 and +dL/dtmax (Fig. 33). These data demonstrate that deficiency of β-arrestinl does not affect positive inotropic and lusitropic responses to SII in isolated cardiomyocytes.
Dependence of ANG Versus SII Effects on Specific GRKs Next, the effects of specific GRK isoform deficiency on ANG- and SII- mediated cardiomyocyte functional responses were assessed. It has been previously shown previously that, in HEK293 cells expressing the ATi A R> GRK5 and GRK6 are individually required for β-arrestin2-mediated ERK activation (Kim et al, Proc. Natl. Acad. Sci. USA 102:1442-1447 (2005)). Consequently, cardiomyocytes from GRK5 KO and GRK6 KO mice were evaluated initially. As shown in Figs. 3OA and 3OB, GRK5 KO cardiomyocytes exhibit equivalent increases in % fractional shortening in response to ANG or SII; similar data were observed for - dL/dtm a x and +dL/dtmax (Fig. 34). In contrast, GRK6 KO cardiomyocytes, like β-arrestin2 KO cardiomyocytes, display augmentations in % fractional shortening in response to ANG, but not to SII [Fig. 3OC - SII versus Basal (p > 0.05), ANG versus SII (p < 0.05); Fig. 3OD - ANG versus SII (p < 0.05)]. Similar data were obtained for -dL/dtmaxand +dL/dtm a x (Fig. 34). These data demonstrate that positive inotropic and lusitropic effects mediated by G protein-independent signaling via the ATi AR require GRK6, but not GRK5. Furthermore, these data are consistent with the findings in HEK293 cells that GRK6 is the predominant GRK isoform required for SII-induced β-arrestin2 recruitment to the ATj AR (see Fig. 27).
Finally, the effects of deficiency of GRK2, originally identified and characterized as being essential to the desensitization of G protein-mediated signals for a variety of 7TMRs (Rockman et al, Nature 10:206-212 (2002)), on cardiomyocyte responsiveness to ANG and SII was assessed. Cardiomyocytes from GRK2 +/- mice [GRK2 KO mice die in utero secondary to systolic
102 ventricular dysfunction/heart failure (Jaber et al, Proc. Natl. Acad. Sci. USA 93:12974-12979 (1996))] exhibited enhanced % fractional shortening in response to both ANG and SII (Figs. 3OE and 30F) and, surprisingly, increased responsiveness to SII in comparison to ANG was observed [Fig. 3OE - ANG versus SII (p < 0.05); Fig. 3OF - ANG versus SII (p < 0.05)]. These data suggest that, in mouse cardiomyocytes, GRK2 appears to negatively regulate β-arrestin- dependent signaling via the ATi AR, thereby controlling the balance between G protein-dependent and -independent signaling.
In conclusion, the physiologic functions of 7TMR signal transduction have traditionally been attributed to activation of heterotrimeric G proteins, historically the first identified effectors of 7TMRs (Gilman, Annu. Rev. Biochem. 56:615-649 (1987)). However, the existence of G protein-independent signal transduction for many 7TMRs suggests that not only might these novel pathways have physiologic consequences, but that the conventional understanding of the physiologic functions of 7TMR signaling may need to be re-assessed for individual 7TMRs. In these studies of adult mouse cardiomyocytes expressing endogenous cell surface receptors, using the biased ATi AR agonist SII, it is shown that: (1) the ATi AR mediates augmentation of cardiomyocyte systolic and diastolic function by two distinct, apparently independent pathways - one is G protein-dependent (presumably, Gαq) and involves PKC, whereas for the other the signal is carried by β-arrestin2 and GRK6; and (2) GRK2 appears to antagonize the β-arrestin- dependent signaling.
It was observed that the effects of ANG on isolated cardiomyocyte function are predominantly mediated via the Gαq/PKC pathway, and are independent of β-arrestin2. This suggests that, under physiologic conditions, with endogenous levels of receptor expression, ANG may exert its inotropic effects primarily via activation of heterotrimeric G proteins. However, approximately 30% of ANG-mediated cardiomyocyte functional responses were insensitive to
103 PKC inhibition, i.e., they were PKC-independent. This component of ANG- induced effects may be β-arrestin-mediated, consistent with previous published work in HEK293 cell systems (Wei et al, Proc. Natl. Acad. Sci. USA 100:10782- 10787 (2003); Kim et al, Proc. Natl. Acad. Sci. USA 102:1442-1447 (2005)). Furthermore, the apparent lack of impairment in ANG-induced positive inotropic and lusitropic responses in β-arrestin2 KO (and GRK6 KO) mice may be due to robust activation of Gαq-dependent signaling pathways by the high concentrations of ANG used in these experiments which overcomes the deficiency in activation of β-arrestin-dependent pathways. Finally, the positive inotropic and lusitropic effects of SII also indicate that β-arrestin-mediated signaling is sufficient for the ATI AR to modulate cardiomyocyte function. Thus, activation of either the Goq- dependent or β-arrestin/GRK-dependent signaling pathways alone may be sufficient to mediate effects on myocardial function in the setting of optimal ATIAR stimulation by either "full" or "biased" agonist ligands. The molecular mechanisms by which β-arrestin2 promotes an AT]AR- mediated inotropic effect on cardiomyocytes are currently completely unknown. Broadly, two possibilities exist: (1) coupling of β-arrestin to the regulation of cytosolic [Ca2+], and/or (2) coupling of β-arrestin to myofilament proteins. These two possibilities encompass the factors governing myocardial contractile function at the molecular level. The first mechanism suggests Ca2+ channel/pump proteins as possible β-arrestin-dependent effectors and/or interaction partners: (1) the L- type Ca2+ channel, (2) the IP3 receptor, (3), the ryanodine receptor, and (4) SERCA. Similarly, several myofilament proteins, such as myosin, actin, troponin, and tropomyosin, may be effectors or interaction partners of β-arrestin. Studies examining β-arrestin interaction with such proteins, or examining the role of β- arrestin in regulating the functions of these various proteins, remain to be performed.
104 These studies were conducted on cardiomyocytes from healthy mice that had not undergone any cardiovascular manipulations. Thus, under pathophysiologic conditions, the roles of G protein-dependent and -independent signal transduction via the ATIAR and other 7TMRs may be considerably different. Numerous lines of evidence suggest that chronic activation of either Gαs or Gαq, the primary Gα proteins to which most myocardial 7TMRs couple, results in deleterious effects on ventricular function in several experimental systems, and even clinically (Adams and Brown, Oncogene 20:1626-1634 (2001)). With respect to the ATi AR, over-expression of Gαq results in ventricular dysfunction (D'Angelo et al, Proc. Natl. Acad. Sci. USA 94:8121-8126 (1997)), whereas inhibition of Gαq by transgenic over-expression of an inhibitory peptide prevents high afterload-induced left ventricular dysfunction (Eposito et al, Circulation 105:85-92 (2002)) and hypertrophy (Akhtar et al, Science 280:574-577 (1998)). In contrast, G protein-independent, β-arrestin/GRK-dependent signaling has been shown to be cytoprotective in several cellular systems, and thus might be beneficial to myocardial function, especially if sustained chronically. Supporting this notion, in the setting of over-expression of a Gαq-uncoupled mutant ATI AR in the myocardium, cardiomyocytes display reduced cell death in comparison to cardiomyocytes from mice that over-express the WT AT)AR (Zhai et al, J. Clin. Invest. 115:3045-3056 (2005)).
Isolated cardiomyocyte function and ventricular function in vivo have been shown to correlate in a variety of experimental studies (Barki-Harrington et al, Circulation 108:1611-1618 (2003); Rockman et al, J. Biol. Chem. 273:18180- 18184 (1998)). Thus, this work raises the possibility of developing drugs for 7TMRs which selectively signal in a β-arrestin-dependent fashion. For example, in either the acute or chronic setting, agents such as SII, which are null with respect to activation of Gαq, will competitively antagonize ANG-mediated Gαq activation; they are thus ARBs. However, they still retain the ability to carry β-
105 arrestin-dependent signals from the ATIAR to mediate beneficial effects on cardiomyocyte function; thus, they are simultaneously "biased agonists" with respect to β-arrestin-dependent signaling. These features distinguish such drugs from the conventional antagonists of many 7TMRs currently used in clinical practice (e.g., βAR blockers, ARBs, etc.), which inhibit all signals from 7TMRs. Based on the biochemical evidence implicating β-arrestins in cytoprotective signaling through activation of effectors such as ERK (Defea et al, Proc. Natl. Acad. Sci. USA 97:11086-11091 (2000)), PI-3-Kinase (Povsic et al, J. Biol. Chem. 278:51334-51339 (2003)), and Akt (Povsic et al, J. Biol. Chem. 278:51334-51339 (2003); Goel et al, J. Biol. Chem. 277:18640-18648 (2002)), these biased ligands can be expected to function as agents that bifunctionally antagonize cytotoxic G protein-mediated signaling while actively inducing cytoprotective β-arrestin/GRKdependent signaling.
EXAMPLE 8
Halothane anesthetized wild-type c57B16 mice fitted with a carotid artery polyethylene catheter connected to a pressure transducer for the real-time detection of mean arterial blood pressure and a jugular catheter were implanted for the delivery of drugs. Blood pressure was then recorded before and after the administration of SII (10 mg/kg i.v.) and after a dose response to angiotensin II (10-7 - 10-5 g/kg). Fig. 35A illustrates the effect of SII on mean arterial blood pressure as both percent and absolute changes from basal. SII significantly decreased mean arterial blood pressure in anesthetized wild-type mice (p<0.01, Student's modified t-test). Fig. 35B illustrates that SII pre-treatment (10 mg/kg) results in a leftward shift in the angiotensin II dose response (p<0.05 control vs. post-SII two-way ANOVA w/ Bonferroni). Thus, SII can lower blood pressure and acts as a competitive antagonist of angiotensin II in vivo.
106 Ketamine/xylazine induced halothane maintained wild-type c57B16 mice were vagotomized and mechanically ventilated. The mice then had a Millar conductance catheter introduced into the common carotid artery and passed retrograde into the LV cavity, for determining cyclic changes in ventricular pressure and volume, while simultaneously recording systemic arterial pressure via a left axillary arterial catheter. An internal jugular venous catheter was also be placed for the administration of drugs. The effects of AngII or SII on dP/dtmax, dP/dtmin and heart rate are shown in Figs. 36-38, respectively. SII produced significant increases in dP/dtmax, dP/dtmin and heart rate. * /?<0.05 response vs. basal, one-way ANOVA and Bonferroni correction for multiple comparisons.
EXAMPLE 9
Experimental Details
Materials - Acebutolol, alprenolol, atenolol, betaxolol, ICI 118,551, isoproterenol, labetalol, metoprolol, nadolol, pindolol, propranolol, sotalol and timolol were obtained from Sigma. Bisoprolol and practolol were obtained from Mutual Pharmaceutical Co. and Ayerst Laboratories Inc., respectively..Racemic carvedilol was generously provided by Dr. Richard Bond (University of Houston). Antibodies recognizing the β2AR and phosphorylated β2AR on phosphoserines 355 or 356 (1 :2,000 for Western blot) were purchased from Santa Cruz
Biotechnology. Rabbit polyclonal β-arrestin antibody (AlCT) was generated as described by Attramadal et al (J. Biol. Chem. 267: 17882-17890 (1992)). Detection of pERK was with a rabbit polyclonal anti-phospho-p44/42 MAPK (Cell Signaling Technology, 1 :3,000 for Western blot). Total ERK1/2 was detected with anti-MAPK 1/2 (Upstate Technology Inc., 1 :6,000 for Western blot). Horseradish peroxidase-conjugated secondary antibodies were from Amersham Biosciences. M2 anti-FLAG affinity agarose beads, G418, mouse
107 monoclonal anti-FLAG M2 antibody, and anti-mouse IgG conjugated to fluorescein isothiocyanate, were obtained from Sigma.
Plasmids - FLAG-β2AR/pcDNA3 (Shenoy et al, J. Biol. Chem. 281 :1261-1273 (2006)) and β-arrestin2-GFP (Shenoy et al, J. Biol. Chem. 281 :1261-1273 (2006)) were generated as described previously. The β2AR-V2R chimera receptor was a generous gift from Dr. Marc Caron (Duke University) and has been described previously (Oakley et al, J. Biol. Chem. 274:17201-17210 (2000)).
Cell Culture - HEK-293 cells were obtained from ATCC and maintained in modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution (Sigma). HEK-293 lines stably expressing 2pmoles/mg of β2AR or β2ARTYY were generated as described previously (Shenoy et al, J. Biol. Chem. 281 :1261-1273 (2006)). HEK-293 cells stably expressing either β2AR or β2ARτγγ were maintained in Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin solution, and 400 μg/ml G418 (Sigma).
ICUE2 cAMP Assay - Neocin-resistant HEK-293 cells stably overexpressing the human β2AR (Shenoy et al, J. Biol. Chem. 281 :1261-1273 (2006)) were stably transfected with a zeocin-resistant plasmid encoding the cAMP biosensor ICUE2 (DiPilato et al, Proc. Natl. Acad. Sci USA 101 :16513- 16518 (2004)). These cells were clonally selected and validated as described (DiPilato et al, Proc. Natl. Acad. Sci USA 101 :16513-16518 (2004)). Intracellular cAMP concentrations were measured as a FRET Ratio: the CFP channel intensity divided by the FRET channel intensity. These channels were detected by excitation through a 438/24 bandpass filter (24 nm bandpass centered at 438 nm) and emission through a 542/27 nm bandpass filter (for the FRET channel) and a 483/32 nm bandpass filter (for the CFP channel). All experiments were performed on a BMG Labtech NOVOstar plate reader using 96-well plates
108 (Costar #3604) seeded with 50,000 cells per well. All filters were from Semrock. Data shown are the differences between the FRET Ratio before and after addition of ligand, or, for the inverse agonism experiments, addition of isobutylmethylxanthine (IBMX). IBMX was used at 250 μM to inhibit PDE activity and allow detection of constituitive β2AR activity.
Phospho-ERK and Phospho-β2AR Assays - pERK and phospho-β2AR assays were carried out as has previously been described (Shenoy et al, J. Biol. Chem. 281 :1261-1273 (2006)).
Immunoblotting - Immunoblotting was carried out as previously described (Ahn et al, J. Biol. Chem. 279:35518-35525 (2004)) except chemiluminescence detection was performed with horseradish peroxidase-coupled secondary antibody (Amersham Biosciences) and SuperSignal West Pico reagent (Pierce). Chemiluminescence was quantified by a charge-coupled device camera (Syngene ChemiGenius2) according to the manufacturer. Metabolic Labeling and β-arrestin Translocation Assays - Metabolic labeling and β-arrestin translocation assays were accomplished according to previously described protocols (Shenoy et al, J. Biol. Chem. 281 :1261-1273 (2006)) except for β-arrestin translocation, HEK-293 cells in 10 cm dishes were transiently transfected with the β2V2R chimera (Oakley et al, J. Biol. Chem. 274:17201-17210 (2000)) (consisting of the first 341 aa of the β2AR and the last 29 aa of the V2R) and β-arrestin-2-GFP using FuGENE (Roche Applied Science). Also, images of GFP fluorescence were taken starting 1 minute post-stimulation and subsequently at 1 -minute intervals up to 10 minutes.
Internalization and siRNA Silencing of Gene Expression - Internalization and siRNA gene silencing were carried out with previously described siRNAs and methods Shenoy et al, J. Biol. Chem. 281 :1261-1273 (2006)), except for the addition of a second siRNA targeting β-arrestin2 which was 5'- CCAACCUCAUUGAAUUUGA-3'. To determine β-arrestin2 protein silencing,
109 endogenous β-arrestinl/2 were detected with AlCT (1 :3000) antibody, as previously described (Attramadal et al, J. Biol. Chem. 267:17882-17890 (1992)). Only experiments with validated silencing were analyzed; average silencing of β- arrestin2 was 69.9% ± 1.6.
Statistical Analysis - Statistical analyses performed were one-way
ANOVA with post-hoc Bonferroni tests, or Student's paired t tests. A P value of < 0.05 was considered statistically significant. GraphPad PRISM software was used for data analyses.
Results cAMP accumulation monitored by ICUE2. Typically receptor ligands have been classified as either agonist (full or partial) or antagonist with respect to G protein coupling efficiency. Work over the last 20 years has expanded this classification to include the concept of inverse agonism and it has been observed that numerous classical neutral antagonists actually act as either partial agonists or inverse agonists (Kenakin, Trends Pharmacol. Sci. 25:186-192 (2004)). Here, a FRET based biosensor was used to monitor cAMP concentration in live cells in order to assess the extent to which known βAR antagonists can stimulate G5- dependent AC activation through the β2AR (DiPilato et al, Proc. Natl. Acad. Sci USA 101 :16513-16518 (2004)). Acebutolol, alprenolol, atenolol, labetalol, oxprenolol, pindolol and practolol displayed weak partial agonism for G5- dependent AC activation (Fig. 39A). Ligands that did not stimulate significant cAMP generation were further analyzed for inverse agonism. Betaxolol, bisoprolol, carvedilol, ICI 118,551, metoprolol, nadolol, propranolol, sotalol and timolol all functioned as inverse agonists decreasing constitutive cAMP accumulation (Fig 39B). It should be noted that, similar to previous reports (Kenakin, Trends Pharmacol. Sci. 25:186-192 (2004)), no neutral antagonists
110 were observed in these assays indicating that this classification may be an artifact of assay sensitivity and that most if not all βAR ligands actually fall on either side of zero with regard to efficacy.
ERK 1/2 activation. Recent work has demonstrated that β-arrestins can serve as scaffolds for signaling networks including the MAPK, ERK 1/2 (DeFea et al, J. Cell. Biol. 148:1267-1281 (2000), Luttrell et al, Proc. Natl. Acad. Sci. USA 98:2449-2454 (2001)) and that this activation can be independent of G protein (Shenoy et al, J. Biol. Chem. 281 :1261-1273 (2006)). While none of the βAR antagonists tested led to ERKl/2 activation in untransfected HEK-293 cells, in HEK-293 cells stably expressing 2pmoles/mg β2AR, a wide range of phosphorylated ERK 1/2 (pERK) responses can be elicited by different βAR antagonists (Fig. 40A). Acebutolol, atenolol, alprenolol, carvedilol, labetalol, oxprenolol, pindolol, practolol and propranolol all stimulate significant pERK to varying degrees. In order to explore the role of G protein stimulation in the ERK 1/2 responses elicited by these compounds, a mutant β2AR was used that does not couple to G proteins but maintains the ability to stimulate MAP kinases: β2ARτγγ (Shenoy et al, J. Biol. Chem. 281 : 1261-1273 (2006)). Of these ligands, only carvedilol-stimulated a significant response in HEK-293 cells stably expressing the mutant β2ARτγγ (Fig. 40B). In both cell lines, carvedilol- stimulated pERK was insensitive to pertussis toxin pretreatment indicating that Gi coupling plays no role in carvedilol stimulated pERK (Fig. 44). Furthermore, carvedilol-stimulated pERK was completely blocked by pretreatment with the β2AR selective antagonist ICI 118,551 (data not shown), demonstrating that this activation occurs specifically through the β2AR. β2AR phosphorylation. The screen was then focused on carvedilol and propranolol, which were the only βAR antagonists that functioned as inverse agonists for Gs-dependent AC activation but were still able to stimulate
111 phosphorylation of ERK 1/2. Previous work has demonstrated that the cytoplasmic tail of the β2AR must be phosphorylated by GRKs in order for β- arrestin to be recruited to the receptor (Lohse et al, J. Biol. Chem. 267:8558-8564 (1992)). Using both a phospho-specific antibody identifying GRK 5 phosphorylation sites as well as 32P metabolic labeling, it was observed that carvedilol but not propranolol, can stimulate significant GRK mediated phosphorylation of the β2AR. A 30 minute treatment with carvedilol stimulated a 1.9 ± 0.09 fold (p < 0.0001) increase in receptor phosphorylation at the known GRK sites, serine 355/356 (Fig. 41A) and a 1.9 ± 0.1 (p < 0.0001) fold increaseo over basal in global receptor phosphorylation (Fig. 41B). Carvedilol mediated receptor phosphorylation was blocked by propranolol pretreatment (data not shown). βarrestin recruitment. The β2AR exhibits a transient, low affinity interaction with β-arrestins and undergoes rapid recycling to the plasma 5 membrane after internalization, a pattern known as "Class A" recruitment. "Class B" receptors such as the vasopressin V2 receptor (V2R) have a more prolonged interaction with β-arrestin and are recycled to the plasma membrane more slowly than Class A receptors (Oakley et al, J. Biol. Chem. 275:17201-17210 (2000)). One previously devised method for increasing β-arrestin affinity, and therefore o assay sensitivity, is to construct chimeric Class A receptors which possess a Class B receptor cytoplasmic tail (Oakley et al, J. Biol. Chem. 274:17201-17210 (2000)). The β2AR-V2R receptor chimera is one such example. Using confocal microscopy, a determination was made as to whether carvedilol or propranolol could stimulate recruitment of β-arrestin2 to the β2AR-V2R in HEK-293 cells. In5 this system, both isoproterenol and carvedilol stimulated recruitment of β- arrestin2-GFP to the receptor whereas propranolol did not (Fig. 42A). Alprenolol, labetalol, and ICI 118,551 were also unable to stimulate recruitment of β- arrestin2-GFP to the β2AR-V2R (data not shown). Furthermore, carvedilol
112 stimulated β-arrestin recruitment was blocked by pretreatment with either ICI 118,551 or propranolol (data not shown), demonstrating β2AR specificity. β2AR internalization. Previous studies have demonstrated that β-arrestins can serve as adaptors for AP-2 and clathrin (Goodman et al, Nature 383:447-450 (1996), Krupnick et al, J. Biol. Chem. 272:15011-15016 (1997), Laporte et al, J. Biol. Chem. 277:9247-9254 (2002)), which bring activated receptors to clathrin- coated pits for endocytosis and facilitate receptor internalization. Carvedilol, but not propranolol, induces receptor internalization as assessed by fluorescence- activated cell sorting. Carvedilol stimulated 5.5% ± 1.7 (p < 0.05) receptor internalization, whereas the full agonist isoproterenol stimulated 38.7% ± 3.5 (p < 0.0001) receptor internalization (Fig. 42B). β-arrestin2 mediated ERK activation. To test the potential role of G proteins in carvedilol stimulated cell signaling, an analysis was made of ERK 1/2 activation after cellular depletion of β-arrestin2 using β-arrestin2 specific siRNA in HEK-293 cells stably expressing β2AR. For isoproterenol, pERK was reduced by 42.3% ± 2.2 at 5 minutes whereas for carvedilol, pERK was reduced by 71.0% ± 4.5 (Fig. 43A). This correlated with the overall effect of β-arrestin2 siRNA to lower cellular levels of β-arrestin2 by -70% (data not shown). In HEK-293 cells stably expressing β2ARτγγ, pERK for isoproterenol was reduced by 38.5% ± 5.2 at 5 minutes whereas for carvedilol, pERK was reduced by 70.1% ± 4.4 after β- arrestin2 silencing (Fig. 43B). This also correlated with the overall efficiency of the β-arrestin2 siRNA to lower cellular levels of β-arrestin2 by ~70% (data not shown). Additionally, a second siRNA targeting β-arrestin2 was used to test
TW specificity and similar results were obtained (data not shown). β2AR stable cells were created in HEK-293 cells that express endogenous levels of wild type β2AR. Consequently at 10 μM, isoproterenol-stimulated ERK activation seen in cells expressing β2ARτγγ in the presence of siRNA targeting β-arrestin2 was
113 largely due to G protein-stimulated ERK activation from endogenous wild type β2ARs on the surface of these cells and is not therefore mediated by the mutant receptors Shenoy et al, J. Biol. Chem. 281 :1261-1273 (2006)).
In summary, amongst a panel of 16 βAR antagonists, a diverse spectrum of efficacies was observed for both Gs-dependent and β-arrestin-dependent cellular signaling (Table 3). Additionally, carvedilol is identified as a compound that possesses the unique signaling profile of negative efficacy for Gs-dependent AC activation while simultaneously stimulating β-arrestin-dependent ERK 1/2 activation. Moreover, carvedilol stimulates phosphorylation of the β2AR, β- arrestin translocation to the receptor, and receptor internalization, all of which are characteristic of β-arrestin-mediated cellular processes. Thus carvedilol acts as a biased ligand (Violin et al, Trends Pharmacol. Sci. 28(8):416-422 (2007)) signaling via β-arrestin-dependent ERK 1/2 activation in the absence of G protein activation. This bias may help explain carvedilol's unique clinical effectiveness in heart failure and other cardiovascular diseases.
114 TABLE 3
Figure imgf000114_0001
The concept of ligand bias challenges the traditional paradigm of 7TMR ligand characterization based on "intrinsic efficacy," which can be defined as a measure of stimulus per receptor molecule elicited by a ligand upon binding (Urban et al, J. Pharmacol. Exp. Ther. 320:1-13 (2007)). Implicit in this classification is the assumption that a given ligand should be equally effective at stimulating all cellular responses for a given receptor (Urban et al, J. Pharmacol. Exp. Ther. 320:1-13 (2007), Benovic et al, J. Biol. Chem. 263:3893-3897 (1988)). However, much work over the last 15 years has demonstrated that a single ligand can have differential intrinsic efficacies for various effector systems downstream of a given receptor (Kenakin, Trends Pharmacol. Sci. 16:232-238 (1995), Urban et al, J. Pharmacol. Exp. Ther. 320:1-13 (2007), Baker et al, MoI. Pharmacol. 63:1312-1321 (2003), Azzi et al, Proc. Natl. Acad. Sci. USA 100:11406-11411 (2003), Galandrin et al, MoI. Pharmacol. 70:1575-1584 (2006)). Consequently a given ligand' s pharmacologic efficacy, or its ability to produce a desired therapeutic effect, may not be fully explained by its ability to stimulate a single receptor-mediated signaling pathway. The recognition that pharmacologic efficacy cannot be encompassed by the simple agonist/antagonist classification has not, to date, been correlated with insight into associated clinical outcomes of biased ligands. Recently, it has been appreciated that many receptors including the β2AR can exist in multiple "active" conformations after ligand binding (Kenakin, Trends Pharmacol. Sci. 16:232-238 (1995), Ghanouni et al, Proc. Natl. Acad. Sci. USA 98:5997-6002 (2001), Swaminath et al, J. Biol. Chem. 280:22165-22171 (2007), Granier et al, J. Biol. Chem. 282:13895-13905 (2007)). These variable conformations may lead to widely differing cellular outcomes and may help explain the diverse signaling profiles observed with a variety of βAR antagonists. Viewed in the context of this report, it seems likely that carvedilol stabilizes distinct receptor conformations from other βAR antagonists. Thus, it is perhaps
116 not surprising that these compounds range in their clinical efficacy as well (Bristow, Circulation 101 :558-569 (2000)). It is postulated that the diverse signaling profiles observed in this study indicate that ligand bias may play a role in determining the effectiveness of pharmacologic agents targeting 7TMRs. One of the major paradigm shifts in cardiovascular medicine was the innovation that βAR antagonists could be an effective therapy for the treatment of heart failure (Waagstein et al, Br. Heart J. 37:1022-1036 (1975)). This originated from the observation that activation of the sympathetic nervous system may be fundamental to the progression of heart failure (Bristow, N. Engl. J. Med. 311 :850-851 (1984)). Consequently, it was hypothesized that pharmacologic blockade of the sympathetic nervous system, in particular of the βl AR and β2AR, could slow the progression of heart failure (Fowler et al, Am. J. Cardiol. 55:120D-124D (1985)). It was recognized quite early that different βAR antagonists possessed differing clinical efficacies in heart failure and their effectiveness could not simply be attributed to a class effect (Cruickshank, Int. J. Cardiol. 120:10-27 (2007), Hunt et al, Circulation 112:3154-e235 (2005)). Currently, only three agents, carvedilol, metoprolol succinate and bisoprolol are approved for the treatment of heart failure in the United States (Hunt et al, Circulation 112:3154-e235 (2005)). Although their relative efficacies are unknown, some evidence suggests that carvedilol may possess survival advantages over other βAR antagonists (Poole- Wilson et al, Lancet 362:7-13 (2003)).
Survival advantages observed with carvedilol treatment in cardiovascular diseases including heart failure (Poole- Wilson et al, Lancet 362:7-13 (2003)) and post- AMI (Kopecky, Am. J. Cardiol. 98:1115-1119 (2006)) have previously been ascribed to a multitude of ancillary properties of carvedilol as outlined earlier (Yue et al, J. Pharmacol. Exp. Ther. 263:92-98 (1992), Packer, Prog. Cardiovasc. Dis. 41 :39-52 (1998), Ohlstein et al, Proc. Natl. Acad. Sci. USA 90:6189-6193
117 (1993), Naccarelli et al, Clin. Cardiol. 28:165-173 (2005), Bristow et al, J. Cardiovasc. Pharmacol. 19 Suppl l :S68-80 (1992)). However, controversy still exists over whether any of these properties can sufficiently explain carvedilol's clinical efficacy. The unique signaling profile exhibited by carvedilol may, in part, explain its distinctive clinical efficacy. This hypothesis is supported by recent animal work demonstrating that β-arrestin-dependent signaling can be cardioprotective in the presence of chronic catecholamine stimulation, as is the case in heart failure, whereas G protein-dependent signaling may be cardiotoxic under these same conditions (Noma et al, J. Clin. Invest., in press (2007)). These in vivo experiments revealed that in these conditions, the loss of β-arrestin- mediated signaling through the betal adrenergic receptor resulted in increased apoptosis and cardiac deterioration. This work provides strong evidence that a biased ligand such as carvedilol, which antagonizes G protein-mediated signaling while simultaneously stimulating β-arrestin-mediated signaling, may have increased therapeutic potential over conventional βAR antagonists.
In the studies described above, only carvedilol possessed this unique profile of inverse agonism for Gs-dependent AC activation while concurrently stimulating β-arrestin-dependent ERK 1/2 activation. Propranolol, which also acted as an inverse agonist for Gs-dependent AC activation, was able to weakly activate ERK 1/2 as has previously been observed by others (Baker et al, MoI. Pharmacol. 63: 1312-1321 (2003), Azzi et al, Proc. Natl. Acad. Sci. USA 100:11406-11411 (2003), Galandrin et al, MoI. Pharmacol. 70:1575-1584 (2006)). In this system, however, propranolol was unable to activate several characteristic processes of β-arrestin-dependent signaling. These observations suggest the possibility of yet another signaling pathway to ERK 1/2 activation that is both G protein and β-arrestin-independent which can be stimulated by propranolol. The functional consequences of such a pathway are unknown but are likely to be different from both the G protein and β-arrestin-dependent pathways.
118 In the above-described studies, only ERK 1/2 activation was monitored as a readout for β-arrestin-dependent signaling. Since β-arrestin is known to signal via a range of other signaling pathways, it is likely that β-arrestin biased ligands could also stimulate β-arrestin-dependent, G protein-independent signaling via JNK3, p38 kinase, PI3K or Akt. It is also possible that biased ligands could stimulate multiple levels of specificity such as GRK subtype-specific receptor phosphorylation and/or stabilization of specific conformations of β-arrestin coupled to distinct functional outcomes. Furthermore, compounds could also be biased in the opposite direction than carvedilol such that they activate G protein mediated pathways while simultaneously antagonizing β-arrestin-dependent signaling pathways (Groer et al, MoI. Pharmacol. 71 : 549-557 (2007)). The physiological and clinical profiles of such novel agents might be distinct from those currently available. Ultimately these biased ligands may possess even greater efficacy in the treatment of various diseases while reducing off-target effects and would represent a new model for the development of 7TMR-targeted therapeutics.
The recognition that intrinsic efficacy is not simply a function of G protein coupling efficacy and can vary greatly depending on the observed effector system has changed the way pharmacologic efficacy is defined. The above-described studies have resulted in the identification of a signaling profile unique to carvedilol which may correlate with, or be responsible for, its unique clinical efficacy. In this study, carvedilol stimulates only very weak activation of β- arrestin dependent signaling processes. Carvedilol can serve as a prototype for the design of novel compounds that, while possessing no efficacy for G protein stimulation, can nevertheless stimulate β-arrestin dependent signaling to a greater extent than carvedilol. Biased ligands of this type represent a new generation of therapeutic agents that can be targeted for any number of receptors. Although the therapeutic potential of these biased ligands remains to be demonstrated, the data
119 presented herein suggest a potential new direction in the treatment of cardiovascular disease and in particular in heart failure.
All documents and other information sources cited above are hereby incorporated in their entirety by reference.
120

Claims

WHAT IS CLAIMED IS:
1. A method of identifying a biased ligand for a G protein coupled receptor (GPCR) comprising: i) determining the effect of a test compound on GPCR-mediated G protein activity, and ii) determining the effect of said test compound on a GPCR-mediated β- arrestin function, wherein a test compound that has a greater positive effect on said GPCR- mediated β-arrestin function than on said GPCR-mediated G-protein activity, relative to a reference agonist for both said GPCR-mediated G-protein activity and said GPCR-mediated β-arrestin function, is a biased ligand.
2. The method according to claim 1 wherein said GPCR is present in a eukaryotic cell.
3. The method according to claim 1 wherein step (i) is effected by measuring the level of calcium, cyclic adenosine monophosphate (cAMP), diacyl glycerol or inositol triphosphate in the presence and absence of said test compound.
4. The method according to claim 1 wherein step (i) is effected by measuring phosphatidyl inositol turnover, GTP-γ-S loading, adenylatecyclase activity or GTP hydrolyis in the presence and absence of said test compound.
5. The method according to claim 1 wherein step (ii) is effected by measuring β-arrestin or GRK recruitment to, or internalization or GRK-mediated
121 phosphorylation of, said GPCR in the presence and absence of said test compound.
6. The method according to claim 5 wherein step (ii) is effected by measuring said β-arrestin recruitment to said GPCR by assaying the physical interaction between β-arrestin and said GPCR.
7. The method according to claim 5 wherein said β-arrestin recruitment is measured by resonance energy transfer, bimolecular fluorescence, enzyme complementation, visual translocation, co-immunoprecipitation, membrane association, or interaction of β-arrestin with a naturally occurring binding partner.
8. The method according to claim 7 wherein said GPCR is present in a eukaryotic cell and said β-arrestin recruitment is measured by resonance energy transfer.
9. The method according to claim 8 wherein said cell co-expresses said GPCR fused to a first fluorescent protein (GPCR fusion protein) and β-arrestin fused to a second fluorescent protein (β-arrestin fusion protein), wherein said first and second fluorescent proteins undergo fluorescence resonance energy transfer (FRET) upon interaction of said β-arrestin fusion protein with said GPCR fusion protein, and wherein said β-arrestin recruitment is determined by measuring the FRET increase in the presence of said test compound.
122
10. The method according to claim 9 wherein said first fluorescent protein is a monomeric cyan variant of Green Fluorescent Protein and said second fluorescent protein is a yellow variant of Green Fluorescent Protein.
11. The method according to claim 1 wherein step (i) effected by measuring the level of cAMP cell in the presence and absence of said test compound and step (ii) is effected by measuring the β-arrestin recruitment to said GPCR in the presence and absence of said test compound.
12. A method of identifying a candidate therapeutic that modulates a physiological process comprising: i) determining the effect of a test compound on G-protein activity mediated by a GPCR relevant to said physiological process, and ii) determining the effect of said test compound on a β-arrestin function mediated by said GPCR, wherein a test compound that has a greater positive effect on said β- arrestin function mediated by said GPCR than on said G-protein activity mediated by said GPCR, relative to a reference agonist for both said G-protein activity mediated by said GPCR and said β-arrestin function mediated by said GPCR, is said candidate therapeutic.
13. The method according to claim 12 wherein said method is a method of identifying a candidate therapeutic for treating a cardiovascular disease or disorder.
123
14. The method according to claim 13 wherein said cardiovascular disease or disorder is hypertension, heart failure, coronary artery disease, pulmonary hypertension, peripheral vasculature disease or arrhythmia.
15. The method according to claim 12 wherein said method is a method of identifying a candidate therapeutic for treating a pulmonary disease or disorder.
16. The method according to claim 15 wherein said pulmonary disease or disorder is asthma, chronic obstructive pulmonary disease (COPD) or pulmonary fibrosis.
17. The method according to claim 12 wherein said method is a method of identifying a candidate therapeutic for treating an ophthalmologic disease or disorder.
18. The method according to claim 17 wherein said ophthalmologic disease is glaucoma.
19. The method according to claim 12 wherein said method is a method of identifying a candidate therapeutic for treating a hematologic disease or disorder.
20. The method according to claim 19 wherein said hematologic disease or disorder is a thrombolytic disease or disorder.
124
21. The method according to claim 12 wherein said method is a method of identifying a candidate therapeutic for treating an endocrine or metabolic disease or disorder.
22. The method according to claim 21 wherein said endocrine or metabolic disease or disorder is diabetes or obesity.
23. The method according to claim 12 wherein said method is a method of identifying a candidate therapeutic for treating a neurological or psychiatric disease or disorder.
24. The method according to claim 23 wherein neurological disease is Parkinsonism or Alzheimer's.
25. A method of modulating a physiological process regulated by a GPCR comprising administering to a patient in need thereof a compound that is an agonist of G-protein activity mediated by said GPCR and that has a greater positive effect on β-arrestin function mediated by said GPCR than on G-protein activity mediated by said GPCR, relative to a reference agonist for both said G- protein activity mediated by said GPCR and said β-arrestin function mediated by said GPCR, wherein said compound is administered in an amount such that said modulation is effected.
26. The method according to claim 25 wherein said method is a method of treating a cardiovascular disease or disorder.
125
27. The method according to claim 26 wherein said cardiovascular disease or disorder is hypertension, heart failure, coronary artery disease, pulmonary hypertension, peripheral vasculature disease or arrhythmia.
28. The method according to claim 25 wherein said method is a method of treating a pulmonary disease or disorder.
29. The method according to claim 28 wherein said pulmonary disease or disorder is asthma, chronic obstructive pulmonary disease (COPD) or pulmonary fibrosis.
30. The method according to claim 25 wherein said method is a method of treating an ophthalmologic disease or disorder.
31. The method according to claim 30 wherein said ophthalmologic disease is glaucoma.
32. The method according to claim 25 wherein said method is a method of treating a hematologic disease or disorder.
33. The method according to claim 32 wherein said hematologic disease or disorder is a thrombolytic disease or disorder.
34. The method according to claim 25 wherein said method is a method of treating an endocrine or metabolic disease or disorder.
35. The method according to claim 34 wherein said endocrine or metabolic disease or disorder is diabetes or obesity.
126
36. The method according to claim 25 wherein said method is a method of treating a neurological or psychiatric disease or disorder.
37. The method according to claim 24 wherein neurological disease is Parkinsonism or Alzheimer's.
38. A method of modulating a physiological process regulated by a GPCR comprising administering to a patient in need thereof a compound that is an antagonist of G-protein activity mediated by said GPCR and that has a greater positive effect on β-arrestin function mediated by said GPCR than on G-protein activity mediated by said GPCR, relative to a reference agonist for both said G- protein activity mediated by said GPCR and said β-arrestin function mediated by said GPCR, wherein said compound is administered in an amount such that said modulation is effected.
39. The method according to claim 38 wherein said method is a method of treating a cardiovascular disease or disorder.
40. The method according to claim 39 wherein said cardiovascular disease or disorder is hypertension, heart failure, coronary artery disease, pulmonary hypertension, peripheral vasculature disease or arrhythmia.
41. The method according to claim 38 wherein said method is a method of treating a pulmonary disease or disorder.
127
42. The method according to claim 41 wherein said pulmonary disease or disorder is asthma, chronic obstructive pulmonary disease (COPD) or pulmonary fibrosis.
43. The method according to claim 38 wherein said method is a method of treating an ophthalmologic disease or disorder.
44. The method according to claim 43 wherein said ophthalmologic disease is glaucoma.
45. The method according to claim 38 wherein said method is a method of treating a hematologic disease or disorder.
46. The method according to claim 45 wherein said hematologic disease or disorder is a thrombolytic disease or disorder.
47. The method according to claim 38 wherein said method is a method of identifying a candidate therapeutic for treating an endocrine or metabolic disease or disorder.
48. The method according to claim 47 wherein said endocrine or metabolic disease or disorder is diabetes or obesity.
49. The method according to claim 38 wherein said method is a method of identifying a candidate therapeutic for treating a neurological or psychiatric disease or disorder.
128
50. The method according to claim 38 wherein neurological disease is Parkinsonism or Alzheimer's.
51. The method according to claim 26 wherein said compound is of formula I
Figure imgf000128_0001
wherein
Ri is an alkyl,
R2 and R3 are the same or different and are -H, alkyl or cycloalkyl, or R2 and R3 together with the nitrogen to which they are attached form a ring, and
R4 is -OH or H, or pharmaceutically acceptable salt thereof, with the proviso that said method is not a method of treating asthma when said compound according to formula I is isoetharine or ethylnorepinephrine.
52. The method according to claim 39 wherein said compound is of formula II
Figure imgf000128_0002
wherein R is a substituted or unsubstituted linear or branched alkyl or a substituted or unsubstituted cycloalkyl, or a substituted or unsubstituted heterocyclic group,
129 with the proviso that said method is not a method of treating cardiovascular disease when said compound of formula II is carvedilol or (+)-l- (carbazol-4-yloxy)-3-(isopropylamine)-2 propanol.
53. The method according to claim 38 wherein said compound is the mutant angiotensin (Ang) II peptide Sar1, He4, Ile8-AngII (II), or derivative of mimetic thereof.
54. The method according to claim 53 wherein said method is a method of treating acute heart failure.
55. A compound selected from (+)-l-(carbazol-4-yloxy)-3-(t- butylamino)-2-propanol and (+)-l-(carazol-4-yloxy)-3-(cyclopentylamino)-2- propanol.
56. A composition comprising the compound of claim 55 and a carrier.
130
PCT/US2007/018394 2006-08-18 2007-08-20 Biased ligands and methods of identifying same WO2008021552A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US83847406P 2006-08-18 2006-08-18
US60/838,474 2006-08-18

Publications (3)

Publication Number Publication Date
WO2008021552A2 true WO2008021552A2 (en) 2008-02-21
WO2008021552A9 WO2008021552A9 (en) 2008-05-29
WO2008021552A3 WO2008021552A3 (en) 2008-11-06

Family

ID=39082807

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2007/018394 WO2008021552A2 (en) 2006-08-18 2007-08-20 Biased ligands and methods of identifying same

Country Status (1)

Country Link
WO (1) WO2008021552A2 (en)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009151542A2 (en) * 2008-05-27 2009-12-17 Duke University Nicotinic acid receptor ligands
WO2016196750A1 (en) * 2015-06-03 2016-12-08 Trevena, Inc. Beta-arrestin effectors and compositions and methods of use thereof
US9789131B1 (en) 2016-04-21 2017-10-17 Astrocyte Pharmaceuticals, Inc. Compounds and methods for treating neurological and cardiovascular conditions
US10765693B2 (en) 2018-09-26 2020-09-08 Astrocyte Pharmaceuticals, Inc. Polymorphic compounds and uses thereof
WO2022192252A1 (en) * 2021-03-09 2022-09-15 Chronic Airway Therapeutics Limited Use of nadolol to treat chronic obstructive pulmonary disease by blockage of the arrestin-2 pathway
US11839615B2 (en) 2018-02-09 2023-12-12 Astrocyte Pharmaceuticals, Inc. Compounds and methods for treating addiction and related disorders
US11866482B2 (en) 2018-02-07 2024-01-09 Duke University System and method for homogenous GPCR phosphorylation and identification of beta-2 adrenergic receptor positive allosteric modulators

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6358990B1 (en) * 1997-10-15 2002-03-19 Boehringer Mannheim Pharmaceuticals Corp. Method for treating Alzheimer's disease
US6673931B2 (en) * 1999-04-27 2004-01-06 Hoffman-La Roche Inc. Naphthalenyl piperidines as renin inhibitors

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6358990B1 (en) * 1997-10-15 2002-03-19 Boehringer Mannheim Pharmaceuticals Corp. Method for treating Alzheimer's disease
US6673931B2 (en) * 1999-04-27 2004-01-06 Hoffman-La Roche Inc. Naphthalenyl piperidines as renin inhibitors

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BORRELLO F. ET AL.: 'Reappraisal of beta-blocker therapy in the acute and chronic post-myocardial infarction period' no. 4, SUPPL. 3, 2003, *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009151542A2 (en) * 2008-05-27 2009-12-17 Duke University Nicotinic acid receptor ligands
WO2009151542A3 (en) * 2008-05-27 2010-02-25 Duke University Nicotinic acid receptor ligands
WO2016196750A1 (en) * 2015-06-03 2016-12-08 Trevena, Inc. Beta-arrestin effectors and compositions and methods of use thereof
US9789131B1 (en) 2016-04-21 2017-10-17 Astrocyte Pharmaceuticals, Inc. Compounds and methods for treating neurological and cardiovascular conditions
US10265338B2 (en) 2016-04-21 2019-04-23 Astrocyte Pharmaceuticals, Inc. Compounds and methods for treating neurological and cardiovascular conditions
US10953031B2 (en) 2016-04-21 2021-03-23 Astrocyte Pharmaceuticals, Inc. Compounds and methods for treating neurological and cardiovascular conditions
US11484545B2 (en) 2016-04-21 2022-11-01 Astrocyte Pharmaceuticals, Inc. Compounds and methods for treating neurological and cardiovascular conditions
US11866482B2 (en) 2018-02-07 2024-01-09 Duke University System and method for homogenous GPCR phosphorylation and identification of beta-2 adrenergic receptor positive allosteric modulators
US11839615B2 (en) 2018-02-09 2023-12-12 Astrocyte Pharmaceuticals, Inc. Compounds and methods for treating addiction and related disorders
US10765693B2 (en) 2018-09-26 2020-09-08 Astrocyte Pharmaceuticals, Inc. Polymorphic compounds and uses thereof
US11324769B2 (en) 2018-09-26 2022-05-10 Astrocyte Pharmaceuticals, Inc. Polymorphic compounds and uses thereof
WO2022192252A1 (en) * 2021-03-09 2022-09-15 Chronic Airway Therapeutics Limited Use of nadolol to treat chronic obstructive pulmonary disease by blockage of the arrestin-2 pathway

Also Published As

Publication number Publication date
WO2008021552A9 (en) 2008-05-29
WO2008021552A3 (en) 2008-11-06

Similar Documents

Publication Publication Date Title
Noma et al. β-arrestin–mediated β 1-adrenergic receptor transactivation of the EGFR confers cardioprotection
Iturrioz et al. Identification and pharmacological properties of E339–3D6, the first nonpeptidic apelin receptor agonist
Bai Dimerization of G-protein-coupled receptors: roles in signal transduction
Kara et al. A phosphorylation cluster of five serine and threonine residues in the C-terminus of the follicle-stimulating hormone receptor is important for desensitization but not for β-arrestin-mediated ERK activation
Shenoy et al. β-Arrestin-dependent, G protein-independent ERK1/2 activation by the β2 adrenergic receptor
Gicquiaux et al. Rapid internalization and recycling of the human neuropeptide Y Y1 receptor
Sjögren et al. Regulators of G protein signaling proteins as targets for drug discovery
Chan et al. Sorting nexin 27 couples PTHR trafficking to retromer for signal regulation in osteoblasts during bone growth
WO2008021552A2 (en) Biased ligands and methods of identifying same
Ge et al. Dual modulation of formyl peptide receptor 2 by aspirin‐triggered lipoxin contributes to its anti‐inflammatory activity
Doan et al. Biochemical and pharmacological characterization of nuclear urotensin‐II binding sites in rat heart
Chen et al. Individual phosphorylation sites at the C-terminus of the apelin receptor play different roles in signal transduction
KR20140099322A (en) Method for detecting protein-protein interaction
Kharmate et al. Role of somatostatin receptor 1 and 5 on epidermal growth factor receptor mediated signaling
Gehlert et al. Co-expression of neuropeptide Y Y1 and Y5 receptors results in heterodimerization and altered functional properties
Liu et al. Cytoophidia coupling adipose architecture and metabolism
Yamada et al. BRD8 is a potential chemosensitizing target for spindle poisons in colorectal cancer therapy
Lamian et al. Characterization of agonist-induced motilin receptor trafficking and its implications for tachyphylaxis
Kishi et al. Identification of a short linear sequence present in the C-terminal tail of the rat follitropin receptor that modulates arrestin-3 binding in a phosphorylation-independent fashion
US20220273751A1 (en) Gpcr heteromer inhibitors and uses thereof
Donnellan et al. Identification of a novel endoplasmic reticulum export motif within the eighth α-helical domain (α-H8) of the human prostacyclin receptor
Hawkes et al. Heterotrimeric G proteins and the single-transmembrane domain IGF-II/M6P receptor: functional interaction and relevance to cell signaling
Jong et al. GPCR signaling from intracellular membranes
EP1865320A2 (en) Cellular antanonist
US20150005237A1 (en) Peptidic inhibitor of signal transmission from g-alpha-s to g-alpha-i-coupled receptor cascades

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07837074

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

NENP Non-entry into the national phase in:

Ref country code: RU

122 Ep: pct application non-entry in european phase

Ref document number: 07837074

Country of ref document: EP

Kind code of ref document: A2