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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
  • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching

Definitions

• the invention relates to detection of cAMP levels.
• FIG. 1 Domain structure and comparison of FRET responses for cAMP reporters. Sandwiched between enhanced CFP (ECFP) and citrine are truncated forms of Epac2 or full length Epacl (with or without an R522E mutation). The construct comprising full- length Epacl generated the biggest FRET response and was designated as ICUEl.
• FIGS. 2A-C Responses of ICUEl to changes in cellular cAMP levels.
• FIG. 2A FRET response of HEK-293 cells transfected with ICUEl. The first image is a YFP -only image. Pseudocolor images depict the FRET response of the reporter to isoproterenol (ISO) stimulation at various time points. Scale bar represents 10 ⁇ m.
• FIG. 2B Representative emission ratio time courses of ICUEl and the R522E mutant stimulated with 10 ⁇ M ISO followed by 10 ⁇ M propranolol and 50 ⁇ M forskolin (FsK).
• FIG. 1A FRET response of HEK-293 cells transfected with ICUEl.
• the first image is a YFP -only image.
• Pseudocolor images depict the FRET response of the reporter to isoproterenol (ISO) stimulation at various time points. Scale bar represents 10 ⁇ m.
• FIG. 2B Representative emission ratio
• FIGS. 3A-E Fusions of ICUEl targeted to various subcellular locations.
• FIG. 3A Domain structures of the fusion constructs.
• FIG. 3B YFP-only images showing plasma membrane and nuclear distributions of various fusions. Scale bars represent 10 ⁇ m.
• Merged pseudocolor images showing co-localization of nuclear localized ICUEl with Hoechst 33342 cell-permeable dye in nucleus and mitochondria-targeted ICUEl with MitoTracker at mitochondria.
• FIG. 3A Domain structures of the fusion constructs.
• FIG. 3B YFP-only images showing plasma membrane and nuclear distributions of various fusions. Scale bars represent 10 ⁇ m.
• Merged pseudocolor images showing co-localization of nuclear localized ICUEl with Hoechst 33342 cell-permeable dye in nucleus and mitochondria-targeted ICUEl with MitoTracker at mitochondria.
• FIG. 3C Representative emission ratio time courses for untagged (ICUEl), plasma membrane-targeted (pm ICUEl), mitochondria-targeted (MitoCOX- and MitoDAKAPl -ICUEl) and nuclear-localized cAMP reporters (NLS- ICUEl) stimulated with ISO (10 ⁇ M).
• FIG. 3D Representative emission ratio time courses for pm ICUEl stimulated with PGE 1 (10 ⁇ M), followed by the removal Of PGE 1 and the addition of ISO (10 ⁇ M).
• FIG. 3E Representative emission ratio time courses for NLS-ICUEl in response to PGE 1 (10 ⁇ M) and ISO (10 ⁇ M) separated by a washing step.
• FIGS. 4A-C Simultaneous imaging of cAMP reporters targeted to different subcellular locations.
• FIG. 4A Cellular distribution of different fusions.
• FIG. 4B Representative emission ratio time courses for the pm ICUEl and nuclear localized PKA activity reporter (NLS-AKAR) in the same cell stimulated with ISO (10 ⁇ M). Identical results were found in four different cells. The AKAR response was plotted using normalized ratio of yellow to cyan emissions.
• FIG. 5 Graph showing emission ratio time courses for ICUE2 and targeted versions of ICUE2.
• Y axis normalized emission ratio (cyan/yellow).
• FIG. 6 Graph showing emission ratio time courses for ICUE2 and ICUE3.
• cAMP reporters also referred to as “reporters” of the invention comprise (a) a donor moiety; (b) a polypeptide linked to the donor moiety and comprising a cAMP-binding domain of an "exchange protein directly activated by cAMP” (Epac); and (c) an acceptor moiety linked to the polypeptide.
• Epac exchange protein directly activated by cAMP
• an acceptor moiety linked to the polypeptide In the absence of cAMP, the donor moiety and the acceptor moiety are in sufficient proximity to each other to exhibit a detectable resonance energy transfer when the donor is excited.
• Binding of cAMP to the cAMP-binding domain causes a conformational change which changes the distance or relative orientation between the donor and acceptor moieties and alters the resonance energy transfer between the moieties.
• the degree of alteration reflects cAMP levels and can be detected qualitatively or quantitatively.
• cAMP reporters of the invention are useful for detecting intracellular cAMP and for assessing intracellular cAMP dynamics, although they also can be used in in vitro assays.
• Nucleic acid molecules encoding cAMP reporters of the invention can be delivered to cells using standard DNA transfection techniques, thereby generating cells which express high levels of the reporters.
• the reporters have advantages over previous methods for assessing cAMP dynamics inside cells.
• the reporters are unimolecular and can be readily targeted to different subcellular locations or fused to signaling components. They can be used to examine compartmentalized Epac activities and their physiological functions.
• a cAMP reporter targeted to plasma membrane, mitochondria, or nucleus revealed differential dynamics of cAMP signaling in response to the activation of the ⁇ -adrenergic receptor ( ⁇ -AR) or the prostanoid receptor.
• ⁇ -AR ⁇ -adrenergic receptor
• cAMP reporters of the invention permit simultaneous imaging of cAMP dynamics and PKA phosphorylation in single living cells using locus-specific reporters.
• Methods of the invention take advantage of spatial separation of subcellular events and provide unambiguous temporal correlation of these events. This methodology complements multi-color imaging (Violin et al, J Cell Biol. 161, 899-909, 2003; DeBernardi & Brooker, Proc. Natl. Acad. Sd. USA 93, 4577-82, 1996) and is well suited for simultaneous monitoring of multiple signaling events and for evaluating the information flow within signaling cascades or crosstalk between different pathways (Zaccolo, CzV. Res. 94, 866-73, 2004).
• Polypeptides used in cAMP reporters of the invention comprise a cAMP-binding domain of an Epac, e.g., Epacl or Epac2.
• Epacl and Epac2 are well-characterized, and the locations of their cAMP -binding domains are known. See de Rooij et al., J. Biol. Chem. 275, 20829-36, 2000.
• Useful polypeptides include full-length, truncated, and mutated Epacl or Epac2 from any species which has an Epac, such as rodents ⁇ e.g., mice, rats) and primates (e.g., humans, orangutans).
• the amino acid sequences of several Epacl and Epac2 proteins are provided in SEQ ID NOS: 1, 3, and 16-20. Nucleic acid sequences which encode SEQ ID NOS: 1, 3, and 20 are shown in SEQ ID NOS:2, 4, and 21, respectively.
• the cAMP-binding domain in a cAMP reporter typically can bind cAMP; however, polypeptides comprising non-functional cAMP-binding domains are also useful, for example, for use in control reporters.
• the polypeptide itself preferably does not substantially emit light or transfer energy to excite the acceptor moiety.
• a "donor moiety” is a fluorophore or a luminescent moiety.
• the absorption spectrum of the "acceptor moiety” overlaps the emission spectrum of the donor moiety.
• the acceptor moiety does not need to be fluorescent and can be a fluorophore, chromophore, or quencher.
• both the donor and acceptor moieties are fluorescent proteins.
• both the donor and acceptor moieties are luminescent moieties.
• either one of the donor or acceptor moieties can be a fluorescent protein while the other moiety is a luminescent moiety.
• the acceptor moiety is a "quencher moiety.”
• Suitable acceptor moieties include, for example, a coumarin, a xanthene, a fluorescein, a fluorescent protein, a circularly permuted fluorescent protein, a rhodol, a rhodamine, a resorufin, a cyanine, a difluoroboradiazaindacene, a phthalocyanine, an indigo, a benzoquinone, an anthraquinone, an azo compound, a nitro compound, an indoaniline, a diphenylmethane, a triphenylmethane, and a zwitterionic azopyridinium compound.
• Suitable donor moieties include, but are not limited to, a coumarin, a xanthene, a rhodol, a rhodamine, a resorufin, a cyanine dye, a bimane, an acridine, an isoindole, a dansyl dye, an aminophthalic hydrazide, an aminophthalimide, an aminonaphthalimide, an aminobenzofuran, an aminoquinoline, a dicyanohydroquinone, a semiconductor fluorescent nanocrystal, a fluorescent protein, a circularly permuted fluorescent protein, and fluorescent lanthanide chelate.
• Fluorescent proteins include, but are not limited to, a coumarin, a xanthene, a rhodol, a rhodamine, a resorufin, a cyanine dye, a bimane, an acridine, an isoind
• either or both of the donor and acceptor moieties is a fluorescent protein.
• Suitable fluorescent proteins include green fluorescent proteins (GFP), red fluorescent proteins (RFP), yellow fluorescent proteins (YFP), and cyan fluorescent proteins (CFP).
• GFP green fluorescent proteins
• RFP red fluorescent proteins
• YFP yellow fluorescent proteins
• CFP cyan fluorescent proteins
• Useful fluorescent proteins also include mutants and spectral variants of these proteins which retain the ability to fluoresce.
• RFPs include Discosoma RFPs, such Discosoma DsRed (SEQ ID NO:9) or a mutant thereof which includes an Ilel25Arg mutation, or a non-oligomerizing tandem DsRed containing, for example, two RFP monomers linked by a peptide linker.
• a non-oligomerizing tandem RFP can contain two DsRed monomers or two mutant DsRed- I125R monomers linked by a peptide (having, for example, the amino acid sequence shown in SEQ ID NO:10).
• Useful GFPs include an Aequorea GFP ⁇ e.g., SEQ ID NO:11), a Renilla GFP, a Phialidium GFP, and related fluorescent proteins for example, a cyan fluorescent protein (CFP), a yellow fluorescent protein (YFP), or a spectral variant of the CFP or YFP.
• CFP (cyan) and YFP (yellow) are color variants of GFP.
• CFP and YFP contain 6 and 4 mutations, respectively. They are Tyr66Try, Phe66Leu, Ser65Thr, Asnl45Ile, Metl53Thr, and Vall63Ala in CFP and Ser65Gly, Vall68Leu, Ser72Ala, and Thr203Tyr.
• Spectral variants include an enhanced GFP (EGFP; SEQ ID NO: 12), an enhanced CFP (ECFP; SEQ ID NO: 13), an enhanced YFP (EYFP; SEQ ID NO: 14), and an EYFP with V68L and Q69K mutations.
• Other examples of fluorescent proteins comprising mutations are Aequorea GFP with one or more mutations at amino acid residues A206, L221 or F223 of SEQ ID NO:11 (e.g., mutations A206K, L221K, F223R, Q80R); mutations L221K and F223R of ECFP (SEQ ID NO: 12), and EYFP-V68L/Q69K of SEQ ID NO:11.
• GFP-related fluorescent proteins include those having one or more folding mutations, and fragments of the proteins that are fluorescent, for example, an A. victoria GFP from which the two N-terminal amino acid residues have been removed.
• these fluorescent proteins contain different aromatic amino acids within the central chromophore and fluoresce at a distinctly shorter wavelength than the wild type GFP species.
• the engineered GFP proteins designated P4 and P4-3 contain, in addition to other mutations, the substitution Y66H; and the engineered GFP proteins designated W2 and W7 contain, in addition to other mutations, Y66W.
• Folding mutations in Aequorea GFP-related fluorescent proteins improve the ability of the fluorescent proteins to fold at higher temperatures and to be more fluorescent when expressed in mammalian cells, but have little or no effect on the peak wavelengths of excitation and emission. If desired, these mutations can be combined with additional mutations that influence the spectral properties of GFP to produce proteins with altered spectral and folding properties, and, particularly, with mutations that reduce or eliminate the propensity of the fluorescent proteins to oligomerize.
• Folding mutations include the substitutions F64L, V68L, S72A, T44A, F99S, Y145F, N1461, M153T, M153A, V163A, I167T, S175G, S205T, andN212K.
• Luminescent moieties useful in a cAMP reporter include lanthanides, which can be in the form of a chelate, including a lanthanide complex containing the chelate (e.g, ⁇ -diketone chelates of cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, or ytterbium).
• Lanthanide chelates are well known in the art. See Soini and Kojola, Clin. Chem. 29, 65, 1983; Hemmila et al, Anal. Biochem.
• Suitable ⁇ -diketones are, for example, 2-naphthoyltrifluoroacetone (2-NTA), 1-naphthoyltrifluoroacetone (1-NTA), p-methoxybenzoyltrifluoroacetone (MO-BTA), p- fluorobenzoyltrifluoroacetone (F-BTA), benzoyltrifluoroacetone (BTA), furoyltrifluoroacetone (FTA), naphthoylfuroylmethane (NFM), dithenoylmethane (DTM), and dibenzoylmethane (DBM). See also US 20040146895.
• Luminescent proteins include, but are not limited to, lux proteins (e.g., luxCDABE from Vibrio fischerii), luciferase proteins (e.g., firefly luciferase, Gaussia luciferase, Pleuromamma luciferase, and luciferase proteins of other beetles, Dinoflagellates (Gonylaulax; Pyrocystis;), Annelids (Dipocardia), Molluscs (Lativa), and Crustacea (Vargula; Cypridina), and green fluorescent proteins of bioluminescent coelenterates (e.g., Aequorea Victoria, Renilla mullerei, Renilla reniformis; see Prendergast et al, Biochemistry 17, 3448-53, 1978; Ward et ah, Photochem.
• lux proteins e.g., luxCDABE from Vibrio fischerii
• Firefly luciferase is available from Sigma, St. Louis, MO, and Boehringer Mannheim Biochemicals, Indianapolis, IN. Recombinantly produced firefly luciferase is available from Promega Corporation, Madison, WI. Jellyfish aequorin and luciferase from Renilla are commercially available from Sealite Sciences, Bogart, GA.
• DNA sequences of the aequorin and other luciferases employed for preparation of some cAMP reporters of the invention can be derived from a variety of sources.
• cDNA can be prepared from mRNA isolated from the species disclosed above. See Faust, et al, Biochem. 18, 1106-19, 1979; De Wet et al, Proc. Natl. Acad. Sd. USA 82, 7870-73, 1985.
• Luciferase substrates are well known and include coelenterazine (available from Molecular Probes, Eugene, OR) and ENDURENTM. These cell-permeable reagents can be directly administered to cells, as is known in the art. Luciferin compounds can be prepared according to the methods disclosed by Hori et al., Biochemistry 14, 2371-76, 1975; Hori et al., Proc. Natl. Acad. ScL USA 74, 4285-87, 1977).
• the acceptor moiety is a quencher moiety, preferably a "dark quencher” (or “black hole quencher”) as is known in the art.
• a "dark quencher” or “black hole quencher”
• the change in conformation which occurs upon cAMP binding eliminates quenching, resulting in an increase in energy emission from the donor moiety.
• "Dark quenchers” themselves do not emit photons. Use of a “dark quencher” reduces or eliminates background fluorescence or luminescence which would otherwise occur as a result of energy transfer from the donor moiety.
• Suitable quencher moieties include dabcyl (4-(4'- dimethylaminophenylazo)-benzoic acid), QSYTM-7 carboxylic acid, succinimidyl ester (N,N'-dimethyl-N,N'-diphenyl-4-((5-t-butoxycarbonylaminopentyl)aminocarbon yl) piperidinylsulfone-rhodamine (a diarylrhodamine derivative from Molecular Probes, Eugene, OR).
• Suitable quencher moieties are disclosed, for example, in US 2005/0118619; US 20050112673; and US 20040146959.
• any suitable fluorophore may be used as the donor moiety provided its spectral properties are favorable for use with the chosen dark quencher.
• the donor moiety can be, for example, a Cy-dye, Texas Red, a Bodipy dye, or an Alexa dye.
• the fluorophore is an aromatic or heteroaromatic compound and can be a pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, thiazole, benzothiazole, cyanine, carbocyanine, salicylate, anthranilate, coumarin, a fluorescein (e.g., fluorescein, tetrachlorofluorescein, hexachlorofluorescein), rhodamine, tetramethyl-rhodamine, or other like compound.
• a fluorescein e.g., fluorescein, tetrachlorofluorescein, hexachlorofluorescein
• rhodamine tetramethyl-rhodamine, or other like compound.
• Suitable fluorescent moieties for use with dark quenchers include xanthene dyes, such as fluorescein or rhodamine dyes, including 6-carboxyfluorescein (FAM), 27'-dimethoxy-4'5 I -dichloro-6-carboxyfluorescein (JOE), tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G), N,N,N;N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX).
• Suitable fluorescent reporters also include the naphthylamine dyes that have an amino group in the alpha or beta position.
• naphthylamino compounds include l-dimethylaminonaphthyl-5-sulfonate, 1- anilino-8-naphthalene sulfonate and 2-p-toluidinyl-6-naphthalene sulfonate, 5-(2'- aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS).
• fluorescent moieties include coumarins, such as 3-phenyl-7- isocyanatocoumarin; acridines, such as 9-isothiocyanatoacridin- e and acridine orange; N-(p-(2-benzoxazolyl)phenyl)maleimide; cyanines, such as indodicarbocyanine 3 (Cy3), indodicarbocyanine 5 (Cy5), indodicarbocyanine 5.5 (Cy5.5), 3-l-carboxy-pentyl)-3'- ethyl-5,5'-dimethy- loxacarbocyanine (CyA); lH,5H,lH,15H-Xantheno[2,3,4-ij:5,6,7- i'j']diquinol- izin-18-ium, 9-[2(or 4)-[[[6-[2,5-dioxo-l-pyrroli
• cAMP reporters of the invention optionally can include a subcellular targeting sequence which can target a cAMP reporter to a subcellular domain such as a plasma membrane, a nuclear membrane, a cytosol, an endoplasmic reticulum, a mitochondria, a mitochondrial matrix, a chloroplast, a medial trans-Golgi cisternae, a lumen of a lysosome, or a lumen of an endosome.
• Many such targeting sequences are known in the art. Examples include the plasma membrane targeting sequence shown in SEQ ID NO: 6, the nuclear localization signal sequence shown in SEQ ID NO:5, the mitochondrial localization sequence shown in SEQ ID NO:7, and the mitochondrial matrix targeting signal shown in SEQ ID NO: 8.
• Targeting sequences can be linked to cAMP reporters using, for example, a tetracysteine motif such as Cys Cys Xaa Xaa Cys Cys (SEQ ID NO: 15). Targeting sequences can be linked at either the N- or C-terminus of a cAMP reporter or at intermediate points in the reporter.
• a tetracysteine motif such as Cys Cys Xaa Xaa Cys Cys (SEQ ID NO: 15).
• Targeting sequences can be linked at either the N- or C-terminus of a cAMP reporter or at intermediate points in the reporter.
• cAMP reporters of the invention do not include those which consist of YFP which is not circularly permuted, CFP which is not circularly permuted, and any of the following polypeptides: amino acids 1-443 of SEQ ID NO:3 (a mouse Epac2), amino acids 1-149 of SEQ ID NO:3, amino acids 29-149 of SEQ ID NO:3, amino acids 285-443 of SEQ ID NO:3, amino acids 304-443 of SEQ ID NO:3, amino acids 310-443 of SEQ ID NO:3, amino acids 285-454 of SEQ ID NO:3, amino acids 285- 460 of SEQ ID NO:3, and amino acidsl57-316 of SEQ ID NO:1 (human Epacl).
• cAMP reporters which are fusion proteins preferably can be expressed recombinantly, and the invention provides nucleic acid molecules for this purpose.
• a nucleic acid molecule encoding a cAMP reporter can comprise any nucleotide sequence which encodes the amino acid sequence of the reporter.
• Nucleic acid molecules of the invention include single- and double-stranded DNA (including cDNA) and mRNA.
• kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, WI), Stratagene (La Jolla, CA), CLONTECH (Mountain View, CA), Santa Cruz Biotechnology (Santa Cruz, CA), MBL International Corporation (MIC; Watertown, MA), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).
• the nucleic acid molecules are expression constructs which contain the necessary elements for the transcription and translation of an inserted coding sequence encoding a cAMP reporter.
• Expression constructs can be used as vectors for introducing cAMP reporters into cells. Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding cAMP reporters and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1989.
• Expression vectors of the invention can be expressed in a variety of host cells. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems, particularly mammalian systems, including human systems. See WO 01/98340, which is incorporated herein by reference in its entirety. The choice of vector components and appropriate host cells is well within the capabilities of those skilled in the art.
• protein or non-protein donor and/or acceptor moieties can be linked to the polypeptide by covalent attachment.
• the attachment can be direct, via a functional group on the polypeptide (e.g., amino, carboxyl and sulfhydryl groups) and a reactive group on the fluorophore. Free amino groups in the polypeptide can be reacted with fluorophores derivatized with isothiocyanate, maleic anhydride, N- hydroxysuccinimide, tetrafiuorylphenyl and pentafmoryl esters.
• Free carboxyl groups in the polypeptide can be reacted with carbodiimides such as l-ethyl-3- [dimethylaminopropyl]carbodiimide hydrochloride to create a reactive moiety that will react with an amine moiety on the donor or acceptor moiety.
• Sulfhydryl groups can be attached to donor or acceptor moities modified with maleimide and iodoacetyl groups, although such linkages are more susceptible to reduction than linkages involving free amino groups.
• the polypeptide can also be linked indirectly via an intermediate linker or spacer group, using chemical groups such as those listed above.
• cAMP reporters of the invention using chemical methods to synthesize the amino acid sequence of the polypeptide and, optionally, one or more fluorescent or luminescent proteins.
• Methods include direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et ah, Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 43 IA Peptide Synthesizer (Perkin Elmer).
• fragments of polypeptide portions of cAMP reporters can be separately synthesized and combined using chemical methods to produce a full-length reporter molecule. See WO 01/98340.
• AU cAMP reporters of the invention can be introduced into cells in vitro using reversible permeabilization techniques. See U.S. Patent 6,127,177; U.S. Patent 6,902,931; Russo et ah, Nature Biotechnology 15, 278-82, March 1997; Santangelo et ah, Nucleic Acids Res. 32, 1-9, April 14, 2004.
• cAMP reporter is a fusion protein
• expression vectors comprising a cAMP reporter- encoding nucleotide sequence can be transfected into any cell in vitro in which it is desired to monitor cAMP levels or distribution.
• Any transfection method known in the art can be used, including, for example, including, but not limited to, transferrin- polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun,” and DEAE- or calcium phosphate-mediated transfection.
• the invention provides various methods for detecting cAMP by detecting conformational changes in a cAMP reporter.
• the methods involve detecting a change in resonance energy transfer of a cAMP reporter of the invention when the reporter is subjected to a change in cAMP concentration.
• cAMP binding to the reporter induces a conformational change that changes resonance energy transfer from the donor moiety to the acceptor moiety.
• a change in resonance energy transfer can readily be detected using methods well known in the art. See, e.g., US 2005/0118619; US 2002/0137115; US 2003/0165920; US 2003/0186229; US 2004/0137479; US 2005/0026234; US 2005/0054573; US 2005/0118619; U.S. Patent 6,773,885; U.S. Patent 6,803,201; U.S. Patent 6,818,420; Ayoub et ah, 2002; Boute et ah, 2002; Domin et ah, Prog. Biomed. Optics and Imaging, Proc.
• RET resonance energy transfer
• cAMP reporters of the invention can be used in cell-free systems, in isolated cells (for example, in primary cell culture or a cell line) or in cells in situ ⁇ e.g., in an isolated tissue sample, an isolated whole organ, or in a mammal). Subcellular distribution of cAMP or changes in cAMP concentration can be detected, for example, as described in Example 2, below. Absolute cAMP levels can be detected by obtaining a RET measurement in the assay system and comparing it to a standard curve obtained in vitro. [45] In some embodiments, steady-state RET measurements are first obtained and then measurements are taken after addition of a test compound to the assay system.
• Test compounds can be used, for example, to increase cAMP concentration to make it easier to detect cAMP in a particular subcellular compartment or to monitor the effect of the test compound on cAMP concentration (e.g., in drug-screening methods).
• Test compounds can be pharmacologic agents already known in the art to affect cAMP levels or can be compounds previously unknown to have such an activity.
• ⁇ -adrenergic receptor agonists e.g., norepinephrine, epinephrine, isoproterenol, sulfonterol, metaproterenol, SB-251023
• ⁇ -adrenergic receptor antagonists e.g., propranolol, butoxamine, practolol, alprenolol, pindolol, nadolol, metaprolol, SR-59230A
• direct or indirect activators of adenylate cyclase e.g., forskolin, prostaglandin E 1
• cAMP analogs e.g., 8-(4-chloro-phenylthio)-2'-O-methyl adenosine 3 ',5 '-monophosphate; N 6 ,2'-Odibutyryl cyclic adenosine
• Test compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, . test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection.
• Mutation R522E was incorporated by the QUICKCHANGE® method (Stratagene). Enhanced cyan fluorescent protein (ECFP) and citrine were fused to the N and C terminal ends of the individual gene constructs (FIG. 1). The constructs were first generated in pRSET B (Invitrogen) and subcloned into pcDNA3 (Invitrogen) behind a Kozak sequence for mammalian expression.
• pRSET B Invitrogen
• pcDNA3 Invitrogen
• the nuclear localization signal PKKKRKVEDA (SEQ ID NO:5) was added to the C terminus. Localization to the mitochondrial matrix was achieved by fusing the first 12 amino acids of subunit IV of human cytochrome oxidase c and a four- residue linker (SEQ ID NO: 8) to the N terminal of the construct.
• the sequence KKKKKSKTKCVM (SEQ ID NO:6) was inserted at the C terminus.
• the signal sequence MAIQLRSLFPLALPGMLALLGWWWFFSRKK (SEQ ID NO: 7) was inserted at the N terminus for targeting ICUE to mitochondria.
• HEK-293, HeLa and PC 12 cells were plated onto sterilized glass coverslips in 35mm dishes and grown to 50-90% confluency in DMEM (10% FBS at 37 0 C, 5% CO 2 ). Cells were then transfected with FuGENE-6 transfection reagent (Roche) or calcium phosphate and allowed to grow for 12-24 hours before imaging. Colocalization studies were performed by incubating transfected HEK-293 cells with MitoTracker Red 580 or Hoechst 33342 cell-permeable dyes (Molecular Probe) for staining mitochondria or nucleic acids, respectively.
• MitoTracker Red 580 or Hoechst 33342 cell-permeable dyes Molecular Probe
• Imaging Cells were washed twice with Hanks' balanced salt solution buffer after 12- to 24-h incubation at 37 0 C culture medium. Cells were maintained in buffer in the dark at room temperature with addition of isoproterenol (Aldrich), forskolin (Calbiochem), Prostaglandin E 1 (PGE 1 ) (Sigma), and 8-(4-chloro-phenylthio)-2'-O-methyl adenosine 3 ' ⁇ '-monophosphate (8-pCPT-2'-O-Me-cAMP) (Axxora Biolog) as indicated.
• isoproterenol Aldrich
• forskolin Calbiochem
• PGE 1 Prostaglandin E 1
• 8-pCPT-2'-O-Me-cAMP 8-pCPT-2'-O-Me-cAMP
• DMNB-cAMP P-(4,5-dimethoxy-2-nitrobenzyl) adenosine 3 ',5 '-monophosphate
• a cAMP reporter in which ECFP and citrine were fused together with a domain (P350- A501; amino acids 350-501 of SEQ ID NO:20) containing the second cyclic nucleotide monophosphate-binding domain from Epac2 and a C-terminal lid was expressed in HEK- 293 cells.
• This reporter showed variable ratios of cyan to yellow emissions which are inversely correlated with expression level of the protein. This concentration dependence indicates intermolecular FRET between different reporter molecules that may occur due to oligomerization or aggregation (Zacharias et ah, Science 296, 913-16, 2002). Upon cAMP elevations, this protein did not show a cAMP-dependent FRET change.
• FIG. 3A To directly monitor cAMP dynamics at different subcellular locations inside cells, we prepared several fusions of ICUEl to various specific targeting motifs (FIG. 3A). To localize the reporter to the plasma membrane, we fused the plasma membrane-targeting signal of small guanosine triphosphatase K-ras4B (Roy et al., Biochem. 39, 8298-307, 2000) to the C terminus of ICUEl. This targeting motif combined a farnesylated cysteine residue with a strongly polybasic sequence and effectively targeted the reporter to the plasma membrane (FIG. 3B).
• Epac localizes to mitochondria in a subpopulation of cells, but monitoring of cAMP accumulation at mitochondria has not been possible with previous methods.
• the first MitoCOX-ICUEl was generated by fusing the targeting sequence of subunit IV of cytochrome c oxidase (COX) to the N-terminus of ICUEl. This COX sequence delivers fused proteins to the mitochondrial matrix (Hurt et al, EMBO J. 4, 2061-68).
• FIG. 3B MitoCOX-ICUEl was partially targeted to mitochondria (Filippin et al., J.
• Variable cellular PDE activities may be responsible for this discrepancy.
• a second response of similar amplitude was induced by stimulation with isoproterenol. Both untargeted and mitochondria targeted ICUEl showed similar responses to PGE 1 and isoproterenol.
• Soluble AC and regulatory and catalytic subunits of protein kinase A coexist in the nucleus of mammalian cells (Zippin et ah, J. Cell Biol. 164, 527-34, 2004).
• the activation of bicarbonate-responsive soluble AC in the nucleus led to a rapid increase in PKA-dependent phosphorylation, which was detectable within two minutes.
• the immediate presence of a nuclear pool of cAMP following ⁇ -AR activation raised the question " whether this pool of cAMP could produce functional PE-A responses in the nucleus.
• AKAR Zhang et al., Proc. Natl. Acad. ScL USA 98, 14997-5002, 2001
• the presence of this nuclear pool of cAMP immediately following cAMP production is not sufficient to generate a detectable phosphorylation of AKAR by PKA within the nucleus.
• This lack of immediate PKA response could be due to either the absence of the PKA holoenzyme in the nucleus or insufficient activation of soluble AC-coupled PKA by this pool of cAMP.
• the slow diffusion of the C subunit rather than the fast diffusion of cAMP as the rate- limiting step may provide the temporal control of ⁇ -AR-stimulated PKA-dependent phosphorylation in the nucleus.
• ICUE2 truncated reporter
• Imaging with ICUE2 in HEK-293 cells revealed a 40-50% increase in cyan/yellow emission ratio upon stimulation of cAMP production with forskolin, compared to a 15- 30% response generated by ICUEl. Maximum FRET response was reached in 1.5-3 minutes upon stimulation with isoproterenol, which is on the same time scale as the ICUEl response. Targeted versions of ICUE2 exhibited the increased dynamic range in cyan/yellow emission ratio as well, therefore improving the signal-to-noise ratio.
• ICUE2 also responded to lower concentrations of isoproterenol. We observed FRET responses upon the addition of 0.1 ⁇ M, IuM, as well as lO ⁇ M isoproterenol, which was the lowest concentration of isoproterenol that generated a FRET response of ICUEl. The ICUE2 response reverses in an average of 9 minutes once it reaches maximum without addition of ⁇ -AR antagonist, propranolol, or washing out of agonist.
• ICUE2 The dynamic range of ICUE2 was increased by replacing citrine with a circularly permuted YFP, cp Venus Ll 95 to form a cAMP reporter termed ICUE3. Circular permutation introduces new N and C termini to a protein and can improve the dynamic range of FRET-based reporters by altering the relative orientation of fluorescent proteins (Nagai et al, 2004).

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