WO1999006830A1 - Procede servant a moduler des recepteurs couples a une proteine g - Google Patents

Procede servant a moduler des recepteurs couples a une proteine g Download PDF

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Publication number
WO1999006830A1
WO1999006830A1 PCT/US1998/015717 US9815717W WO9906830A1 WO 1999006830 A1 WO1999006830 A1 WO 1999006830A1 US 9815717 W US9815717 W US 9815717W WO 9906830 A1 WO9906830 A1 WO 9906830A1
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rdgc
phosphatase
receptors
cell
sample
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PCT/US1998/015717
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English (en)
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Charles Zuker
Javier Vinos
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The Regents Of The University Of California
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Priority to AU86690/98A priority Critical patent/AU8669098A/en
Publication of WO1999006830A1 publication Critical patent/WO1999006830A1/fr

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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • 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
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to methods of screening for modulators, of G-protein coupled receptor signal transduction activity, and methods for screening for mutant RDGC phosphatases associated with G-protein coupled receptor signal transduction disease.
  • G-protein coupled receptors mediate responses to a wide range of extracellular stimuli, including hormones, neurotransmitter, peptides, and sensory signals such as odorants, light, and taste ("GPCRs"; see, e.g. , Neer, Cell 80:249-257 (1995); Fong, Cell Signal. 8:217 (1996); and Baldwin, Curr. Opin. Cell Biol. 6: 180 (1994)).
  • GPCRs mediate signal transduction across a cell membrane upon activation, e.g., the binding of a ligand to an extracellular portion of the GPCR or activation by other stimulus such as light.
  • the intracellular portion of a GPCR interacts with a G-protein to modulate signal transduction from outside to inside a cell.
  • a GPCR is therefore said to be "coupled” to a G-protein.
  • G-proteins are heterotrimers composed of three polypeptide subunits: an subunit, which binds and hydrolyzes GTP, and a dimeric ⁇ y subunit. In the basal, inactive state, the G-protein exists as a heterotrimer of the and ⁇ y subunits. When the G-protein is inactive, guanosine diphosphate (GDP) is associated with the subunit of the G-protein. When a GPCR is bound and activated, e.g. , by a ligand, the GPCR binds to the G-protein and decreases the affinity of the G ⁇ subunit for GDP.
  • GDP guanosine diphosphate
  • the G ⁇ subunit In its active state, the G ⁇ subunit exchanges GDP for guanine triphosphate (GTP) and then active G ⁇ subunit disassociates from both the receptor and the dimeric ⁇ y subunit.
  • GTP guanine triphosphate
  • the disassociated, active G ⁇ subunit transduces signals to effectors that are "downstream" in the G-protein signaling pathway within the cell.
  • effectors include enzymes like adenylate cyclase and phospholipase C.
  • the G-protein's endogenous GTPase activity returns active Go. subunit to its inactive state, in which it is associated with GDP and the dimeric ⁇ y subunit.
  • GPCRs share desensitization and inactivation mechanisms, such as the -adrenergic receptor and rhodopsin (see, e.g. , Hein & Kobilka, Neuropharmacology 34:357 (1995); Lefkowitz et al, Adv. Second Messenger Phosphoprotein Res. 28: 1 (1993); Nathans, Biochemistry 31:4923 (1992); Hargrave & McDowell, Int. Rev. Cytol. 137B:49 (1992); and Lagnado & Baylor, Neuron 8:995 (1992)).
  • Light or ligand activation results in phosphorylation of the GPCR by a GPCR kinase.
  • Phosphorylation results in a slight decrease in receptor activity, but also causes the receptor to become a high affinity substrate for arrestin. Arrestin binding terminates the active state of the receptor by preventing its coupling to G protein. Receptor dephosphorylation is essential for completion the signaling cycle (see, e.g. , Hurley, Curr. Opin. Neurobiol. 4:481
  • the retinal degeneration C gene (rdgC gene, encoding RDGC protein) encodes an unusual type of serine/threonine phosphatase, consisting of an N-terminal domain that has Egh sequence similarity to the catalytic domain of protein phosphatases 1, 2A and 2B, and a C-terminal domain containing multiple EF-hand motifs.
  • RDGC has been proposed to be a calcium regulated phosphatase, and has been shown to be required for efficient dephosphorylation of rhodopsin in vitro.
  • RDGC has been proposed to be a calcium regulated phosphatase, and has been shown to be required for efficient dephosphorylation of rhodopsin in vitro.
  • the molecular identification of a G protein coupled receptor phosphatase has been difficult.
  • the fact that GPCR's share a number of structural features, including a carboxyl terminal phosphorylation region, indicates that GPCR dephosphorylation is a common theme in controlling receptor function. There is a need to identify mechanisms by which GPCR phosphorylation events can be controlled in vivo.
  • the present invention thus identifies for the first time a G-protein coupled receptor phosphatase.
  • RDGC phosphatase participates in regulation of G-protein coupled receptor signal transduction by regulating activity of the receptor via desphosphorylation.
  • the invention therefore provides cells and methods for identifying modulators of signal transduction based on GPCR phosphatases that modulate GPCR activity by dephosphorylation. By targeting GPCR phosphatases in the assays, as described herein, modulators of signal transduction can be identified.
  • the invention includes methods and kits for identifying modulators of GPCR signal transduction comprising contacting a GPCR phosphatase in a biochemical assay, cell assay or animal assay, with a test compound and detecting a functional effect on GPCR mediated signal transduction.
  • the invention includes animals (e.g. , mice and flies) and cells (e.g. , mammalian and insect) comprising GPCR phosphatases, modified GPCR phosphatases and defective GPCR phosphatases to use in assays of signal transduction.
  • the invention also includes proteins (as well as polynucleotides encoding the same) corresponding to GPCR phosphatases, modified GPCR phosphatases or defective GPCR phosphatases to use in assays of signal transduction.
  • FIGURE IA shows an autoradiogram of an SDS-PAGE of 32 P0 4 in vivo labeled retinal proteins.
  • FIGURE IB shows an autoradiogram of an SDS-PAGE of 32 P0 4 in v vo-labeled retinal proteins after a 20 s pulse of light.
  • B refers to flies exposed to 20 s of blue light
  • BO refers to flies exposed to 20 s of blue light followed by 20 s of orange light.
  • Rhl stands for rhodopsin 1 (36 kD).
  • RMD356 is the truncation of the carboxy-terminal tail of rhodopsin.
  • the bottom panel shows the same gel blotted and probed with anti-Rhl antibodies.
  • FIGURE IC shows a histogram of the relative level of 32 P incorporation into Rhl rhodopsin. Samples were normalized to control flies exposed to blue and orange light sequentially (BO) and corrected for amount of rhodopsin loaded. Data are means +SEM of triplicate determinations.
  • FIGURE 2A shows a 1 ⁇ m thick cross section of a wild type adult retinas after 6 days of light exposure.
  • the retinas display normal retinal morphology, with ommatidial clusters organized in a well patterned array.
  • FIGURE 2B shows a 1 ⁇ m thick cross section of mutant retinas from rdgC flies displaying dramatic retinal degeneration.
  • FIGURE 2C shows a 1 ⁇ m thick cross section of mutant retinas from Dgq mutants which lack the ⁇ -subunit of the heterotrimeric G protein that couples rhodopsin to PLC and do not display light-dependent degeneration.
  • FIGURE 2D shows a 1 ⁇ m thick cross section of mutant Dgq retinas indicating that this mutant is unable to suppress the degeneration of the Dgq; rdgC double mutants.
  • FIGURE 2E shows a 1 ⁇ m thick cross section of mutant RMD356 transgenic animals displaying normal retinal morphology.
  • FIGURE 2F shows a 1 ⁇ m thick cross section of mutant RhlD356 which drastically suppresses rdgC degeneration in RhlD356; rdgC double mutants.
  • FIGURE 3A shows electroretinograms (ERGS) of w, rdgC, RhlD356 and RhlD356; rdgC mutant flies.
  • Photoreceptors were given a 0.5 s pulse of orange light.
  • rdgC mutants display strong defects in the deactivation kinetics (arrow), and this phenotype is suppressed by the R D356 truncation. Traces from 15 independent measurements were averaged.
  • FIGURE 3B shows intracellular recordings of light-activated responses from control and rdgC photoreceptors.
  • This recording configuration demonstrates the photoreceptor specificity of the phenotype; note the strong deactivation defect phenotype in rdgC (arrow).
  • t50 34 ⁇ 2 ms
  • data are means ⁇ SEM.
  • FIGURE 3C shows a histogram of the deactivation time for the different ERG phenotypes.
  • t85 0. 17 + 0.03 s
  • rdgC t85 1.8 ⁇ 0.3 s
  • RhlD356 t85 0. 1 6 ⁇ 0.03 s
  • data represents means + SD.
  • FIGURE 4A shows an ERG recording of a white eyed control fly showing a prototypical prolonged depolarizing afterpotential (PDA) response. 0, orange light (M'R conversion); B, blue light (R'M conversion).
  • PDA prototypical prolonged depolarizing afterpotential
  • FIGURE 4B shows a histogram of relative amounts of blue light required to enter a PDA for the different genotypes. Values are normalized to control flies and represent means ⁇ SEM of triplicate determinations. Arr2, a known mutant with a PDA defect is shown for comparative purposes.
  • the present invention provides for the first time the molecular identity of a G-protein coupled receptor phosphatase.
  • RDGC has been identified as a phosphorylase for a GPCR substrate, rhodopsin.
  • the molecular identification of the first GPCR phosphatase provides a means for identifying novel therapeutic agents that modulate GPCR signal transduction. Such agents are useful for treating diseases related to GPCR signal transduction.
  • modulators of GPCR signal transduction can be identified using high throughput assay techniques, using the assays described herein.
  • the invention includes means for identifying modulators of GPCR signal transduction comprising contacting a GPCR phosphatase such as RDGC in a biochemical assay, cell assay or animal assay, with a test chemical and detecting a change in signal transduction.
  • the assays of the invention include animals (e.g., mice and flies) and cells (e.g. , mammalian and insect) comprising GPCR phosphatase, modified GPCR phosphatase, or defective GPCR phosphatase.
  • the invention also includes proteins (as well as polynucleotides encoding the same) corresponding to GPCR phosphatases, modified GPCR phosphatases or defective GPCR phosphatases to use in assays of signal transduction.
  • the Drosophila phototransduction system is described herein as an example of GPCR signal transduction.
  • This phototransduction cascade is a G-protein coupled, PLC-signaling pathway that shares many features with other signaling cascades (see, for example, Ranganathan et al , Annual Review of Neuroscience, 18:283-317 (1995), which is incorporated herein by reference).
  • Light activation of rhodopsin activates a Gq ⁇ (a G-protein), which in turn activates a PLC- (phospholipase C) (see, for example, Pak, Invest. Ophthalmol. Vis. Sci. , 36:2340-2357 (1995), which is incorporated herein by reference).
  • PLC ⁇ catalyzes the breakdown of phosphatidylinositol bisphosphate (PIP 2 ) into the two intracellular messengers inositol triphosphate (IP 3 ) and diacylglycerol (DAG), which leads to the eventual opening (and modulation) of the TRP and TRPL light-activated channels (see, for example, Niemyer et al , Cell, 85:651-659 (1996), which is incorporated herein by reference).
  • PIP 3 inositol triphosphate
  • DAG diacylglycerol
  • InaD is one of the many loci that have been identified in genetic screens designed to dissect this signaling cascade.
  • a single mutant allele, InaD 215 photoreceptor deactivation see, for example, Pak, Mutants affecting the vision in Drosophila melaogaster 1-703-733 (Plenum, New York/London, 1975); Shieh et al , Neuron, 14:201-210 (1995), which are incorporated herein by reference).
  • the INAD protein was shown to contain at least two PDZ domains (see, for example, Shieh et al.
  • phototransduction in Drosophila was used as a model to study the activation and deactivation of GPCRs in vivo and in vitro (see Examples 1-4).
  • this signaling pathway photoreceptor neurons report activity with extraordinarily sensitivity and specificity.
  • photoreceptor s achieve superb temporal resolution by ensuring that the transduction machinery is reset quickly after generating a response.
  • Phototransduction in Drosophila is the fastest known G protein-coupled cascade, taking just a few tens of milliseconds to go from light activation of rhodopsin to the generation of a receptor potential, and less than 100 ms to shut-off following termination of the stimulus (see, for example, Rancanathan et al. , (1995), supra).
  • This model of signal transduction, as well as other examples of Drosophila biology applies to signal transduction in humans.
  • the invention also includes test compounds identified by the methods described herein.
  • the invention includes chemicals identified by screening for the activity of a RDGC phosphatase.
  • the present invention also relates to the use of test compounds identified using the methods described herein to treat GPCR associated diseases.
  • a G protein-coupled receptor associated disorder refers to a disorder characterized by the failure of a cell(s) of a mammal to produce a level of a wild-type G-protein comparable to the level produced in a mammal not having the disorder.
  • Such disorders include those in which a G-protein(s) is absent (e.g. , as the result of a null mutation), produced at low levels, or dysfunctional (e.g.
  • treatment of the disorder entails expressing in a cell(s) of the mammal a polypeptide having the biological activity of a promiscuous G-protein; the polypeptide thus compensates for the abnormal endogenous G-protein.
  • RDGC phosphatase is a protein that has phosphatase activity for G- protein coupled receptors.
  • the term “RDGC phosphatase” includes naturally occurring RDGC, recombinant RDGC, and functional RDGC equivalents that have GPCR phosphatase activity, e.g., interspecies homologs, alleles, polymorphic variants and the like.
  • the term RDGC therefore refers to RDGC homologs such as polymorphic variants, alleles, mutants, and closely related interspecies variants that have about at least 60% amino acid sequence identity to RDGC phosphatase (e.g.
  • RDGC are substantially identical to RDGC; see the sequence of RDGC in Steele et al, Cell 69:669- 676 (1992), herein incorporated by reference) and have GPCR phosphatase activity, as determined using the assays described herein.
  • RDGC GPCR phosphatase activity is the activity of an RDGC phosphatse for a he GPCR substrate.
  • other GPCR phosphatases have the same activity for a GPCR substrate. This activity can be measured with endogenously or recombinantly expressed RDGC or GPCR phosphatases and signal transduction proteins using the assays described herein.
  • GPCR and RDGC phosphatases permit enhanced signal transduction by signal transduction proteins compared to signal transduction in the absence of a GPCR.
  • Mammalian GPCR phosphatases for use with mammalian signal transduction proteins are preferred, particularly human GPCR phosphatases.
  • GPCR phosphatase refers to a protein with the GPCR phosphatase activity of RDGC phosphatase or another structurally similar phosphatase regulating the phosphorylation state of a G-protein coupled receptor phosphatase protein. Such phosphatases can regulate G-protein coupled receptor phosphorylation levels which in turn regulates signal transduction activity and its associated proteins. Examples of signal transduction-proteins include GPCRS, tyrosine kinase receptors, tyrosine phosphatase receptors, ion channels, phospholipases, adenylate cyclases, kinases and G-proteins.
  • An "RDGC mimetic” refers to a compound or molecule, e.g.
  • RDGC mimetics thus include RDGC homologs.
  • RDGC mimetics would also include small RDGC peptides that retained the RDGC active site, and conservatively modified variants thereof, as well as truncated versions of RDGC that retained RDGC phosphatase activity.
  • Small chemical molecules that mimic the RDGC active site are also RDGC mimetics. RDGC mimetics are tested using assays for RDGC activity, e.g.
  • RDGC mimetic When testing for an RDGC mimetic, wild-type RDGC is typically used as a positive control for RDGC activity. A relative activity value is assigned to RDGC, e.g., 100. Mimetic activity is achieved when mimetic RDGC activity relative to the control is about 25, more preferably 50-100.
  • a "G-protein coupled receptor" or “GPCR” is a receptor with seven transmembrane regions.
  • the GPCR has "G-protein coupled receptor activity, " e.g., it binds to G-proteins in response to extracellular stimuli and promotes production of second messengers such as IP3, cAMP, and Ca 2+ via stimulation of enzymes such as phospholipase C and adenylate cyclase (for a description of the structure and function of GPCRs, see, e.g. , Fong, supra, and Baldwin, supra).
  • G-protein coupled receptor activity e.g., it binds to G-proteins in response to extracellular stimuli and promotes production of second messengers such as IP3, cAMP, and Ca 2+ via stimulation of enzymes such as phospholipase C and adenylate cyclase (for a description of the structure and function of GPCRs, see, e.g. , Fong, supra, and Baldwin, supra).
  • sample as used herein is a sample of tissue, cells, membranes, or fluid in aqueous solution or solid phase or a combination of both that comprises a GPCR and RDGC phosphatase or a nucleic acid encoding a GPCR or RDGC phosphatase.
  • samples include, but are not limited to, samples isolated from humans, mice, rats, Drosophila.
  • Biological samples may also include sections of tissues such as frozen sections taken for histological purposes.
  • a biological sample is typically obtained from a eukaryotic organism, such as insects, protozoa, birds, fish, reptiles, and preferably a mammal such as rat, mouse, cow, dog, guinea pig, or rabbit, and most preferably a primate such as chimpanzees or humans.
  • a eukaryotic organism such as insects, protozoa, birds, fish, reptiles, and preferably a mammal such as rat, mouse, cow, dog, guinea pig, or rabbit, and most preferably a primate such as chimpanzees or humans.
  • an "animal” refers to any non-human animal.
  • detecting refers to the means by which RDGC phosphatase activity is identified. The term thus encompasses any assay, direct or indirect, used to ascertain the presence or absence or level of RDGC phosphatase activity in a sample or animal.
  • detection of RDGC phosphatase activity involves a determination of any parameter that is indirectly or directly under the influence of RDGC phosphatase activity and its role as the phosphatase for a GPCR substrate, e.g. , any parameter that has a functional effect on GPCR signal transduction.
  • Such assays include changes in ion flux, membrane potential, voltage, conductance, current flow, transcription, signal transduction, receptor-ligand interactions, second messenger concentrations (e.g., cAMP, IP3, or intracellular Ca 2+ ), patch clamping, voltage-sensitive dyes, whole cell currents, radioisotope efflux, inducible markers, oocyte expression; tissue culture cell expression; ligand binding assays; in vitro, in vivo, and ex vivo and also includes other physiologic effects such increases or decreases of neurotransmitter or hormone release.
  • Any suitable label or means of detection can be used in the above assays.
  • a preferred label or detectable moiety in the above assays is a fluorescent label, which is detected by an "detector" apparatus designed to detect fluorescence.
  • Inhibitors refer to inhibitory or activating molecules identified using in vitro and in vivo assays for that have a functional effect on GPCR signal transduction as described above, e.g. , ligands, agonists, antagonists, and their homologs and mimetics.
  • Inhibitors are compounds that decrease, block, prevent, delay activation, inactivate, desensitize, or down regulate signal transduction, e.g. , antagonists.
  • Activators are compounds that increase, open, activate, facilitate, enhance activation, sensitize or up regulate signal transduction, e.g., agonists.
  • Modulators include genetically modified versions of RDGC phosphatase, e.g. , with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like.
  • Such assays for inhibitors and activators include, e.g, expressing RDGC phosphatase in cells or cell membranes, applying putative modulator compounds, and then determining the functional effects on taste transduction, as described above. Samples or assays comprising RDGC phosphatase that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition.
  • Control samples (untreated with inhibitors) are assigned a relative RDGC phosphatase activity value of 100% .
  • Inhibition of RDGC phosphatase is achieved when the RDGC phosphatase activity value relative to the control is about 90%, preferably 50%, more preferably 25% .
  • Activation of RDGC phosphatase is achieved when the RDGC phosphatase activity value relative to the control is 110%, more preferably 150%, more preferable 200% higher.
  • Modulators of GPCR signal transduction refers to activating or inhibitory molecules identified using in vitro and ex vivo assays for RDGC phosphatase activity, which in turn have a functional effect on GPCR signal transduction.
  • Potential modulators include peptides, polypeptides, small chemical molecules, synthetic molecule (e.g. , nucleic acid, protein, non-peptide, or organic molecules), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues.
  • test compound refers to a compound to be tested by one or more screening method(s) of the invention as a putative modulator.
  • test compound also does not include chemicals known to be unsuitable for a therapeutic use for a particular indication due to toxicity of the subject.
  • various predetermined concentrations are used for screening test compounds, such as .01 ⁇ M, .1 ⁇ M, 1.0 ⁇ M, and 10.0 ⁇ M.
  • Signal transduction refers to the coupling of an extracellular signal (the signal) to an intracellular response (the response); this term also refers to the response or individual steps of the response.
  • the response need not occur intracellular ly, it may also be measured in an non-cellular reaction.
  • the signal can also be measured in a non-cellular reaction.
  • an activity directly associated with signal transduction is the activity or physical property of either the receptor (e.g. , GPCR), or a coupling protein (e.g., a G ⁇ protein).
  • Signal transduction detection systems for monitoring an activity directly associated with signal transduction include GTPase activity, ion channel activity and conformational changes.
  • An activity indirectly associated with signal transduction is the activity produced by a molecule other than by either the receptor (e.g., GPCRs, tyrosine kinase receptors, or tyrosine phosphatase receptors), ion channel or a coupling protein (e.g. , a G ⁇ protein) associated with a receptor (e.g., GPCRs, tyrosine kinase receptors, or tyrosine phosphatase receptors), or a coupling protein (e.g., a G ⁇ protein).
  • Such indirect activities include changes in intracellular levels of molecules (e.g. , ions (e.g.
  • Signal transduction assays are further described in commonly owned U.S. applications by Negulescu et al. , 60/020,234, filed June 21, 1996.
  • Signal transduction detection systems for monitoring an activity indirectly associated with signal transduction include transcriptional-based assays, enzymatic assays, intracellular ion assays and second messenger assays.
  • the signal for signal transduction can be any signal compatible with the assay being used.
  • the signal may be a test chemical itself or a known activator of signal transduction.
  • the activating step can include activating signal transduction with a signal selected from the group consisting of a chemical signal found in blood, a chemical signal found in a synaptic cleft, a chemical signal found in interstitial fluid, light or a chemical signal found in air or other signals known the art or developed in the future.
  • GPCR mobility assay refers to an assay for RDGC GPCR phosphatase activity that tests for changes in GPCR electrophoretic mobility after treatment with RDGC and optionally an inhibitor.
  • GPCR phosphorylation assay refers to an assay for RDGC GPCR phosphatase activity that tests for dephosphorylation after treatment with RDGC.
  • a signal transduction assay refers to any assay that detects GPCR signal transduction, including changes in ion flux, membrane potential, voltage, conductance, current flow, transcription, signal transduction, receptor-ligand interactions, second messenger concentrations (e.g. , cAMP, IP3, or intracellular Ca 2+ ), patch clamping, voltage-sensitive dyes, whole cell currents, radioisotope efflux, inducible markers, oocyte expression; tissue culture cell expression; ligand binding assays; in vitro, in vivo, and ex vivo and also includes other physiologic effects such increases or decreases of neurotransmitter or hormone release.
  • second messenger concentrations e.g. , cAMP, IP3, or intracellular Ca 2+
  • patch clamping voltage-sensitive dyes, whole cell currents, radioisotope efflux, inducible markers, oocyte expression; tissue culture cell expression; ligand binding assays; in vitro, in vivo, and ex vivo and also includes other physiologic effects such
  • contacting a cell refers to any method whereby RDGC or an RDGC mimetic or homolog is introduced into a cell or sample, e.g., by transduction of a nucleic acid encoding RDGC or an RDGC homolog or mimetic, by administering RDGC to the cell medium or sample, by injecting RDGC or an RDGC homolog or mimetic into the cell or sample, by conjugating RDGC or an RDGC homolog or mimetic to a molecule, e.g.
  • a receptor ligand that allows RDGC or the RDGC homolog or mimetic to be translocated into a cell or sample, and by introducing RDGC or the RDGC homolog or mimetic into a cell or sample using a vehicle such as a liposome.
  • Transduction refers to any method whereby a nucleic acid is introduced into a cell, e.g., by transfection, electroporation, passive uptake, lipid: nucleic acid complexes, viral vector transduction, injection, and the like.
  • an "expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell.
  • the expression vector can be part of a plasmid, virus, or nucleic acid fragment.
  • the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
  • In vivo refers to assays that are performed using a whole non-human animal.
  • In vitro refers to assays that do not require the presence of a whole animal, e.g. , that are performed using a cell, membrane, tissue sample, fluid sample, protein, expression vector, e.g., explants, cultured cell lines, transformed cell lines, primary cell lines, insect eyes, insect heads, extracted tissue, e.g. , blood, oocytes, tissue slices, recombinant or naturally occurring proteins, expression vectors encoding proteins, and the like.
  • In vitro assays are typically performed in aqueous solution, or with at least one component of the assay bound covalently or non-covalently to a solid phase such as a plate, microchip, bead, column, membrane, dipstick, etc.
  • Fluorescent donor moiety refers to the radical of a fluorogenic compound, which can absorb energy and is capable of transferring the energy to another fluorogenic molecule or part of a compound.
  • Suitable donor fluorogenic molecules include, but are not limited to, coumarins and related dyes xanthene dyes such as fluoresceins, rhodols, and rhodamines, resorufins, cyanine dyes, bimanes, acridines, isoindoles, dansyl dyes, aminophthalic hydrazides such as luminol and isoluminol derivatives, aminophthalimides, aminonaphthalimides, aminobenzofurans, aminoquinolines, dicyanohydroquinones, and europium and terbium complexes and related compounds.
  • Quencher refers to a chromophoric molecule or part of a compound, which is capable of reducing the emission from a fluorescent donor when attached to the donor. Quenching may occur by any of several mechanisms including fluorescence resonance energy transfer, photoinduced electron transfer, paramagnetic enhancement of formation of dark complexes.
  • Acceptor refers to a quencher that operates via fluorescence resonance energy transfer. Many acceptors can re-emit the transferred as energy as fluorescence. Examples include coumarins and related fluorophores, xanthenes such as fluoresceins, rhodols, and rhodamines, resorufins, cyanines, difluoroboradiazaindacenes, and phthalocyanines. Other chemical classes of acceptors generally do not re-emit the transferred energy. Examples include indigos, benzoquinones, anthraquinones, azo compounds, nitro compounds, indoanilines, di- and triphenylrnethanes.
  • Binding pair refers to two moieties (e.g. chemical or biochemical) that have an affinity for one another.
  • binding pairs include antigen/antibodies, lectin/avidin, target polynucleotide/probe oligonucleotide, antibody/antiantibody, receptor/ligand, enzyme/ligand and the like.
  • One member of a binding pair refers to one moiety of the pair, such as an antigen or ligand.
  • Dye refers to a molecule or part of a compound that absorbs specific frequencies of light, including but not limited to ultraviolet light.
  • the terms “dye” and chromophore” are synonymous.
  • “Fluorophore” refers to a chromophore that fluoresces.
  • Membrane-permeant derivative refers a chemical derivative of a compound that has enhanced membrane permeability compared to an underivativized compound. Examples include ester, ether and carbamate derivatives. These derivatives are made better able to cross cell membranes, i.e. membrane permeant, because hydrophilic groups are masked to provide more hydrophobic derivatives. Also, masking groups are designed to be cleaved from a precursor (e.g. , fluorogenic substrate precursor) within the cell to generate the derived substrate intracellular ly. Because the substrate is more hydrophilic than the membrane permeant derivative it is now trapped within the cells.
  • a precursor e.g. , fluorogenic substrate precursor
  • Nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
  • PNAs peptide-nucleic acids
  • nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
  • polypeptide and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g. , hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group. , e.g.
  • amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • nucleotides likewise, may be referred to by their commonly accepted single-letter codes.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res .
  • nucleic acid variations are "silent variations, " which are one species of conservatively modified variations.
  • Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. The following six groups each contain amino acids that are conservative substitutions for one another:
  • a “detectable moiety” or label” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include 32 P, fluorescent dyes and molecules, electron-dense reagents, enzymes (e.g. , as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available.
  • Preferred detectable moieties or labels are fluorescent dyes and molecules.
  • a protein that is "linked to a detectable moiety" is one that is bound, either covalently, through a linker, or through ionic, van der Waals or hydrogen bonds to a label such that the presence of the protein may be detected by detecting the presence of the label or detectable moiety bound to the protein.
  • recombinant when used with reference, e.g. , to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
  • heterologous when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature.
  • the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source.
  • a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
  • a “promoter” is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
  • a “constitutive” promoter is a promoter that is active under most environmental and developmental conditions.
  • An “inducible” promoter is a promoter that is active under environmental or developmental regulation.
  • operably linked refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
  • An "expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell.
  • the expression vector can be part of a plasmid, virus, or nucleic acid fragment.
  • the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • This definition also refers to the complement of a test sequence, which has a designated percent sequence or subsequence complementarity when the test sequence has a designated or substantial identity to a reference sequence.
  • a designated amino acid percent identity of 70% refers to sequences or subsequences that have at least about 70% amino acid identity when aligned for maximum correspondence over a comparison window as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • the percent identity exists over a region of the sequence that is at least about 25 amino acids in length, more preferably over a region that is 50 or 100 amino acids in length.
  • the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated according to, e.g. , the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California, USA).
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated or default program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 25 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment.
  • PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol 35:351-360 (1987). The method used is similar to the method described by Higgins & Sharp, CABIOS 5: 151-153 (1989).
  • the program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids.
  • the multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments.
  • the program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters.
  • PILEUP a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
  • PILEUP can be obtained from the GCG sequence analysis software package, e.g, version 7.0 (Devereaux et al, Nuc. Acids Res. 12:387-395 (1984).
  • BLAST algorithm Another example of algorithm that is suitable for determining percent sequence identity (i.e., substantial similarity or identity) is the BLAST algorithm, which is described in Altschul et al, J. Mol. Biol 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra).
  • HSPs high scoring sequence pairs
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues, always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as default parameters a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g. , Karlin & Altschul, Proc. Nat 'I. Acad. Sci. USA 90:5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. III. In vitro and in vivo assays for modulators and mimetics of RDGC phosphatase activity
  • the present invention provides assays to identify modulators of RDGC phosphatase and RDGC phosphatase mimetics of this activity.
  • the activity of RDGC phosphatase can be assessed using a variety of in vitro and in vivo assays, e.g., measuring GPCR phosphorylation, GPCR electrophoretic mobility, current, membrane potential, ion flux, e.g., sodium, measuring sodium concentration, second messengers (e.g., cAMP, IP3, or Ca 2+ ) and transcription levels, neurotransmitter levels, using e.g., voltage-sensitive dyes, radioactive tracers, patch-clamp electrophysiology, transcription assays, and the like.
  • Modulators can also be genetically altered versions of RDGC phosphatase. Such modulators of RDGC phosphatase activity and GPCR signal transduction activity are useful for identifying compounds that can be used to treat disease that are associated with GPCRs.
  • RDGC phosphatase modulators samples or non-human animals that are with treated RDGC phosphatase or RDGC phosphatase homologs or mimetics and with potential RDGC phosphatase inhibitors or activators are compared to control samples or non-human animals without the test compound, to examine the extent of modulation. Modulators of RDGC phosphatase activity are tested using biologically active RDGC phosphatase, either recombinant or naturally occurring.
  • the protein can be isolated, present in a fluid, membrane, cell or tissue sample, expressed in a cell, expressed in a membrane derived from a cell, or expressed in a whole animal.
  • the assay can be in vitro, e.g. , isolated protein (recombinant or naturally occurring), membranes, tissues, fluids, or cells, in an aqueous or solid phase or a combination of both, or in vivo in a whole animal.
  • Modulation is tested using one of the in vitro or in vivo assays described herein.
  • Samples or non-human animals that are treated with a potential RDGC phosphatase inhibitor or activator are compared to control samples or animals without the test compound, to examine the extent of modulation.
  • Control samples or animals (untreated with activators or inhibitors) are assigned a relative RDGC phosphatase activity value of 100.
  • Inhibition of RDGC is achieved when the RDGC phosphatase activity value relative to the control is about 90%, preferably 50%, more preferably 25% .
  • Activation of RDGC is achieved when the RDGC phosphatase activity value relative to the control is 110%, more preferably 150%, more preferable 200% higher.
  • the compounds tested as modulators of RDGC phosphatase can be any small chemical compound, or a biological entity, such as a protein, sugar, nucleic acid or lipid. Typically, test compounds will be small chemical molecules and peptides.
  • modulators can be genetically altered versions of RDGC phosphatase.
  • the mimetics or modulators are added to the assays in test concentrations, by any suitable means.
  • test compounds When assaying for RDGC phosphatase modulators and RDGC phosphatase mimetics, the test compounds are added in test concentrations to the cell or oocyte, as described above.
  • modulators can be added to cell media in aqueous solutions or organic solvents such as DMSO, for cellular uptake.
  • Modulators and mimetics can also be administered by injection, by fusion proteins, by liposome delivery, by viral transduction, by transfection, by expression vectors, etc.
  • RDGC phosphatase, and RDGC phosphatase homologs and mimetics can be administered to a cell, e.g., by transduction with an expression vector encoding RDGC phosphatase or an RDGC phosphatase homolog or mimetic; by injection of RDGC phosphatase or an RDGC phosphatase homolog or mimetic; by administering RDGC phosphatase or an RDGC phosphatase homolog or mimetic in a liposome; by creating a targeted fusion protein with RDGC phosphatase or an RDGC phosphatase homolog or mimetic.
  • recombinant or naturally occurring RDGC phosphatase and GPCR can be used.
  • recombinant RDGC phosphatase can be used in combination with a cell extract or membrane expresses a GPCR, or a transgenic animal can be engineered to express RDGC.
  • recombinant or naturally occurring GPCRs and RDGC phosphatase or RDGC phosphatase homologs or mimetics are incubated together under standard reaction conditions, for direct detection of GPCR dephosphorylation.
  • G-protein-coupled receptors are muscarinic acetylcholine receptors (mAChR), adrenergic receptors, serotonin receptors, dopamine receptors, angiotensin receptors, adenosine receptors, bradykinin receptors, metabotropic excitatory amino acid receptors, taste cell receptors and the like.
  • mAChR muscarinic acetylcholine receptors
  • neuronal nicotinic acetylcholine receptors e.g. , the human ⁇ 2 , ⁇ 3 , and ⁇ 2 , subtypes.
  • the human ⁇ 5 subtype Choini, et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:1572-1576), the rat ⁇ 2 subunit (Wada, et al. , (1988) Science 240, pp. 330- 334); the rat ⁇ 3 subunit (Boulter, et al., (1986) Nature 319, pp.
  • the rat ⁇ 4 subunit Goldman, et al. (1987) Cell 48, pp. 965-973); the rat ⁇ 5 subunit (Boulter, et al. (1990) I. Biol. Chem. 265, pp. 4472-4482); the chicken ⁇ 7 subunit (Couturier et al. (1990) Neuron 5:847-856); the rat ⁇ 2 subunit (Deneris, et al. (1988) Neuron 1, pp. 45-54) the rat ⁇ 3 subunit (Deneris, et al. (1989) J. Biol. Chem. 264, pp.
  • the rat ⁇ 4 subunit Duvoisin, et al. (1989) Neuron 3, pp. 487-496
  • combinations of the rat ⁇ subunits, and s ⁇ subunits and a and p subunits GAB A receptors, e.g. , the bovine x, and ⁇ x , subunits (Schofield, et al. (1987) Nature 328, pp. 221-227); the bovine X 2 , and X 3 , subunits (Levitan, et al. (1988) Nature 335, pp. 76-79); the ⁇ -subunit (Pritchett, et al. (1989) Nature 338, pp.
  • ⁇ 2 , and ⁇ 3 , subunits Ymer, et al. (1989) EMBO J. 8, pp. 1665-1670); the 8 subunit (Shivers, B.D. (1989) Neuron 3, pp. 327-337); and the like; glutainate receptors, e.g., rat GluRl receptor (Hollman, et al. (1989) Nature 342, pp. 643-648); rat GluR2 and GluR3 receptors (Boulter et al. (1990) Science 249: 1033-1037; rat GluR4 receptor (Keinanen et al.
  • glutainate receptors e.g., rat GluRl receptor (Hollman, et al. (1989) Nature 342, pp. 643-648); rat GluR2 and GluR3 receptors (Boulter et al. (1990) Science 249: 1033-1037; rat GluR4 receptor (Keinanen
  • rat NMDAR2A, NMDAR2B and NMDAR2C receptors (Monyer et al. (1992) Science 256: 1217-1221); rat metabotropic mGluRl receptor (Houamed et al. (1991) Science 252: 1318-1321); rat metabotropic mGluR2, mGluR3 and mGluR4 receptors (Tanabe et al. (1992) Neuron 8: 169-179); rat metabotropic mGluR5 receptor (Abe et al. (1992) I. Biol. Chem.
  • adrenergic receptors e.g., human ⁇ l (Frielle, et al. (1987) Proc. Natl. Acad. Sci. 84, pp. 7920-7924); human ⁇ 2 (Kobilka, et al. (1987) Science 238, pp. 650-656); hamster ⁇ 2 (Dixon, et al. (1986) Nature 321, pp. 75-79); and the like; dopamine receptors, e.g. , human D2 (StorTnann, et al. (1990) Molec. Pharm. 37, pp.
  • mammalian dopamine D2 receptor U.S. Patent No. 5,128,254
  • rat Bunzow, et al. (1988) Nature 336, pp. 783-787
  • serotonin receptors e.g. , human 5HTla (Kobilka, et al. (1987) Nature 329, pp. 75-79); serotonin 5HT1C receptor (U.S. Patent No. 4,985,352); human 5HT1D (U.S. Patent No. 5,155,218); rat 5HT2 (Julius, et al. (1990) PNAS 87, pp. 928-932); rat 5HTlc (Julius, et al. (1988) Science 241, pp. 558-564), and the like.
  • Ion channels can also be used with the invention and include, but are not limited to, calcium channels comprised of the human calcium channel ( ⁇ 2 ⁇ and/or ⁇ -subunits (see also, W089/09834; human neuronal ⁇ 2 subunit); rabbit skeletal muscle al subunit (Tanabe, et al. (1987) Nature 328, pp. 313-E318); rabbit skeletal muscle ⁇ 2 subunit (Ellis, et al. (1988) Science 241, pp. 1661-1664); rabbit skeletal muscle p subunit (Ruth, et al. (1989) Science 245, pp. 1115-1118); rabbit skeletal muscle ⁇ subunit (Jay, et al. (1990) Science 248, pp.
  • human calcium channel ⁇ 2 ⁇ and/or ⁇ -subunits
  • rabbit skeletal muscle al subunit Teanabe, et al. (1987) Nature 328, pp. 313-E318
  • potassium ion channels e.g., rat brain (BK2) (McKinnon, D. (1989) J. Biol Chem. 264, pp. 9230-8236); mouse brain (BK1) (Tempel, et al. (1988) Nature 332, pp. 837-839); and the like; sodium ion channels, e.g. , rat brain I and II (Noda, et al. (1986) Nature 320, pp. 188-192); rat brain III (Kayano, et al. (1988) FEBS Lett. 228, pp. 187-1.94); human II (ATCC No.
  • the effects of the test compounds upon the function of the phosphatase can be measured by examining any of the parameters described above. Any suitable physiological change that affects phosphatase activity can be used to assess the influence of a test compound on the phosphatases of this invention.
  • any suitable physiological change that affects phosphatase activity can be used to assess the influence of a test compound on the phosphatases of this invention.
  • the functional consequences are determined using intact cells or animals, one can also measure a variety of effects such as transmitter release, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as Ca 2+ , IP3 or cAMP.
  • RDGC phosphatase activity can be measured directly, e.g., by examining dephosphorylation of an RDGC GPCR substrate such as rhodopsin. Dephosphorylation can be measured e.g., by examining transfer of a label associated with a phosphate, or by examining GPCR mobility.
  • cells, membranes, tissues, or samples comprising RDGC phosphatase and a GPCR substrate are contacted with a test compound.
  • the samples are subjected to assays that measure dephosphorylation, e.g., ELISAs, immunoassays, and western blots are probed with specific antibodies against phosphorylated or dephosphorylated GPCRs.
  • samples can be probed with specific antibodies using in situ techniques.
  • the assays can be performed either in an aqueous phase or in a mixture of solid and aqueous phase.
  • RDGC phosphatase activity can be detected by examining increased electrophoretic mobility of an RDGC.
  • samples are subjected to electrophoresis, and western blots are probed with a suitable antibody to detect dephosphorylated or phosphorylated GPCR.
  • Transformed cell lines can also be used in vitro to identify RDGC phosphatase modulators and mimetics.
  • RDGC phosphatase activity can also be indirectly measured, by examining GPCR signal transduction activity.
  • Preferred assays for G-protein coupled receptor signal transduction activity include cells that are loaded with ion or voltage sensitive dyes to report receptor and activity. Assays for determining activity of such receptors can also use known agonists and antagonists for other G-protein coupled receptors as negative or positive controls to assess activity of tested compounds.
  • Assays for identifying modulatory compounds e.g., agonists, antagonists
  • changes in the level of ions in the cytoplasm or membrane voltage will be monitored using an ion sensitive or membrane voltage fluorescent indicator, respectively.
  • ion-sensitive indicators and voltage probes that may be employed are those disclosed in the Molecular Probes 1997 Catalog.
  • promiscuous G-proteins such as G15 and G16 can be used in the assay of choice (Wilkie et al, PNAS USA 88: 10049-10053 (1991)). Such promiscuous G-proteins allow coupling of a wide range of receptors.
  • Changes in ion flux may be assessed by determining changes in polarization (i.e., electrical potential) of the cell or membrane expressing GPCR.
  • polarization i.e., electrical potential
  • One means to determine changes in cellular polarization is by measuring changes in current (thereby measuring changes in polarization) with voltage-clamp and patch-clamp techniques, e.g., the "cell-attached" mode, the "inside-out” mode, and the "whole cell” mode (see, e.g. , Ackerman et al, New Engl J. Med. 336: 1575-1595 (1997)).
  • Whole cell currents are conveniently determined using the standard methodology (see, e.g. , Hamil et al, PFlugers. Archiv. 391:85 (1981).
  • radiolabeled ion flux assays include: radiolabeled ion flux assays and fluorescence assays using voltage-sensitive dyes (see, e.g., Vestergarrd-Bogind et al , J. Membrane Biol. 88:67-75 (1988); Gonzales & Tsien, Chem. Biol. 4:269-277 (1997); Daniel et al, J. Pharmacol. Meth. 25: 185-193 (1991); Holevinsky et al, J. Membrane Biology 137:59-70 (1994)).
  • the compounds to be tested are present in the range from 1 pM to 100 mM.
  • cells expressing RDGC phosphatases may be loaded with ion or voltage sensitive dyes to report receptor or ion channel activity, such as calcium channels or N-methyl-D-aspartate (NMD A) receptors, GABA receptors, kainate/AMPA receptors, nicotinic acetylcholine receptors, sodium channels, calcium channels, potassium channels excitatory amino acid (EAA) receptors, and nicotinic acetylcholine receptors.
  • NMD A N-methyl-D-aspartate
  • GABA receptors GABA receptors
  • kainate/AMPA receptors kainate/AMPA receptors
  • nicotinic acetylcholine receptors sodium channels
  • calcium channels calcium channels
  • potassium channels excitatory amino acid (EAA) receptors and nicotinic acetylcholine receptors.
  • Receptor activation typically initiates subsequent intracellular events, e.g. , increases in second messengers such as IP3, which releases intracellular stores of calcium ions.
  • IP3 inositol triphosphate
  • IP3 phospholipase C-mediated hydrolysis of phosphatidylinositol (Berridge & Irvine, Nature 312:315-21 (1984)).
  • IP3 in turn stimulates the release of intracellular calcium ion stores.
  • a change in cytoplasmic calcium ion levels, or a change in second messenger levels such as IP3 can be used to assess G-protein coupled receptor function.
  • Cells expressing such G-protein coupled receptors may exhibit increased cytoplasmic calcium levels as a result of contribution from both intracellular stores and via activation of ion channels, in which case it may be desirable although not necessary to conduct such assays in calcium-free buffer, optionally supplemented with a chelating agent such as EGTA, to distinguish fluorescence response resulting from calcium release from internal stores.
  • Other assays can involve determining the activity of receptors which, when activated, result in a change in the level of intracellular cyclic nucleotides, e.g., cAMP or cGMP, by activating or inhibiting enzymes such as adenylate cyclase.
  • cyclic nucleotide-gated ion channels e.g. , rod photoreceptor cell channels and olfactory neuron channels that are permeable to cations upon activation by binding of cAMP or cGMP (see, e.g. , Altenhofen et al, Proc. Natl Acad. Sci. U.S.A.
  • a receptor-activating compound e.g., forskolin
  • Cells for this type of assay can be made by co-transfection of a host cell with DNA encoding a cyclic nucleotide-grated ion channel, GPCR phosphatase and DNA encoding a receptor (e.g.
  • ion channels PCT publication WO 93/13423
  • cell surface receptors U.S. Patents 5,401,629, and 5,436,128 and PCT Application WO 93/13423 (Akong et al)
  • intracellular receptors PCT publication WO 96/41013, U.S. Patent 5,548,063, U.S. Patent 5,171,671, U.S. Patent 5,274,077, U.S. Patent 4,981,784, EP 0 540 065 Al, U.S. Patent 5,071,773, and U.S. 5,298,429).
  • New chemical or recombinant RDGC phosphatase mimetics, homologs, and modulators are generated by identifying compounds with RDGC phosphatase activity or the ability to modulate RDGC phosphatase activity, using the assays described above. These compounds are often referred to as lead compounds. Once a lead compound is identified, variants are typically created and evaluated for use as a therapeutic agent.
  • An example of an RDGC phosphatase mimetic is a small peptide containing the RDGC phosphatase active site, or a small chemical molecule that has the same chemical structure as the RDGC phosphatase active site.
  • An example of an RDGC phosphatase homolog is a naturally-occurring or recombinant variant of RDGC phosphatase that has increased stability or increased activity.
  • An example of an RDGC phosphatase modulator is a small chemical or peptide that inhibits or activates RDGC phosphatase activity.
  • a wide variety of RDGC phosphatase homologs can be tested for RDGC phosphatase activity, e.g., conservative modifications, truncations, targeted fusion proteins, etc. Such molecules are typically isolated from naturally occurring sources or made using standard recombinant technology, described below, or designed by computer assisted drug design, described below.
  • the RDGC phosphatase active site can be identified using computer assisted drug design, by site-directed mutagenesis of conserved RDGC phosphatase domains, by screening nested deletions or by screening linker scanner deletions of RDGC phosphatase.
  • the RDGC phosphatase active site and conserved domains can also be identified by comparing the amino acid sequences of RDGC phosphatase alleles. RDGC phosphatase homologs can then be designed that include the essential components of the active site and are conservatively modified in other regions.
  • Standard recombinant techniques are typically used to make RDGC phosphatase homologs, e.g., site-directed mutagenesis, random mutagenesis, nested deletions, linker scanner deletions, truncations, fusions, isolation of alleles, etc.
  • Combinatorial libraries also provide a source of potential RDGC phosphatase homologs, modulators, and mimetics, particularly mimetics and modulators.
  • a library containing a large number of potential therapeutic compounds is provided.
  • Such "combinatorial chemical libraries” are then screened in one or more RDGC phosphatase activity assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity.
  • the compounds thus identified can serve as conventional "lead compounds” or can themselves be used as potential or actual therapeutics.
  • a preferred method of screening for compounds is described in U.S. Serial NO. 08/858,016 to Stylie et al , filed May 16, 1997.
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991); Houghton et al , Nature 354:84-88 (1991)).
  • Peptide synthesis is by no means the only approach envisioned and intended for use with the present invention.
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (PCT Publication No.
  • Patent 5,539,083) antibody libraries (see, e.g., Vaughn et al , Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/ 10287), carbohydrate libraries (see, e.g., Liang et al , Science, 274: 1520- 1522 (1996) and U.S. Patent 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent 5,569,588; thiazolidinones and metathiazanones, U.S. Patent 5,549,974; pyrrolidines, U.S.
  • Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA).
  • High throughput methodology Any of the assays for compounds that modulate or mimic RDGC phosphatase activity, described herein, are amenable for use in high throughput screening.
  • High throughput assays for the activity of a particular product e.g. , RDGC phosphatase or RDGC phosphatase mimetics or homologs, are well known to those of skill in the art.
  • high throughput screening systems are commercially available (see, e.g., Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.).
  • a molecule such as a GPCR is labeled with a detectable moiety.
  • a GPCR can be labeled at the C-terminus or N-terminus.
  • the particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with RDGC phosphatase activity.
  • detectable labels have been well-developed in the field of immunoassays and, in general, most any label useful in such methods can be applied to the present invention.
  • a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
  • a preferred label is a fluorescent label.
  • Suitable labels are any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include magnetic beads (e.g. , DYNABEADSTM), fluorescent dyes and molecules (e.g. , fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g. , 3 H, 125 I, 35 S, 14 C, or 32 P), enzymes (e.g. , horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g.
  • Preferred labels include fluorescent molecules and dyes, Green Fluorescent Protein (GFP; see, e.g. , U.S. 5,625048; WO96/23810; W097/28261; PCT/UX97/12410; PCT/US97/ 14593), /3-lactamase (see, e.g., U.S. 5,741,657; and WO96/30540), fluorescent membrane potential indicators, also known as fluorescent membrane voltage sensors (see, e.g., U.S. 5,661,035 and W096/41166), and FRET (fluorescence resonance energy).
  • GFP Green Fluorescent Protein
  • the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art.
  • Non-radioactive labels are often attached by indirect means.
  • a ligand molecule e.g., biotin
  • the ligand then binds to another molecules (e.g. , streptavidin) molecule, which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • the ligands and their targets can be used in any suitable combination with antibodies that recognize the RDGC phosphatase substrate, or secondary antibodies that recognize anti-RDGC phosphatase-substrates.
  • the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore.
  • Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidotases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
  • Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g. , luminol.
  • Means of detecting labels are well known to those of skill in the art.
  • means for detection include a scintillation counter or photographic film as in autoradiography.
  • the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
  • CCDs charge coupled devices
  • enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
  • simple colorimetric labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
  • E. Computer assisted drug design Yet another assay for compounds that modulate RDGC phosphatase activity involves computer assisted drug design, in which a computer system is used to generate a three-dimensional structure of a protein based on the structural information encoded by the amino acid sequence. The three dimensional structure of the protein is then used to identify potential ligands that bind to the protein, or to identify molecules that are mimetics of the protein of interest. For example, the three-dimensional structure of RDGC phosphatase can be used to identify RDGC phosphatase mimetics and modulators that bind to RDGC phosphatase. The structure can also be used to identify the RDGC phosphatase protease active site.
  • the three-dimensional structure of GPCRs such as rhodopsin can be used to identify RDGC phosphatase mimetics that bind to GPCRs, or RDGC phosphatase modulators that bind to GPCRs.
  • the input amino acid sequence interacts directly and actively with a preestablished algorithm in a computer program to yield secondary, tertiary, and quaternary structural models of the protein.
  • the models of the protein structure are then examined to identify regions of the structure that have the ability to bind, e.g. , RDGC phosphatase or a GPCR.
  • the three-dimensional structural model of the protein is generated by entering channel protein amino acid sequences of at least 10 amino acid residues or corresponding nucleic acid sequences encoding RDGC phosphatase or GPCR into the computer system.
  • the amino acid sequence represents the primary sequence or subsequence of the protein, which encodes the structural information of the protein.
  • the sequence is entered into the computer system from computer keyboards, computer readable substrates that include, but are not limited to, electronic storage media (e.g.
  • RDGC phosphatase or GPCR is then generated by the interaction of the amino acid sequence and the computer system, using software known to those of skill in the art.
  • the amino acid sequence represents a primary structure that encodes the information necessary to form the secondary and tertiary structure of RDGC phosphatase or GPCR.
  • the software looks at certain parameters encoded by the primary sequence to generate the structural model. These parameters are referred to as "energy terms,” and primarily include electrostatic potentials, hydrophobic potentials, solvent accessible surfaces, and hydrogen bonding. Secondary energy terms include van der Waals potentials. Biological molecules form the structures that minimize the energy terms in a cumulative fashion. The computer program is therefore using these terms encoded by the primary structure or amino acid sequence to create the secondary structural model.
  • the tertiary structure of the protein encoded by the secondary structure is then formed on the basis of the energy terms of the secondary structure.
  • the user at this point can enter additional variables such as whether the protein is membrane bound or soluble, its location in the body, and its cellular location, e.g. , cytoplasmic, surface, or nuclear. These variables along with the energy terms of the secondary structure are used to form the model of the tertiary structure.
  • the computer program matches hydrophobic faces of secondary structure with like, and hydrophilic faces of secondary structure with like.
  • potential ligand binding regions are identified by the computer system.
  • Three-dimensional structures for potential ligands are generated by entering amino acid or nucleotide sequences or chemical formulas of compounds, as described above.
  • the three-dimensional structure of the potential ligand is then compared to that of the RDGC phosphatase or GPCR protein to identify ligands that bind to RDGC phosphatase or GPCR. Binding affinity between the protein and ligands is determined using energy terms to determine which ligands have an enhanced probability of binding to the protein.
  • RDGC phosphatase and GPCRs and homologs and mimetics thereof can be used in the assays of the invention.
  • Recombinant RDGC phosphatase and GPCRs are conveniently used for in vitro and in vivo assays.
  • recombinant RDGC phosphatase homologs and fusion proteins can be prepared for testing as RDGC phosphatase mimetics and potential therapeutic agents. The preparation of recombinant RDGC phosphatase and GPCRs is described below, as well as methods for isolating naturally occurring proteins.
  • recombinant proteins are used in the assays of the present invention, e.g., recombinant RDGC phosphatase, GPCRs, mutants thereof, and functional equivalents.
  • this invention relies on routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al , Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A
  • nucleic acids sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences.
  • kb kilobases
  • bp base pairs
  • proteins sizes are given in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.
  • Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et. al , Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Reanier, /. Chrom. 255: 137-149 (1983).
  • sequence of the cloned genes and synthetic oligonucleotides can be verified after cloning using, e.g., the chain termination method for sequencing double-stranded templates of Wallace et al, Gene 16:21-26 (1981).
  • nucleic acid sequences encoding RDGC phosphatase, GPCRs and related nucleic acid sequence homologs are cloned from cDNA and genomic DNA libraries or isolated using amplification techniques with oligonucleotide primers.
  • RDGC phosphatase sequences are typically isolated from Drosophila nucleic acid (genomic or cDNA) libraries, while genes for GPCRs (e.g. , rhodopsin, etc.) can be cloned from mammalian libraries, preferably human libraries.
  • Amplification techniques using primers can also be used to amplify and isolate RDGC phosphatase and GPCRs from DNA or RNA (see U.S.
  • Methods such as polymerase chain reaction (PCR) and ligase chain reaction (LCR) can be used to amplify nucleic acid sequences of RDGC phosphatase directly from mRNA, from cDNA, from genomic libraries or cDNA libraries, and from plasmids.
  • Degenerate oligonucleotides can be designed to amplify homologs. These primers can be used, e.g., to amplify a probe of several hundred nucleotides, which is then used to screen a human library for full-length RDGC phosphatase. Alternatively, the nucleic acid for RDGC phosphatase can be directly amplified. Similar procedures can be used to isolate sequences encoding GPCRs.
  • Nucleic acids encoding RDGC phosphatase and GPCRs can also be isolated from expression libraries using antibodies as probes. Such polyclonal or monoclonal antibodies can be raised using recombinant or naturally occurring RDGC phosphatase or GPCRs as antigens. Synthetic oligonucleotides can be used to construct recombinant RDGC phosphatase genes for use as probes, for expression of protein, and for construction of polymo ⁇ hic variants or mutants such as deletion mutants. This method is performed using a series of overlapping oligonucleotides usually 40-120 bp in length, representing both the sense and nonsense strands of the gene. These DNA fragments are then annealed, ligated and cloned.
  • Polymo ⁇ hic variants, alleles, and interspecies homologs that are substantially identical to RDGC phosphatase or GPCRs can be isolated using RDGC phosphatase and GPCR nucleic acid probes and oligonucleotides under stringent hybridization conditions, by screening libraries using probes, or using amplification techniques as described above.
  • expression libraries can be used to clone polymo ⁇ hic variants, alleles, and interspecies homologs, by detecting expressed homologs immunologically with antisera or purified antibodies, which also recognize and selectively bind to the homolog.
  • the gene encoding Drosophila RDGC phosphatase has been cloned and sequenced (Steele et al, supra)
  • the nucleic acids of interest are typically cloned into intermediate vectors before transformation into prokaryotic or eukaryotic cells for replication and/or expression.
  • These intermediate vectors are typically prokaryote vectors, e.g. , plasmids, or shuttle vectors, as described below.
  • a cloned gene such as those cDNAs encoding RDGC phosphatase and GPCRs
  • Suitable bacterial promoters are well known in the art and described, e.g. , in Sambrook et al. and Ausubel et al.
  • Bacterial expression systems for expressing the RDGC phosphatase protein are available in, e.g., E.
  • Kits for such expression systems are commercially available.
  • Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.
  • the promoter used to direct expression of a heterologous nucleic acid depends on the particular application and is not critical. Exemplary promoters include the SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells, as well as prokaryotic promoters.
  • the promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
  • the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the nucleic acid in host cells.
  • a typical expression cassette thus also contains signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination.
  • the nucleic acid sequence encoding the gene of choice may typically be linked to a cleavable signal peptide sequence to promote secretion of the encoded protein by the transformed cell.
  • signal peptides would include, among others, the signal peptides from tissue plasminogen activator, insulin, and neuron growth factor, and juvenile hormone esterase of Heliothis virescens.
  • Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.
  • the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination.
  • the termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
  • Additional elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences.
  • some expression systems have markers that provide gene amplification such as thymidine kinase, hygromycin B phosphotransf erase, and dihydrofolate reductase.
  • the particular antibiotic resistance gene chosen is not critical, any of the many resistance genes known in the art are suitable.
  • the prokaryotic sequences are preferably chosen such that they do not interfere with the replication of the DNA in eukaryotic cells, if necessary.
  • the particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and fusion expression systems such as GST and LacZ.
  • exemplary eukaryotic vectors include pMSG, pAV009/A + , pMTO10/A + , pMAMneo-5, baculovirus pDSVE.
  • Tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g. , c-myc, or hexahistidine. Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of RDGC phosphatase protein, which are then purified using standard techniques (see, e.g. , Colley et al , J. Biol. Chem.
  • Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques, e.g. , calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors (see, e.g. , Morrison, J. Bad. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al , eds, 1983). After the expression vector is introduced into the cells, the transfected cells are cultured under conditions favoring expression of the gene of choice, which is recovered from the culture using standard techniques identified below.
  • Naturally occurring or recombinant RDGC phosphatase and GPCRs can be purified for use in the assays of the invention.
  • Naturally occurring RDGC phosphatase is purified, e.g., from Drosophila.
  • Naturally occurring GPCRs are purified from a variety of knowbn sources.
  • Recombinant RDGC phosphatase and GPCR are purified from any suitable expression system.
  • RDGC phosphatase and GPCRs may be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, irnmunopurification methods, and others (see, e.g.
  • proteins having established molecular adhesion properties can be reversible fused to the protein of choice.
  • the protein can be selectively adsorbed to a purification column and then freed from the column in a relatively pure form. The fused protein is then removed by enzymatic activity.
  • the protein of choice can be purified using affinity or immunoaffmity columns.
  • A. Purification of protein from recombinant bacteria Recombinant proteins are expressed by transformed bacteria in large amounts, typically after promoter induction; but expression can be constitutive. Promoter induction with IPTG is a one example of an inducible promoter system. Bacteria are grown according to standard procedures in the art. Fresh or frozen bacteria cells are used for isolation of protein. Proteins expressed in bacteria may form insoluble aggregates ("inclusion bodies"). Several protocols are suitable for purification of recombinant inclusion bodies. For example, purification of inclusion bodies typically involves the extraction, separation and/or purification of inclusion bodies by disruption of bacterial cells, e.g.
  • the cell suspension can be lysed using 2-3 passages through a French Press, homogenized using a Polytron (Brinkman Instruments) or sonicated on ice. Alternate methods of lysing bacteria are apparent to those of skill in the art (see, e.g. , Sambrook et al, supra; Ausubel et al, supra).
  • the inclusion bodies are solubilized, and the lysed cell suspension is typically centrifuged to remove unwanted insoluble matter.
  • Proteins that formed the inclusion bodies may be renatured by dilution or dialysis with a compatible buffer.
  • suitable solvents include, but are not limited to urea (from about 4 M to about 8 M), formamide (at least about 80%, volume/ volume basis), and guanidine hydrochloride (from about 4 M to about 8 M).
  • Other suitable buffers are known to those skilled in the art.
  • the protein of choice is separated from other bacterial proteins by standard separation techniques, e.g., with Ni-NTA agarose resin.
  • the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to skill in the art.
  • the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgS0 4 and kept in an ice bath for approximately 10 minutes, for cell lysis to occur.
  • the cell suspension is centrifuged and the supernatant decanted and saved.
  • the recombinant proteins present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art.
  • an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the protein of interest.
  • the preferred salt is ammonium sulfate.
  • Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations.
  • a typical protocol includes adding saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This concentration will precipitate the most hydrophobic of proteins.
  • the precipitate is then discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest.
  • the precipitate is then solubilized in buffer and the excess salt removed if necessary, either through dialysis or diafiltration.
  • Other methods that rely on solubility of proteins, such as cold ethanol precipitation, are well known to those of skill in the art and can be used to fractionate complex protein mixtures.
  • the molecular weight of the protein of choice can be used to isolated it from proteins of greater and lesser size using ultrafiltration through membranes of different pore size (for example, Amicon or Millipore membranes).
  • membranes of different pore size for example, Amicon or Millipore membranes.
  • the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cut-off than the molecular weight of the protein of interest.
  • the retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest.
  • the recombinant protein will pass through the membrane into the filtrate.
  • the filtrate can then be chromatographed as described below.
  • RDGC phosphatase can be purified using a PA63 heptamer affinity column (Singh et al, J. Biol. Chem. 269:29039- 29046 (1994)).
  • kits for screening for modulators of RDGC phosphatase can be prepared from readily available materials and reagents.
  • such kits can comprise any one or more of the following materials: biologically active RDGC phosphatase, reaction tubes, and instructions for testing RDGC phosphatase activity.
  • a wide variety of kits and components can be prepared according to the present invention, depending upon the intended user of the kit and the particular needs of the user.
  • the kit can be tailored for in vivo or in vitro phosphorylation of a GPCR such as rhodopsin, determination of electrophoretic mobility, or GPCR signal transduction activity.
  • RDGC was analyzed biochemically, physiologically, and genetically to determine its activity as a GPCR phosphatase (see Vinos et al, Science 277:687-690 (1997, herein inco ⁇ orated by reference). First, light dependent phosphorylation of rhodopsin in wild-type and rdgC mutant photoreceptor neurons were examined.
  • the following stocks were used in the phosphorylation experiments: wl, wlll8;; ninaE234, wlll8;; rdgC306 cu red, wlll8;; P[ninaED356 ry+] ry ninaEI17 es (RhlD356), wlll8; P[ninaED356 ry+]; rdgC306 cu red ry ninaEI17 es (RhlD356; rdgC).
  • the amount of label ingested was monitored by adding a green food colorant to the radioactive food.
  • Rhl is phosphorylated in a blue-light dependent manner, while subsequent exposure to orange light promotes its dephosphorylation (Fig. IA; Fig. IB, lanes 1 and 2). As expected, Rhl is missing in ninaE mutants (lane 3).
  • Rhl is dramatically hyper-phosphorylated in rdgC mutants, and remains in this state even after exposure to orange light (lane 4). This is consistent with the loss of a major rhodopsin phosphatase activity in rdgC mutant photoreceptor cells. No other proteins were found to have altered phosphorylation profiles in rdgC.
  • FIGURE 1(A) shows an autoradiogram of SDS-polyacrylamide gel electrophoresis (PAGE) of 32 P0 4 in vivo labeled retinal proteins from flies that were exposed to blue light (B) or orange light (O) for 15 min. Blue light promoted phosphorylation of Rhl, whereas orange light promoted dephosphorylation (W, white-eyed control flies).
  • PAGE SDS-polyacrylamide gel electrophoresis
  • transgenic flies were generated that express a truncated rhodopsin molecule, R1I1 ⁇ 356.
  • This mutation eliminates the last 18 amino acid residues of rhodopsin, including the serines and threonines in the cytoplasmic tail. These residues are phosphorylated by GRKs.
  • This truncated molecule was expressed in a ninaE mutant background such that the only rhodopsin present in photoreceptor s was the one directed by the transgene.
  • the truncated receptor was expressed in near normal amounts and the cells displayed normal light response. Rhodopsin was not hype ⁇ hosphorylated in Rh 356 flies (Fig. IB).
  • FIGURE 2 indicates that R ⁇ 356 suppresses rdgC light-dependent retinal degeneration.
  • flies were exposed to white light [30 W white lamp, attenuated 100 fold, at 15 cm] for six days. Fly heads were cut, fixed and embedded in resin as described in Smith et al. , Science 254: 1478-84 (1991). 1 ⁇ m thick sections were stained with methylene blue/borax prior to analysis. Shown are cross sections (1 ⁇ m thick) through adult retinas after 6 days of light exposure.
  • Figure 2(A) shows control flies with normal retinal mo ⁇ hology, with ommatidial clusters organized as a patterned array.
  • Figure 2(B) shows retinas from rdgC flies show marked degeneration.
  • Figure 2(C) shows dgq mutants that lack the ex subunit of the G protein that couples rhodopsin to PLC do not show light-dependent degeneration.
  • dgq is unable to suppress the degeneration of the Dgq; rdgC double mutants; in figure 2(E) R1I1 ⁇ 356 transgenic flies display normal retinal mo ⁇ hology; in figure 2(F), R1I1 ⁇ 356 suppresses rdgC degeneration in R1I1 ⁇ 356; rdgC double mutants. If kept in the dark, all genotypes display normal retinal mo ⁇ hology. Scale bar, 40 ⁇ m.
  • Example 3 In wild-type photoreceptor cells, termination of the light response results from the concerted action of regulatory events at multiple steps. Because rdgC mutants accumulate phosphorylated rhodopsin, alterations in the kinetics of photoreceptor cell inactivation were examined. Examination of photoreceptor light responses by electroretinograms (ERGs) and intracellular recordings showed that the rdgC mutant exhibited a notable decrease in the rate of deactivation relative to control flies.
  • ERPs electroretinograms
  • Electroretinograms (ERGs) of 4 days old adult, dark-raised flies were recorded. Stimulating light was delivered with a 450 W Xenon lamp (Osram) and filtered with either 480 nm bandpass (Oriel, 53850) filter for blue light or 570 nm longpass (Oriel, 51310) filter for orange light. Recordings in Fig. 3A were performed with orange light attenuated 1585 fold. For intracellular recordings, a coronal section was made through the compound eye. Dorsal hemispheres of the head were mounted upside down on a coverslip and immediately immersed in Schneiders medium (Gibco). Photoreceptor cells were impaled with 90-150 mV electrodes filled with 2M KC1. Maximal differences in deactivation time are found when 0.5 s or longer stimuli are used.
  • Figure 3(A) shows that rdgC mutants have defective deactivation kinetics: ERGs of wt, rdgC, R1I7 ⁇ 356, and R1I1 ⁇ 356; rdgC mutant flies. Photoreceptors were given a 0.5 s pulse of orange light. rdgC mutants display strong defects in the deactivation kinetics (arrow), and this phenotype is suppressed by the Rhl ⁇ 356 truncation. Traces from 15 independent measurements were averaged.
  • Figure 3(B) shows intracellular recordings of light-activated responses from control and rdgC photoreceptors.
  • Arrestin binding is required for termination of the active state of GPCRs.
  • the formation of a rhodopsin-arrestin complex is largely determined by the phosphorylation state of rhodopsin.
  • rdgC mutants accumulate hype ⁇ hosphorylated rhodopsin, the deactivation defect may result from a defect in the rhodopsin-arrestin interaction.
  • a manifestation of this interaction in vivo is the prolonged depolarizing afte ⁇ otential (PDA) . This sustained photoresponse occurs when a substantial amount of rhodopsin has been photoconverted from R to the active M state.
  • a PDA results when metarhodopsin is produced in excess of free arrestin.
  • Wild-type photoreceptors require -20% conversion of R to M to trigger a PDA.
  • This amount of rhodopsin isomerization approximately equals the total number of arrestin molecules in the photoreceptor cell.
  • Mutants expressing small amounts of arrestin enter a PDA with very little light (because only a small amount of M would readily saturate arrestin availability), whereas mutants that reduce amounts of Rhl to less than those of arrestin prevent entry into a PDA (there could never be an excess of M over arrestin).
  • dark raised rdgC mutants the amounts of arrestin and rhodopsin are the same as in the wild- type.
  • rdgC photoreceptors entered a PDA with approximately on-eight as much light as did photoreceptors in control flies (see Fig. 4B).
  • R1I1 ⁇ 356; rdgC double mutants displayed a normal PDA.
  • RDGC is required for normal rhodopsin function.
  • the deactivation defect may be attributable to impaired arrestin function that results from rhodopsin hype ⁇ hosphorylation. Defects in the PDA response of rdgC flies are shown in FIGURE 4.
  • O orange light (M-R conversion); B, blue light (R-M conversion).
  • Figure 4(B) shows a histogram of relative amount of blue light required to enter a PDA for the different genotypes. Values are normalized to control flies and represent means +SEM of triplicate determinations. Aur2, a known mutant with a PDA defect (Dolph et al, Science 260: 1910 (1993)), is shown for comparative pu ⁇ oses.

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Abstract

L'invention concerne des procédés servant à rechercher par criblage des modulateurs de l'activité de transduction de signal de récepteurs couplés à une protéine G, ainsi que des procédés servant à rechercher par criblage des RDCG phosphatases mutantes associées à une maladie de la transduction de signal de récepteurs couplés à une protéine G.
PCT/US1998/015717 1997-07-30 1998-07-29 Procede servant a moduler des recepteurs couples a une proteine g WO1999006830A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU86690/98A AU8669098A (en) 1997-07-30 1998-07-29 Method for modulating g-protein coupled receptors

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US5416597P 1997-07-30 1997-07-30
US60/054,165 1997-07-30
US5449297P 1997-08-01 1997-08-01
US60/054,492 1997-08-01

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WO1999006830A1 true WO1999006830A1 (fr) 1999-02-11

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001057240A2 (fr) * 2000-02-01 2001-08-09 Agy Therapeutics, Inc. Interaction du recepteur de n-methyl-d-aspartate et de la tyrosine phosphatase
WO2003000929A2 (fr) * 2001-06-26 2003-01-03 Sankyo Company, Limited Polynucleotides, polypeptides et cancer
US7314725B2 (en) 2001-07-20 2008-01-01 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Phenylthiocarbamide (PTC) taste receptor
US7579453B2 (en) 2003-06-19 2009-08-25 The United States Of America As Represented By The Secretary Of The Dapartment Of Health And Human Services Variants of human taste receptor genes

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
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DATABASE MPSRCH GENBANK 1 January 1900 (1900-01-01), ZUKER C S, ET AL: "D. Melanogaster Opsin (Rh3) Gene, Complete Cds", XP002912875, Database accession no. M17718 *
PITCHER J. A., ET AL.: "THE G-PROTEIN-COUPLED RECEPTOR PHOSPHATASE: A PROTEIN PHOSPHATASE TYPE 2A WITH A DISTINCT SUBCELLULAR DISTRIBUTION AND SUBSTRATE SPECIFICITY.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 92., 1 August 1995 (1995-08-01), US, pages 8343 - 8347., XP002912880, ISSN: 0027-8424, DOI: 10.1073/pnas.92.18.8343 *
STEELE F. R., ET AL.: "DROSOPHILA RETINAL DEGENERATION C (RDGC) ENCODES A NOVEL SERINE/THREONINE PROTEIN PHOSPHATASE.", CELL, CELL PRESS, US, vol. 69., 15 May 1992 (1992-05-15), US, pages 669 - 675., XP002912876, ISSN: 0092-8674, DOI: 10.1016/0092-8674(92)90230-A *
STEELE F. R., O'TOUSA J. E.: "THE DROSOPHILA RDGC PROTEIN PHOSPHATASE AND PROTEIN PHOSPHORYLATION/DEPHOSPHORYLATION EVENTS IN VISION.", ADVANCES IN PROTEIN PHOSPHATASES., XX, XX, vol. 07., 1 January 1993 (1993-01-01), XX, pages 515 - 527., XP002912878 *
VINOS J., ET AL.: "A G PROTEIN-COUPLED RECEPTOR PHOSPHATASE REQUIRED FOR RHODOPSIN FUNCTION.", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 277., 1 August 1997 (1997-08-01), US, pages 687 - 690., XP002912879, ISSN: 0036-8075, DOI: 10.1126/science.277.5326.687 *
ZUKER C. S.: "THE BIOLOGY OF VISION IN DROSOPHILA.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 93., no. 02., 23 January 1996 (1996-01-23), US, pages 571 - 576., XP002912877, ISSN: 0027-8424, DOI: 10.1073/pnas.93.2.571 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001057240A2 (fr) * 2000-02-01 2001-08-09 Agy Therapeutics, Inc. Interaction du recepteur de n-methyl-d-aspartate et de la tyrosine phosphatase
WO2001057240A3 (fr) * 2000-02-01 2002-04-25 Thorsten Melcher Interaction du recepteur de n-methyl-d-aspartate et de la tyrosine phosphatase
WO2003000929A2 (fr) * 2001-06-26 2003-01-03 Sankyo Company, Limited Polynucleotides, polypeptides et cancer
WO2003000929A3 (fr) * 2001-06-26 2003-11-27 Sankyo Co Polynucleotides, polypeptides et cancer
US7314725B2 (en) 2001-07-20 2008-01-01 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Phenylthiocarbamide (PTC) taste receptor
US7666601B2 (en) 2001-07-20 2010-02-23 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Phenylthiocarbamide (PTC) taste receptor
US8148082B2 (en) 2001-07-20 2012-04-03 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Phenylthiocarbamide (PTC) taste receptor
US7579453B2 (en) 2003-06-19 2009-08-25 The United States Of America As Represented By The Secretary Of The Dapartment Of Health And Human Services Variants of human taste receptor genes
US8309701B2 (en) 2003-06-19 2012-11-13 The United States of America as represented by the Secrectary of the Department of Health and Human Services Variants of human taste receptor genes
US9783590B2 (en) 2003-06-19 2017-10-10 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Variants of human taste receptor genes

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