AGS proteins and nucleic acid molecules and uses therefor Number:7,144,711 from the United States Patent and Trademark Office (PTO) owispatent A screening assay in yeast is disclosed wherein G-protein coupled-receptor independent activators and inhibitors of the pheromone pathway can be identified using a mammalian cDNA library. Novel Activator of G protein Signaling ("AGS") proteins, which are Ras-related proteins that stimulate G protein activity in a receptor-independent manner, are disclosed, as well as nucleic acid molecules encoding AGS proteins. In addition to isolated AGS proteins, the invention further provides isolated AGS fusion proteins, antigenic peptides and anti-AGS antibodies. The invention also provides isolated AGS nucleic acid molecules, recombinant expression vectors containing a nucleic acid molecule of the invention, host cells into which the expression vectors have been introduced and non-human transgenic animals in which an AGS gene has been introduced or disrupted. Diagnostic, screening and therapeutic methods utilizing compositions of the invention are also provided.<H molecules, therefor, AGS, proteins, an, 7144711.html, Patent, pto, 7144711

Title: AGS proteins and nucleic acid molecules and uses therefor

Abstract: A screening assay in yeast is disclosed wherein G-protein coupled-receptor independent activators and inhibitors of the pheromone pathway can be identified using a mammalian cDNA library. Novel Activator of G protein Signaling ("AGS") proteins, which are Ras-related proteins that stimulate G protein activity in a receptor-independent manner, are disclosed, as well as nucleic acid molecules encoding AGS proteins. In addition to isolated AGS proteins, the invention further provides isolated AGS fusion proteins, antigenic peptides and anti-AGS antibodies. The invention also provides isolated AGS nucleic acid molecules, recombinant expression vectors containing a nucleic acid molecule of the invention, host cells into which the expression vectors have been introduced and non-human transgenic animals in which an AGS gene has been introduced or disrupted. Diagnostic, screening and therapeutic methods utilizing compositions of the invention are also provided.

Patent Number: 7,144,711 Issued on 12/05/2006 to Cismowski, et al.

Inventors: Cismowski; Mary (San Diego, CA), Duzic; Emir (Nanuet, NY)
Assignee: Osi Pharmaceuticals, Inc. (Melville, NY)

Appl. No.: 10/804,491

Filed: March 19, 2004

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
09709103	Nov., 2000	6733991
PCT/US99/10151	May., 1999
60103355	Oct., 1998
60084842	May., 1998

Current U.S. Class: 435/29 ; 435/254.11; 435/254.21; 435/4; 435/7.31

Current International Class: C12Q 1/02 (20060101); C12Q 1/00 (20060101); G01N 33/53 (20060101); C12N 1/15 (20060101); C12N 1/16 (20060101)

Field of Search: 435/4,7.1,29,254.11,255.2

References Cited [Referenced By]

U.S. Patent Documents


4613572	September 1986	MacKay et al.
4736866	April 1988	Leder et al.
4870009	September 1989	Evans et al.
4873191	October 1989	Wagner et al.
4873316	October 1989	Meade et al.
4987071	January 1991	Cech et al.
5096815	March 1992	Ladner et al.
5116742	May 1992	Cech et al.
5223409	June 1993	Ladner et al.
5270181	December 1993	McCoy et al.
5283317	February 1994	Saifer et al.
5482835	January 1996	King et al.
5573908	November 1996	Allen et al.
5654145	August 1997	Fukuda
5691188	November 1997	Pausch et al.

Foreign Patent Documents


0264166	Apr., 1987	EP
WO 9011354	Oct., 1990	WO
WO 9101140	Feb., 1991	WO
WO 9304169	Mar., 1993	WO
WO 9410300	May., 1994	WO
WO 9413802	Jun., 1994	WO
WO 9423025	Oct., 1994	WO
WO 9918211	Apr., 1999	WO

Other References

Cismowski et al. Receptor-independent activators of heterotrimeric G-proteins..quadrature..quadrature.Life Sci. Apr. 6, 2001;68(19-20):2301-8. cited by examiner .
Alton and Vapnek, (1979) "Nucleotide Sequence Analysis of The Chloramphenicol Resistance Transposon Tn9", Nature 282: 864-869. cited by other .
Amann et al., (1988) "Tightly Regulated Tac Promoter Vectors Useful For The Expression of Unfused and Fused Proteins In Escherichia coli", Gene 69:301-315. cited by other .
Appeltauer and Achstetter, (1989) "Hormone-induced Expression of The Chsl Gene From Saccharomyces cerevisiae", European Journal of Biochemistry 181:243-247. cited by other .
Arkin and Yourvan, (1992) "An Algorithm For Protein Engineering: Simulations of Recursive Ensemble Mutagenesis", Proceedings of The Natural Academy of Sciences 89:7811-7815. cited by other .
Baldari, et al., (1987) "A Novel Leader Peptide Which Allows Efficient Secretion of a Fragment of Human Interleukin 1.beta. in Saccharomyces cerevisiae", The Embo Journal 6:229-234. cited by other .
Banerji et al., (1983) "A Lymphocytes-Specific Cellular Enhancer Is Located Downstream of the Joining Region in Immunoglobulin Heavy Chain Genes", Cell 33:729-740. cited by other .
Barbas et al., (1992) "Semisynthetic Combinatorial Antibody Libraries: a Chemical Solution to the Diversity Problem", Proceedings of The National Academy Sciences 89:4457-4461. cited by other .
Bardwell et al., (1994) "Signal Propagation and Regulation in the Mating Pheromone Response Pathway of the Yeast Saccharomyces cerevisiae", Developmental Biology 166:363-379. cited by other .
Bartel et al., (1993) "Elimination of False Positives That Arise in Using the Two-Hybrid System", Biotechniques, 14: 920-924. cited by other .
Bartel, D.P. and Szostak, J.W., (1993) "Isolation of New Ribozymes from a Large Pool of Random Sequences", Science 261:1411-1418. cited by other .
Berman and Gilman, (1998) "Mammalian RGS Proteins: Barbarians at The Gate", The Journal of Biological Chemistry 273: 1269-1272. cited by other .
Bourne et al., (1991) "The GTPase superfamily: Conserved Structure and Molecular Mechanism", Nature 349: 117-127. cited by other .
Bradley, A., (1987) "Production And Analysis of Chimaeric Mice", Teratocarcinomas and Embryonic Stem Cells: A Practical Approach pp. 113-151. cited by other .
Bradley, A., (1991) "Modifying the Mammalian Genome by Gene Targeting", Current Opinion in Biotechnology 2:823-829. cited by other .
Byrne and Ruddle, (1989) "Multiplex Gene Regulation: A Two-tiered Approach to Transgene Regulation in Transgenic Mice", Proceedings of The National Academy of Sciences 86:5473-5477. cited by other .
Calame and Eaton, (1988) "Transcriptional Controlling Elements in the Immunoglobulin and T Cell Receptor Loci", Advances in Immunology 43:235-275. cited by other .
Camper and Tilghman, (1989) "Postnatal Repression of the .varies.-fetoprotein Gene Is Enhancer Independent", Genes and Development 3:537-546. cited by other .
Chen et al., (1997) "Characterization of a Novel Mammalian Rgs Protein That Binds to G.varies. Proteins and Inhibits Pheromone Signaling in Yeast", The Journal of Bilogical Chemistry 272:8679-8685. cited by other .
Chirgwin et al., (1979) "Isolation of Biologically Active Ribonucleic Acid from Sources Enriched in Ribonuclease", Biochemistry 18: 5294-5299. cited by other .
Cismowski et al., (1998) "A Yeast-based Approach to Functional Cloning of Novel Mammalian Proteins in the G-protein Signaling Pathway", Archives of Pharmacology 358:8679-8685 (Abstract Only). cited by other .
Clackson et al., (1991) "Making Antibody Fragments Using Phage Display Libraries", Nature 352:624-628. cited by other .
Cull et al., (1992) "Screening for Receptor Ligands Using Large Libraries of Peptides Linked to the C Terminus of the Lac Repressor", Proceedings of the National Academy of Sciences 89: 1865-1869. cited by other .
Cwirla et al., (1990) "Peptides on Phage: A Vast Library of Peptides for Identifying Ligands", Proceedings of the National Academy of Sciences 87: 6378-6382. cited by other .
Daunt et al., (1997) "Subtype-Specific Intracellular Trafficking of .alpha.2-Andrenergic Receptors", Molecular Pharmacolgy 51: 711-720. cited by other .
Delgrave et al., (1993) "Recursive Ensemble Mutagenesis" Protein Engineering 6(3):327-331. cited by other .
Del Villar, K. et al., (1996) C-terminal Motifs Found in Ras-superfamily G-proteins: Caax and C-seven Motifs, Biochemical Society Transactions 24: 709-713. cited by other .
DeWet et al., (1987) "Firefly Luciferase Gene: Structure and Expression in Mammalian Cells", Molecular and Cellular Biology 7: 725-737. cited by oth- er .
Druey et al., (1996) "Inhibition of G-Protein-Mediated Map Kinase Activation by a New Mammalian Gene Family", Nature 379: 742-746. cited by other .
Edlund et al., (1985) "Cell-Specific Expression of the Rat Insulin Gene: Evidence for Role of Two Distinct 5' Flanking Elements", Science 230:912-916. cited by other .
Foster et al., (1996) "Identification of a Novel Human Rho Protein with Unusual Properties: Gtpase Deficiency and in Vivo Farnesylation", Molecular and Cellular Biology 16: 2689-2699. cited by other .
Gautier et al., (1987) ".varies.-DNA IV: .varies.-Anomeric Tetrathymidylates Covalently Linked to Intercalating Oxazolopyridocarbazoloe" Nucleic Acids Research 15:6625-6641. cited by other .
Gottesman, S., (1990) "Minimizing Proteolysis in Escherichia coli : Genetic Solutions", Gene Expression Technology 119-129. cited by other .
Griffiths et al., (1993) "Human Anti-self Antibodies With High Specificity From Phage Display Libraries", The EMBO Journal 12:725-734. cited by othe- r .
Hagen et al., (1991) "Pheromone Response Elements Are Necessary and Sufficient for Basal and Pheromone-Induced Transcription of the FUS1 Gene of Saccharomyces cerevisiae", Molecular and Cellular Biology 11:2952-2961. cited by other .
Haseloff and Gerlach, (1988) "Simple RNA Enzymes with New and Highly Specific Endoribonuclease Activities", Nature 334:585-591. cited by other .
Helene, C., (1991) "The Anti-gene Strategy: Control of Gene Expression by Triplex-forming-oligonucleotides", Anticancer Drug Design 6(6):569-584. cited by other .
Helene, C. et al., (1992) "Control of Gene Expression by Triple Helix-Forming Oligonucleotides", Annals of the New York Academy Sciences 660:27-36. cited by other .
Houghten et al., (1992) "The Use of Synthetic Peptide Combinatorial Libraries for the Identification of Bioactive Peptides", Biotechniques 13: 412-421. cited by other .
Inoue et al., (1987) "Synthesis and Hybridization Studies on Two Complementary Nona (2'-O-methyl)Ribonucleotides", Nucleic Acids Research 15:6131-6148. cited by other .
Inoue et al., (1987) "Sequence-dependent Hydrolysis of RNA Using Modified Oligonucleotide Splints and R Nase H", FEBS Letters 215:327-330. cited by other .
Iwabuchi et al., (1993) "Use of the Two-hybrid System to Identify the Domain of P53 Involved in Oligomerization", Oncogene 8: 1693-1696. cited by other .
Kang et al., (1990) "Effects of Expression of Mammalian Galpha and Hybrid Mammalian Yeast G.alpha. Proteins on the Yeast Pheromones Response Signal Traduction Pathway", Molecular and Cellular Biology 10: 2582-2590. cited by other .
Kaufman et al., (1987) "Translational Efficiency of Polycistronic Mrnas and Their Utilization to Express Heterologous Genes in Mammalian Cells", The EMBO Journal 6:187-195. cited by other .
Kemppainen et al., (1998) "Dexamethasone Rapidly Induces a Novel Ras Superfamily Member-related Gene in Att-20 Cells", The Journal of Biological Chemistry 273: 3129-3131. cited by other .
Kessel and Gruss, (1990) "Murine Developmental Control Genes", Science 249:374-379. cited by other .
Kurjan, (1993) "The Pheromone Response Pathway In Saccharomyces cerevisiae" Annual Review of Genetics 27:147-179. cited by other .
Kurjan and Herskowitz, (1982) "Structure of a Yeast Pheromone Gene (MF.varies.): A Putative .varies.-Factor Precursor Contains Four Tandem Copies of Mature .varies.-Factor", Cell 30:933-943. cited by other .
Lam et al., (1991) "A New Type of Synthetic Peptide Library for Identifying Ligand-binding Activity", Nature 354: 82-84. cited by other .
Lam, Kit S., (1997) "Application of Combinatorial Library Methods in Cancer Research and Drug Discovery", Anticancer Drug Design 12: 145-167. cited by other .
Lee, E. et al., (1992) "The G226a Mutant of G.sub.s.alpha.Highlights the Requirement for Dissociation of G Protein Subunits", The Journal of Biological Chemistry 267: 1212-1218. cited by other .
Li, E. et al., (1992) "Targeted Mutation of the DNA Methyltransferase Gene Results in Embryonic Lethality", Cell 69:915-926. cited by other .
Li, E. et al., (1998) "Substitutions in the Pheromone-Responsive G.sub..beta. Beta Protein of Saccharomyces cerevisiae Confer a Defect in Recovery from Pheromone Treatment", Genetics 148: 947-961. cited by other .
Logan, J. et al., (1995) "Cationic Lipids For Reporter Gene and CFTR Transfer to Rat Pulmonary Epithelium", Gene Therapy, 2:38-49. cited by other .
Luckow and Summers, (1989) "High Level Expression of Nonfused Foreign Genes with Autographa californica Nuclear Polyhedrosis Virus Expression Vectors", Virology 170:31-39. cited by other .
Madura et al., (1993) "N-recognin/Ubc2 Interactions in the N-end Rule Pathway", The Journal of Biological Chemistry 268:12046-12054. cited by other .
Magee and Newman, (1992) "The Role of Lipid Anchors for Small G Proteins in Membrane Traficking", Trends in Cell Biology 2: 318-323. cited by othe- r .
Maher, L.J., (1992) "DNA Triple-Helix Formation: An Approach to Artificial Gene Repressors?", BioEssays 14(12):807-815. cited by other .
Marks et al., (1992) "Molecular Evolution of Proteins on Filamentous Phage", The Journal of Biological Chemistry 267: 16007-16010. cited by other .
Marra et al., (1998) "The WashU-HHMI Mouse EST Project", EMEST 3: Gen Bank Accession NO. AA790463. cited by other .
Neer, E. J., (1995) "Heterotrimeric G Proteins Organizers of Transmembrane Signals", Cell 80 ; 249-257. cited by other .
Nobes, et al., (1998) "A New Member of the Rho Family, Rndl, Promotes Disassembly of Actin Filament Structures and Loss of Cell Adhesion", The Journal of Cell Biology 141: 187-197. cited by other .
Odagaki, et al., (1998) "Receptor-mediated and Receptor-independant Activation of G-proteins in Rat Brain Membranes" Life Sciences 62: 1537-1541. cited by other .
O'Gorman et al., (1991) "Recombinant-Mediated Gene Activation and Site-Specific Integration in Mammalian Cells", Science 251:1351-1355. cit- ed by other .
Okaoto et al., (1995) "Ligand-dependant G Protein Coupling Function of Amloid Transmembrane Receptor" The Journal of Biological Chemistry 270: 4205-4208. cited by other .
Pinkert et al., (1987) "An Albumin Enhancer Located 10 kb Upstream Functions Along With Its Promoter to Direct Efficient, Liver-specific Expression in Transgenic Mice", Genes and Development 1:268-276. cited by other .
Queen and Baltimore; (1983) "Immunoglobulin Gene Transcription Is Activated by Downstream Sequence Elements", Cell 33:741-748. cited by oth- er .
Rens-Domiano and Hamm, (1995) "Structural and Functional Relationships of Heterotrimeric G-proteins", FASEB Journal 9: 1059-1066. cited by other .
Sapperstein et al., (1994) "Nucleotide Sequence of the Yeast STE14 Gene, Which Encodes Farnesylcysteine Carboxyl Methyltransferase, and Demonstration of its Essential Role in a-Factor Export", Molecular and Cellular Biology 14: 1438-1449. cited by other .
Sato et al., (1996) "Characterization of a G-protein Activator in the Neuroblastoma-Glioma Cell Hybrid NG108-15", The Journal of Biological Chemistry 271: 30052-30060. cited by other .
Schultz et al., (1987) "Expression and Secretion in Yeast of a 400-kDa Envelope Glycoprotein from Epstein-Barr Virus", Gene 54:113-123. cited by other .
Scott and Smith, (1990) "Searching for Peptide Ligands with an Epitope Library", Science 249: 386-390. cited by other .
Seed, B., (1987) "An LFA-3 CDNA a Phospholipid-linked Membrane Protein Homologous to its Receptor CD2", Nature 329:840-842. cited by other .
Sheng et al., (1990) "The Regulation an Function of c-fos and Other Immediate Early Genes in the Nervous System", Neuron 4: 477-485. cited by other .
Sikorski and Hieter, (1989) "A System of Shuttle Vectors and Yeast Host Strains Designed for Efficient Manipulation of DNA in Saccharomyces cerevisiae", Genetics 122:19-27. cited by other .
Simon et al., (1991) "Diversity of G Proteins in Signal Transduction", Science 252: 802-808. cited by other .
Simonsen et al., (1994) "Cloning by Function: Expression Cloning in Mammalian Cells", Trends Pharmacol. Sci. 15: 437-441. cited by other .
Smith et al., (1983) "Production of Human Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vector", Mol. Cell Biol. 3:2156-2165. cited by other .
Smith, D.B. and Johnson, K.S., (1988) "Single-step Purification of Poypeptides Expressed in Escherichia coli as Fusions wtih Glutathione S-transferase" Gene 67:31-40. cited by other .
Stevenson et al., (1992) "Constitutive Mutants of the Protein Kinase STE11 Activate the Yeast Pheromone Response Pathway in the Absence of the G Protein", Genes and Development 6: 1293-1304. cited by other .
Stevenson et al., (1995) "Mutation of RGA1, Which Encodes Putative GTPase-activating Protein for the Polarity-establishment Protein Cdc42p, Activates the Pheromone-response Pathway in the Yeast Saccharomyces cerevisiae", Genes and Development 9: 2949-2963. cited by other .
Strittmatter et al., (1993) "GAP-43 Augments G Protein-coupled Receptor Transduction in Xenopus laevis Oocytes", Proceedings of the National Academy of Sciences 90: 5327-5331. cited by other .
Studier et al., (1990) "Use of T7 RNA Polymerase to Direct Expression of Cloned Genes", Gene Expression Technology 185:60-89. cited by other .
Takesono et al., (1999) "Stimulus Input to Heterotimeric G-protein Signalling Pathways", FASEB Journal 13: A796 (Abstract only). cited by other .
Thomas, K.R. and Capecchi, M. R., (1987) "Site-Directed Mutagenesis by Gene Targeting in Mouse Embryo-Derived Stem Cells", Cell 51: 503-512. cit- ed by other .
Toh et al., (1989) "Isolation and Characterization of a Rat Liver Alkaline Phosphatase Gene", European Journal of Biochemistry 182: 231-238. cited by other .
Valencia et al., (1991) "The ras Protein Family Evolutionary Tree and Role of Conserved Amino Acids", Biochemistry 30: 4637-4648. cited by other .
Wada et al., (1992) "Codon Usage Tabulated from the GenBank Genetic Sequence Data", Nucleic Acids Research 20:2111-2118. cited by other .
Whitney et al., (1998) "A Genome-wide Functional Assay of Signal Transduction in Living Mammalian Cells", Nature Biotechnology 16: 1329-1333. cited by other .
Wilmut, I. et al., (1997) "Viable Offspring from Fetal and Adult Mammalian Cells", Nature 385:810-813. cited by other .
U.S. Appl. No. 09/439,410, filed Nov. 11, 1999, Cismowski et al. cited by other .
U.S. Appl. No. 09/709,103, filed Nov. 8, 2000, Cismowski et al. cited by other .
Winoto and Baltimore, (1989) "A Novel, Inductible and T Cell-specific Enhancer Located at the 3' End of the T Cell Receptor .varies. Locus" The EMBO Journal 8:729-733. cited by other .
Zervos et al., (1993) "Mxil, a Protein That Specifically Interacts with Max to Bind Myc-Max Recognition Sites", Cell 72: 223-232. cited by other .
International Search Report for PCT International Application No. PCT/US99/10151, filed Jul. 5, 1999. cited by other .
Chiu et al., (1998) "Optimizing Energy Potentials for Success in Protein Tertiary Structure Prediction", Folding and Design 3:223-228. cited by other .
Ngo et al. (1994) "Computational Complexity, Protein Structure Prediction, and the Levinthal Paradox", The Protein Folding Problem and Tertiary Structure Prediction pp. 492-494. cited by other .
Verma et al. (1997) "Gene Therapy--Promises, Problems and Prospects", Nature 389: 239-242. cited by other .
Anderson (1998) "Human Gene Therapy", Nature 392: 25-30. cited by other .
Mountain (2000) "Gene Therapy: The First Decade", TIBTECH 18:119-128. cite- d by other.

Primary Examiner: Sullivan; Daniel M.
Attorney, Agent or Firm: Lee; Shu M. Stewart; Alexander A. Nguyen; Kim T.

Parent Case Text

This application is a continuation of U.S. application Ser. No. 09/709,103, filed Nov. 8, 2000, now U.S. Pat. No. 6,733,991, which is a continuation of PCT International Application No. PCT/US99/10151, filed May 7, 1999, designating the United States of America, which claims priority of U.S. Provisional Application No. 60/084,842, filed May 8, 1998 and U.S. Privisional Application No. 60/103,355, filed Oct. 7, 1998, the entire contents of which are hereby incorporated by reference.

Claims

What is claimed is:

1. A method for identifying a compound that modulates signal transduction in a cell, comprising: contacting a cell that expresses an Activator of G protein Signaling ("AGS'") protein with a test compound; determining the effect of the test compound on an activity of the AGS protein; and identifying the test compound as a modulator of signal transduction based on the ability of the test compound to modulate the activity of the AGS protein in the cell, wherein the AGS protein comprises an amino acid sequence having at least 97% identity to SEQ ID NO:2 and simulates G protein activity in a receptor-independent manner.

2. The method of claim 1, wherein the AGS protein is isolated from human cells.

3. The method of claim 1, wherein the AGS protein comprises the amino acid sequence of SEQ ID NO: 2.

4. The method of claim 1, wherein the cell has been engineered to express the AGS protein by introducing into the cell an expression vector encoding the AGS protein.

5. The method of claim 1, wherein the cell has been engineered to express a G protein .alpha. subunit.

6. The method of claim 1, wherein the cell is a yeast cell that has been engineered to express a mammalian or chimeric G protein .alpha. subunit and the effect of the test compound on the activity of the AGS protein is determined by monitoring a pheromone response pathway in the yeast cells.

7. The method of claim 6, wherein the yeast cell has been engineered to express a Gpal-G.alpha.i2 chimeric G protein .alpha. subunit.

8. The method of claim 6, wherein the pheromone response pathway in the yeast cells is monitored by measuring the activity of a pheromone responsive promoter in the yeast cells.

9. The method of claim 1, wherein the effect of the test compound on an activity of the AGS protein is determined by monitoring the ability of the test compound to bind to the AGS protein.

10. The method of claim 1, wherein the effect of the test compound on an activity of the AGS protein is determined by monitoring the ability of the test compound to modulate the interaction of the AGS protein with a target molecule.

11. The method of claim 10, wherein the target molecule is a G protein.

12. The method of claim 1, wherein the compound is a nucleic acid encoding a polypeptide capable of inhibiting an activity of the AGS protein, and wherein said nucleic acid comprises the sequence provided in SEQ ID NO: 24.

13. The method of claim 1, wherein the compound is a nucleic acid encoding a polypeptide capable of inhibiting an activity of the AGS protein, and wherein said nucleic acid encodes the polypeptide having the amino acid sequence provided in SEQ ID NO: 25.

14. The method of claim 1, wherein the cell further comprises a nucleic acid encoding an inhibitor of the AGS protein, and wherein said nucleic acid comprises the nucleotide sequence provided in SEQ ID NO: 24.

15. The method of claim 1, wherein the cell further comprises a nucleic acid encoding an inhibitor of the AGS protein, and wherein said nucleic acid encodes the polypeptide having the amino acid sequence provided in SEQ ID NO: 25.

Description

BACKGROUND OF THE INVENTION

Heterotrimeric G protein-mediated signal transduction is a tightly regulated event. All known G protein-coupled receptor (GPCR) mediated signaling pathways rely on multiple regulatory mechanisms in order to prevent inappropriate induction of the signal and to facilitate recovery during chronic stimulation (Gilman (1987) Ann. Rev. Biochem. 56:615 649, reviewed in Simon et al. (1991) Science 252:802 808; Conklin and Bourne (1993) Cell 73:631 641; Neer (1995) Cell 80:249 257; Rens-Domiano and Hamm (1995) FASEB J. 9:1059 1066). These regulatory mechanisms function at every level of the signaling cascade. Regulation of GPCR activation is believed to involve phosphorylation of the receptor C-terminus and subsequent receptor internalization (Palczewski and Benkovic (1991) Trends Biol. Sci. 16:387 391; Goodman et al. (1996) Nature 383:447 450; Chen and Konopka (1996) Mol. Cell. Biol. 16:247 257) though this does not appear to be a universal mechanism (Daunt et al. (1997) Mol. Pharm. 51:711 720). Known mechanisms of regulation of signal transduction at the level of the heterotrimeric G protein include receptor-mediated facilitation of GTP/GDP exchange on G.alpha. (reviewed in Simon et al. (1991) Science 252:802 808; Conklin and Bourne (1993) Cell 73:631 641; Neer (1995) Cell 80:249 257; Rens-Domiano and Hamm (1995) FASEB J. 9:1059 1066) and enhancement of the intrinsic GTPase activity of G.alpha. proteins by RGS-like proteins (reviewed in Berman and Gilman (1998) J. Biol. Chem. 273:1269 1272). Activation of PAKs, serine/threonine kinases that transduce signals from heterotrimeric G proteins to the MAP kinase cascade, has been shown to occur through interaction with either the small G proteins Cdc42 and Rac, or through interaction with heterotrimeric G proteins (reviewed in Sells and Chernoff (1997) Trends Cell Biol. 7:162 167). GPCR-coupled MAP kinase cascades and their downstream transcription factors, in turn, are regulated through phosphorylation/dephosphorylation cycles that may or may not require small G proteins (reviewed in Cobb and Goldsmith (1995) J. Biol. Chem. 270:14843 14846). Non-receptor activators of G-proteins have also been identified. These include both protein activators (Strittrnatter et al. (1993) Proc. Nat'l Acad. Sci., USA 90:5327 5331; Okamoto et al. (1995) J. Biol. Chem. 270:4205 4208; Sato et al. (1996) J. Biol. Chem. 271:30052 30060) and non-protein activators (summarized in Odagaki et al. (1998) Life Sciences 62:1537 1541.

Even with the identification of these diverse regulatory systems, an in-depth understanding of the temporal and spatial regulation of GPCR mediated signaling remains elusive. In fact, cellular variations in the efficiency and/or specificity of coupling observed for many specific receptor-heterotrimeric G protein complexes suggest the presence of additional unidentified, cell-specific regulators of the signaling process.

SUMMARY OF THE INVENTION

In an attempt to identify novel factors involved in regulating signaling through GPCR-mediated signal transduction pathways, a screening system was devised in the yeast Saccharomyces cerevisiae designed to identify receptor-independent activators of the pheromone response pathway. These functional screens can be designed to target not only specific regulatory pathways in yeast, but also an introduced mammalian component or components. Yeast strains containing an intact signal transduction cascade but lacking a functional GPCR were made conditional for growth upon either pheromone pathway activation (activator screen) or pheromone pathway inactivation (inhibitor screen). In addition, the activator yeast strain carries an integrated FUS1p-HIS3 construct, making histidine prototrophy conditional upon pheromone pathway activation. The inhibitor yeast strain carries an episomal FUS1p-CAN1 construct. Adult human liver cDNAs expressed in a yeast vector under control of a galactose-inducible promoter were introduced into these strain, and those cDNAs that conferred growth in a galactose- and insert-dependent fashion were identified. Provided herein is the characterization of one member of these activator cDNAs, encoding a protein of 281 amino acids with significant homology to ras-related G proteins and containing alterations in conserved amino acids consistent with a deficiency in GTP hydrolysis activity (i.e., a constitutively active form of ras-related G protein). Genetic epistasis tests in yeast were consistent with this activator functioning at the level of the heterotrimeric G-protein and facilitating GTP exchange on the chimeric G.alpha.i2. This protein is referred to herein as Activator of G protein Signaling, or "AGS", or AGS1AGS1 also shows G.alpha. selectivity, as measured by growth assays in yeast expressing various mammalian G.alpha. constructs and tissue specific expression, as measured by Northern blot analysis. A cDNA product identified from the inhibitor screen encodes a previously identified regulator of G-protein signaling, human RGS5.

In one embodiment, an isolated nucleic acid molecule of the present invention encodes an AGS protein (e.g., the nucleic acid molecule has a nucleotide sequence having at least 86% identity to the nucleotide sequence of SEQ ID NO: 1 or the complement thereof). In another embodiment, an isolated nucleic acid molecule of the present invention has a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or the complement of SEQ ID NO: 1 or SEQ ID NO: 3. In yet another embodiment, the isolated nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 1, or the complement thereof. In another embodiment the isolated nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 3, or the complement thereof. In yet another embodiment, an isolated nucleic acid molecule of the present invention encodes a protein that activates G protein-coupled signal transduction in a G protein-coupled receptor independent manner.

In another embodiment, an isolated nucleic acid molecule of the present invention has a nucleotide sequence which encodes a protein having an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 2. In another embodiment the isolated nucleic acid molecule encodes a protein having the amino acid sequence of SEQ ID NO: 2. In yet another embodiment, an isolated nucleic acid molecule of the present invention encodes a protein which activates G protein-coupled signal transduction in a G protein-coupled receptor independent manner.

The present invention also provides vectors including nucleic acid sequences which encode all or a portion of an AGS protein as well as host cells including such vectors. The invention further provides methods for producing an AGS protein including culturing host cells which express an AGS protein. The invention also provides transgenic animals which contain cells carrying a transgene encoding AGS protein.

In another embodiment, the present invention provides isolated AGS proteins (e.g., an isolated AGS protein having an amino acid sequence at least 97% identity to the amino acid sequence of SEQ ID NO: 2.) In another embodiment, the protein has the amino acid sequence of SEQ ID NO: 2. The present invention also provides fusion proteins having at least a portion of an AGS protein operatively linked to a non-AGS polypeptide as well as antibodies that specifically bind to an AGS protein (e.g., monoclonal or polyclonal antibodies). The invention further provides pharmaceutical compositions including AGS proteins or AGS-antibodies.

The present invention further provides methods for identifying compounds that modulate cellular signal transduction. In one embodiment, the method includes the steps of (a) contacting a cell that expresses an AGS protein with a test compound; (b) determining the effect of the test compound on the activity of the AGS protein; and (c) identifying the test compound as a modulator of signal transduction based on the ability of the compound to modulate the activity of the AGS protein in the cell. In another embodiment, the AGS proteins utilized in the subject methods have an amino acid sequence which is at least 97% identical to SEQ ID NO: 2 and stimulates G protein activity in a receptor-independent manner. In yet another embodiment, the AGS protein used in the subject methods has the amino acid sequence of SEQ ID NO: 2.

In yet another embodiment, the compound identified by the above method is a nucleic acid encoding a polypeptide capable of inhibiting the activity of the AGS protein. In still another embodiment, the above method further comprises a nucleic acid encoding an inhibitor of the AGS protein. In a preferred embodiment, the above method is suitable for identifying a test compound capable of modulating the activity of the AGS protein by modulating the inhibitor of the AGS protein.

In a preferred embodiment, cells used in the screening methods of the present invention have been engineered to express the AGS protein. Preferred cells for use in the screening methods are yeast cells. In another preferred embodiment, the yeast cells have further been engineered to express a G protein .alpha. subunit, a chimeric G protein .alpha. subunit, or a Gpa1-G.alpha.i2 chimeric G protein .alpha. subunit. The activity of a test compound on a cell-associated activity (e.g., a G-protein mediated activity) can be determined by monitoring a pheromone response pathway in the yeast cells (e.g., by measuring the activity of a pheromone responsive promoter in the yeast cells), by monitoring the ability of the test compound to bind to the AGS protein, or by monitoring the ability of the test compound to modulate the interaction of the AGS protein with a target molecule (e.g., a G protein).

The present invention further provides methods for modulating G protein-coupled signal transduction in a cell (e.g., by contacting a cell with an agent which modulates AGS protein activity or AGS nucleic acid expression). Methods for treating a subject having a disorder characterized by aberrant AGS protein activity or nucleic acid expression are also provided as well as methods for detecting the presence of AGS in a biological sample.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict diagrams of the yeast pheromone response pathway with major signaling components, (Abbr. indicated are: .alpha., Gpa1; .beta., Ste4; .gamma., Ste18; P, phosphorylation) and the modifications made to the pheromone response pathway (FIG. 1B) for the Activator screen (i.e., strains CY1316/1183) and the Inhibitor screen (e.g., strains CY1141/1451-AGS1/2440).

FIG. 2 depicts the major steps in carrying out the yeast Activator (left panel) and Inhibitor (right panel) screens.

FIGS. 3A and 3B depict the coding region of the cDNA sequence (FIG. 3A) and predicted amino acid sequence of human AGS (FIG. 3B). The nucleotide sequence corresponds to SEQ ID NO: 1. The amino acid sequence corresponds to SEQ ID NO:2. The composite insert region of cDNA clone pYES2-L1 is available from GenBank, Accession No. #AF069506. Consensus sequences for ras-like G proteins (Valencia, et al. (1991) Biochemistry 30:4637) are underlined. Regions unique to AGS1 are indicated in italics. Three residues that are normally conserved in ras-like G proteins but divergent in AGS1 and Rnd1 3 (Nobes, et al. (1998) J. Cell Biol. 141:187) are indicated with asterisks below the residue. These divergent residues were shown to confer GTPase deficiency in Rnd1.

FIGS. 4A and 4B depict the alignment of AGS with representatives of all major classes of small G proteins in humans indicating that AGS is likely to be the founding member of a novel class of small G proteins in humans. G-protein sequences: AGS1 (SEQ ID NO: 46); C-HA-RAS1 (SEQ ID NO: 47); RALA (SEQ ID NO: 48); RAB-1A (SEQ ID NO: 49); RHOHP1 (SEQ ID NO: 50); CDC42 (SEQ ID NO: 51); RAC2 (SEQ ID NO: 52); ARL1 (SEQ ID NO: 53): RND3/RHOE (SEQ ID NO: 54).

FIG. 5 depicts the alignment of residues in the P region and in the G' region of various small G proteins. Asterisks indicate the location of three highly conserved residues that are altered in AGS, Rnd1, Rnd2, and Rnd3. Numbers indicate the positions of the amino acid sequence of AGS. G-protein P-region sequences: RhoE/Rnd3 (SEQ ID NO: 55); Rnd2 (SEQ ID NO: 56); Rnd1 (SEQ ID NO: 57); RhoA (SEQ ID NO: 58); RhoB (SEQ ID NO: 59); Cdc42 (SEQ ID NO: 60); Rac1 (SEQ ID NO: 61); H-ras (SEQ ID NO: 62); AGS (SEQ ID NO: 63). G-protein G'-region sequences: RhoE/Rnd3 (SEQ ID NO: 64); Rnd2 (SEQ ID NO: 65); Rnd1 (SEQ ID NO: 66); RhoA (SEQ ID NO: 67); RhoB (SEQ ID NO: 68); Cdc42 (SEQ ID NO: 69); Rac1 (SEQ ID NO: 70); H-ras (SEQ ID NO: 71); AGS (SEQ ID NO: 72).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of nucleic acid and protein molecules referred to herein as Activator of G protein Signaling ("AGS") proteins and nucleic acid molecules which play a role in or function in G protein-mediated signal transduction in the absence of receptor stimulation. These molecules were identified using an assay of the invention that employs yeast-based functional screens using the pheromone response pathway. In part, the assay relies on the observation that a G-protein coupled receptor (GPCR) signaling pathway is required for mating in haploid yeast. Moreover, there is functional redundancy between this pathway and the mammalian GPCR signaling pathway and all of the major signaling components and regulatory systems in GPCR-mediated signal transduction in mammalian systems appears to be conserved in the yeast pheromone response pathway (FIG. 1A) (Kurjan (1993) Annu. Rev. Genet. 27:147 179; Bardwell et al., Dev. Biol. 166:363 379). Thus, this assay can be used to search for mammalian regulators of this system. Normally, upon receptor activation by pheromone, the GPCR associated heterotrimeric G-protein undergoes subunit dissociation into GTP-bound activated G.alpha. (Gpa1) and G.beta..gamma. (Ste4/Ste18). Free G.beta..gamma. dimer then transduces a signal through a p21-activated kinase (Ste20) to a MAP kinase cascade, leading to the transcriptional activation of mating-specific genes by the transcription factor Ste12, as well as Far1-mediated growth arrest in the G.sub.1 phase of the cell cycle. For the screening assay of the present inventions, the pheromone response pathway was engineered to create yeast strains that could be made conditional for growth upon either pheromone pathway activation or suppression (FIG. 1B). Using these strains functional screens were developed to identify mammalian cDNAs whose expression either activates or down-regulates the yeast pheromone response pathway independent of the presence of receptor. A human AGS cDNA was isolated in a functional cloning screen in yeast based upon its ability to activate G protein signaling in a manner independent of G protein-coupled receptor stimulation. Genetic evidence (described in detail in Examples 1 3) indicates that this AGS-dependent activation occurs at the level of the heterotrimeric G protein. Thus, in one embodiment, the AGS molecules stimulate the activity of one or more G proteins involved in a G protein-mediated signal transduction pathway, e.g., a pheromone response cascade in yeast, to thereby activate G protein-mediated signal transduction independent of G protein coupled receptor stimulation. In a preferred embodiment, the AGS molecules of the present invention stimulate the activity of one or more G proteins involved in a G protein-mediated signal transduction pathway, such that G protein coupled receptor-mediated signal transduction is amplified. In a particularly preferred embodiment, the AGS molecules are capable of modulating the activity of G.alpha. subunits, such as a mammalian G.alpha.i2 subunit or a chimeric G.alpha. subunit comprising a portion of the yeast Gpa1 protein (e.g., the amino-terminal 41 amino acids) linked to a mammalian G.alpha.i2 subunit.

Since the AGS proteins of the invention can function in activation of the pheromone response cascade in yeast cells, and potentially modulate the MEK pathway in mammalian cells, the AGS molecules of the present invention can be used in methods for identifying antagonists of G protein signaling, either receptor-dependent or receptor independent, in screening assays in host cells, such as mammalian or yeast host cells.

A particularly preferred AGS nucleic acid and protein, depicted in FIG. 5 (and shown in SEQ ID NO: 1 and 2, respectively), is isolated from human cells. FIG. 5 depicts the nucleotide sequence of the coding region of an AGS cDNA which was isolated from a human liver cDNA library. An AGS cDNA nucleotide sequence that includes 5' and 3' untranslated regions is shown in SEQ ID NO: 3. The cDNA sequence encodes a predicted protein which is 281 amino acid residues in length and which exhibits homology to ras-related G proteins. The AGS protein also contains alterations in amino acids that typically are conserved among ras-related G proteins that are consistent with AGS having a deficiency in GTP hydrolysis activity.

The AGS proteins of the invention contain several motifs characteristic of Ras superfamily members, including the phosphate/magnesium binding regions GXXXXGK(S/T)(SEQ ID NO: 18) (the P-loop) and DXXG (SEQ ID NO: 19) (the G' region), as well as the guanine base binding loops NKXD (SEQ ID NO: 20) and EXSAK (SEQ ID NO: 21) (Bourne et al. (1991) Nature 349:117 127; Valencia et al. (1991) Biochemistry 30:4637 4648). Additionally, the motif regions G-1 through G-5, characteristic of GTPases (Bourne et al. (1991) Nature 349:117 127), are present in AGS. Moreover, the C-terminal region of AGS has a typical CAAX (SEQ ID NO: 22) motif (Bourne et al. (1991) Nature 349:117 127; Valencia et al. (1991) Biochemistry 30:4637 4648; Del Villar et al. (1996) Biochem. Soc. Trans. 24:709 713). The CAAX motif is immediately preceded by a basic region QAKDKER (SEQ ID NO: 23), thought to be important in anchoring ras-like G proteins to the phospholipid bilayer (Magee and Newman (1992) Trends Cell Biol. 2:318 323).

Various aspects of the invention are described in further detail in the following subsections:

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid molecules that encode an AGS protein or biologically active portion thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify AGS-encoding nucleic acid (e.g., AGS mRNA). As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (e.g., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated AGS nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO: 3, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, a human AGS cDNA can be isolated from a human liver cDNA library using all or portion of SEQ ID NO:1 or SEQ ID NO: 3 as a hybridization probe and standard hybridization techniques (e.g., as described in Sambrook, J., Fritsh. E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Moreover, a nucleic acid molecule encompassing all or a portion of SEQ ID NO:1 or SEQ ID NO: 3 can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon the sequence of SEQ ID NO:1 or SEQ ID NO:3. For example, mRNA can be isolated from liver cells (e.g., by the guanidinium-thiocyanate extraction procedure of Chirgwin et al. (1979) Biochemistry 18: 5294 5299) and cDNA can be prepared using reverse transcriptase (e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, Fla.). Synthetic oligonucleotide primers for PCR amplification can be designed based upon the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3. A nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to an AGS nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO:1. The sequence of SEQ ID NO:1 corresponds to the coding region of an AGS cDNA isolated from human liver cells. Another preferred AGS cDNA sequence is shown in SEQ ID NO: 3, which includes 5' and 3' untranslated regions. This cDNA comprises sequences encoding the AGS protein (e.g., "the coding region", from nucleotides 154 to 999), as well as 5' untranslated sequences (nucleotides 7 to 153) and 3' untranslated sequences (nucleotides 1000 to 1801).

In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3, or a portion of any of these nucleotide sequences. A nucleic acid molecule which is the complement of the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3 is one which has a nucleotide sequence that directly pairs with that of SEQ ID NO: 1 or 3, according to the rules of Watson and Crick base pairing, wherein A pairs with T and G pairs with C. For example, the complement of the sequence 5'GGATGC 3' would be 3'CCTACG 5'' (which, written in the standard 5' to 3' direction, would be 5'GCATCC 3').

In still another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleotide sequence which is at least 60%, preferably at least 70%, more preferably at 80%, and even more preferably at least 90%, or 95%, or 96%, or 97%, or 98%, or 99% homologous to the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3, or a portion of any of these nucleotide sequences. In an additional preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3, or a portion of any of these nucleotide sequences.

Moreover, the nucleic acid molecule of the invention can comprise only a portion of the coding region of SEQ ID NO:1 or SEQ ID NO:3, for example a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of an AGS protein. The nucleotide sequence determined from the cloning of the AGS genes from a mammal, e.g., humans, allows for the generation of probes and primers designed for use in identifying and/or cloning AGS homologues in other cell types, e.g. from other tissues, as well as AGS homologues from other mammals, e.g., mice, rats, etc. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of SEQ ID NO:1 or SEQ ID NO:3 sense an anti-sense sequence of SEQ ID NO:1 or SEQ ID NO:3, or naturally occurring mutants thereof. Primers based on the nucleotide sequence in SEQ ID NO:1 or SEQ ID NO:3 can be used in PCR reactions to clone AGS homologues. Probes based on the AGS nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress an AGS protein, such as by measuring a level of an AGS-encoding nucleic acid in a sample of cells from a subject e.g., detecting AGS mRNA levels or determining whether a genomic AGS gene has been mutated or deleted.

In one embodiment, the nucleic acid molecule of the invention encodes a protein or portion thereof which includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of SEQ ID NO:2 such that the protein or portion thereof maintains the ability to modulate a G-protein mediated response in a cell. As used herein, the language "sufficiently homologous" refers to proteins or portions thereof which have amino acid sequences which include a minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain as an amino acid residue in SEQ ID NO:2) amino acid residues to an amino acid sequence of SEQ ID NO:2 such that the protein or portion thereof is able to modulate a G-protein mediated response in a cell. In another embodiment, the protein is at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95%, or 96%, or 97%, or 98%, or 99% homologous to the entire amino acid sequence of SEQ ID NO:2.

Portions of proteins encoded by the AGS nucleic acid molecule of the invention are preferably biologically active portions of the AGS protein. As used herein, the term "biologically active portion of AGS" is intended to include a portion, e.g., a domain/motif, of AGS that has one or more of the following activities: 1) it can interact with (e.g., bind to) a G protein; 2) it can modulate the activity of a G protein; 3) it can interact with (e.g., bind to) a G protein target molecule; 4) it can modulate the activity of a G protein target molecule; 5) it can modulate a G protein-mediated response in a cell, independent of G protein-coupled receptor activation; and 6) it can augment G protein-coupled receptor signaling by modulating a G protein-mediated response in a cell. Standard binding assays, e.g., immunoprecipitations, yeast two-hybrid assays, and in vitro column binding assays, as described herein, can be performed to determine the ability of an AGS protein or a biologically active portion thereof to interact with (e.g., bind to) a G protein. To determine whether an AGS protein or biologically active portion thereof can modulate G-protein mediated response in a cell, the AGS protein or biologically active portion thereof can be introduced into a cell (e.g., transformed or transfected) which has been engineered to grow only in the presence of an AGS protein or biologically-active portion thereof, e.g., yeast cell strain 1316/1183 (described in the Examples) and the ability of the AGS protein or biologically active portion thereof to facilitate growth determined. Alternatively, a cell can be transformed or transfected with a G-protein mediated signal transduction responsive reporter construct (e.g., FUS1-luciferase) which responds to G-protein mediated signaling by expressing luciferase, and a nucleic acid encoding the AGS protein or biologically active portion thereof. The cells can be harvested and lysed and reporter activity, e.g., luciferase activity, can be measured and compared to reporter activity in a control cell. Examples of control cells include cells which include the G-protein mediated signal transduction responsive reporter construct. An alteration in reporter activity in the cells which include nucleic acid encoding the AGS protein, as compared to reporter activity in the cells without nucleic acid encoding the AGS protein is indicative of a modulation of a G-protein mediated response in the cell.

In addition to the AGS nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:3, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of AGS may exist within a population. Such genetic polymorphism in the AGS gene may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding an AGS protein, preferably a mammalian AGS protein. Such natural allelic variations can typically result in 1 5% variance in the nucleotide sequence of the AGS gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in AGS that are the result of natural allelic variation and that do not alter the functional activity of AGS are intended to be within the scope of the invention. Moreover, nucleic acid molecules encoding AGS proteins from other species and thus which have a nucleotide sequence which differs from the sequence of SEQ ID NO:1 or SEQ ID NO:3, are intended to be within the scope of the invention. For example, non-human homologues of the AGS cDNAs of the invention can be isolated based on their homology to the AGS nucleic acid disclosed herein using the human cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 15 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3. In other embodiments, the nucleic acid is at least 30, 50, 100, 250 or 500 nucleotides in length. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences at least about 65%, more preferably at least about 70%, and even more preferably at least about 75% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 6.3.6. A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6.times. sodium chloride/sodium citrate (SSC) at about 45.degree. C., followed by one or more washes in 0.2.times.SSC. 0.1% SDS at 50 65.degree. C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:1 or SEQ ID NO:3 corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein). In one embodiment, the nucleic acid encodes a natural human AGS.

In addition to naturally-occurring allelic variants of the AGS sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, thereby leading to changes in the amino acid sequence of the encoded AGS proteins, without altering the functional activity of the AGS proteins. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO:1 or SEQ ID NO:3. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequences of AGS (e.g., the sequence of SEQ ID NO:2) without altering the activity of AGS, whereas an "essential" amino acid residue is required for AGS activity. For example, conserved amino acid residues in the following motifs that are conserved among Ras family members are most likely important for the activity of an AGS protein and are thus essential residues of AGS: the phosphate/magnesium binding regions GXXXXGK(S/T)(SEQ ID NO: 18) (the P-loop) and DXXG (SEQ ID NO: 19), the guanine base binding loops NKXD (SEQ ID NO: 20) and EXSAK (SEQ ID NO: 21), the motif regions G-1 through G-5, characteristic of GTPases, and the C-terminal CAAX (SEQ ID NO: 22) motif. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved family of ras-related small G proteins) may not be essential for activity and thus are likely to be amenable to alteration without altering AGS activity.

Accordingly, another aspect of the invention pertains to nucleic-acid molecules encoding AGS proteins that contain changes in amino acid residues that are not essential for AGS activity. Such AGS proteins differ in amino acid sequence from SEQ ID NO:2, yet retain at least one of the AGS activities described herein. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 60% homologous to the amino acid sequence of SEQ ID NO:2.