WO1999058670A1 - Molecules d'acides nucleiques et proteines ags, et utilisations correspondantes - Google Patents

Molecules d'acides nucleiques et proteines ags, et utilisations correspondantes Download PDF

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WO1999058670A1
WO1999058670A1 PCT/US1999/010151 US9910151W WO9958670A1 WO 1999058670 A1 WO1999058670 A1 WO 1999058670A1 US 9910151 W US9910151 W US 9910151W WO 9958670 A1 WO9958670 A1 WO 9958670A1
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protein
nucleic acid
seq
cell
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PCT/US1999/010151
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Mary Cismowski
Emir Duzic
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Cadus Pharmaceutical Corporation
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Priority to AU39776/99A priority Critical patent/AU3977699A/en
Priority to US09/439,410 priority patent/US6746852B1/en
Publication of WO1999058670A1 publication Critical patent/WO1999058670A1/fr
Priority to US09/709,103 priority patent/US6733991B1/en
Priority to US10/804,491 priority patent/US7144711B2/en

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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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G01N2333/39—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts
    • G01N2333/395—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts from Saccharomyces

Definitions

  • 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.
  • MAP kinase cascade 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. 1: 162- 167).
  • GPCR-coupled MAP kinase cascades and their downstream transcription factors 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.
  • 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).
  • the activator yeast strain carries an integrated FUSlp- HIS3 construct, making histidine prototrophy conditional upon pheromone pathway activation.
  • AGS 1 also shows G ⁇ selectivity, as measured by growth assays in yeast expressing various mammalian G ⁇ 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.
  • 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).
  • 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.
  • the isolated nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 1, or the complement thereof.
  • the isolated nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 3, or the complement thereof.
  • 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.
  • 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.
  • the isolated nucleic acid molecule encodes a protein having the amino acid sequence of SEQ ID NO: 2.
  • 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.
  • 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.)
  • the protein has the amino acid sequence of SEQ ID NO: 2.
  • the present invention further provides methods for identifying compounds that modulate cellular signal transduction.
  • 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.
  • 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.
  • the AGS protein used in the subject methods has the amino acid sequence of SEQ ID NO: 2.
  • the compound identified by the above method is a nucleic acid encoding a polypeptide capable of inhibiting the activity of the AGS protein.
  • the above method further comprises a nucleic acid encoding an inhibitor of the AGS protein.
  • 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.
  • 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.
  • the yeast cells have further been engineered to express a G protein ⁇ subunit, a chimeric G protein ⁇ subunit, or a Gpal-G ⁇ i2 chimeric G protein ⁇ subunit.
  • the activity of a test compound on a cell-associated 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.
  • Figures 1A and IB depict diagrams of the yeast pheromone response pathway with major signaling components, (Abbr. indicated are: ⁇ , Gpal; ⁇ , Ste4; ⁇ , Stel8; P, phosphorylation) and the modifications made to the pheromone response pathway (Fig. IB) for the Activator screen (i.e., strains CY1316/1183) and the Inhibitor screen (e.g., strains CY1141/1451-AGS1/2440).
  • Figure 2 depicts the major steps in carrying out the yeast Activator (left panel) and Inhibitor (right panel) screens.
  • 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.
  • AGS Activator of G protein Signaling
  • GPCR G- protein coupled receptor
  • Free G ⁇ 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 Stel2, as well as Far 1 -mediated growth arrest in the G, phase of the cell cycle.
  • the pheromone response pathway was engineered to create yeast strains that could be made conditional for growth upon either pheromone pathway activation or suppression ( Figure IB). 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.
  • 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.
  • 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.
  • the AGS molecules are capable of modulating the activity of G ⁇ subunits, such as a mammalian G ⁇ i2 subunit or a chimeric G ⁇ subunit comprising a portion of the yeast Gpal protein (e.g., the amino-terminal 41 amino acids) linked to a mammalian G ⁇ i2 subunit.
  • 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 Figure 5 (and shown in SEQ ID NO: 1 and 2, respectively), is isolated from human cells.
  • Figure 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.
  • CAAX SEQ ID NO: 22
  • 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).
  • 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).
  • 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.
  • 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.
  • the isolated AGS nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 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.
  • 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.
  • 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.
  • nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to an AGS nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • 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).
  • 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:l or SEQ ID NO:3, or aportion 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.
  • an isolated nucleic acid molecule of the invention comprises a nucleotide sequence which is at least 60%, preferably at least 10%, more preferably at 80%, and even more preferably at least 90%, or 95%, or 96%, or 91%, or 98%, or 99% homologous to the nucleotide sequence shown in SEQ ID NO:l or SEQ ID NO:3, or a portion of any of these nucleotide sequences.
  • 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:l or SEQ ID NO:3, or a portion of any of these nucleotide sequences.
  • 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:l or SEQ ID NO:3 sense, an anti-sense sequence of SEQ ID NO:l or SEQ ID NO:3, or naturally occurring mutants thereof.
  • Primers based on the nucleotide sequence in SEQ ID NO:l 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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%o, or 96%o, 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.
  • 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.
  • 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.
  • a cell e.g., transformed or transfected
  • yeast cell strain 1316/1183 described in the Examples
  • 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.
  • reporter activity e.g. , luciferase activity
  • 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.
  • AGS nucleotide sequences shown in SEQ ID NO:l 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.
  • 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.
  • nucleic acid molecules encoding AGS proteins from other species are intended to be within the scope of the invention.
  • 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.
  • 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:l or SEQ ID NO:3.
  • the nucleic acid is at least 30, 50, 100, 250 or 500 nucleotides in length.
  • 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.
  • the conditions are such that sequences at least about 65%, more preferably at least about 10%, and even more preferably at least about 75% or more homologous to each other typically remain hybridized to each other.
  • 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 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C.
  • an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:l or SEQ ID NO:3 corresponds to a naturally-occurring nucleic acid molecule.
  • 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).
  • the nucleic acid encodes a natural human AGS.
  • 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.
  • 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.
  • 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.
  • 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-l through G-5, characteristic of GTPases, and the C-terminal CAAX (SEQ ID NO: 22) motif.
  • Other amino acid residues may not be essential for activity and thus are likely to be amenable to alteration without altering AGS activity.
  • nucleic acid molecules encoding AGS proteins that contain changes in amino acid residues that are not essential for AGS activity.
  • AGS proteins differ in amino acid sequence from SEQ ID NO:2, yet retain at least one of the AGS activities described herein.
  • 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, and is capable of modulating a G-protein mediated response in a cell.
  • the protein encoded by the nucleic acid molecule is at least about 70% homologous to SEQ ID NO:2, more preferably at least about 80-85% homologous, even more preferably at least about 90-95%) homologous, and most preferably at least about 95, 96, 97, 98, or 99% homologous to SEQ ID NO:2.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first or second sequence for optimal alignment).
  • the length of a reference sequence aligned for comparison purposes is at least 30%>, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • the comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm.
  • a preferred, non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Nati. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Nati. Acad. Sci. USA 90:5873-77.
  • Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • a PAM250 weight residue table When using the Lipman-Pearson algorithm, a PAM250 weight residue table, a gap length penalty of 12, a gap penalty of 4, and a Ktuple of 2 can be used.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the alignment of nucleic acid sequences is the Wilbur-Lipman algorithm (Wilbur and Lipman (1983) Proc. Nati Acad. Sci. USA 80:726-730).
  • a window of 20 When using the Wilbur- Lipman algorithm, a window of 20, gap penalty of 3, Ktuple of 3 can be used.
  • Both the Lipman-Pearson algorithm and the Wilbur-Lipman algorithm are incorporated, for example, into the MEGALIGN program (e.g., version 3.1.7) which is part of the DNASTAR sequence analysis software package.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding an AGS protein.
  • coding region refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding AGS.
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (e.g., also referred to as 5' and 3' untranslated regions).
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of an AGS mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of an AGS mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of an AGS mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g.
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave AGS mRNA transcripts to thereby inhibit translation of AGS mRNAs.
  • a ribozyme having specificity for an AGS-encoding nucleic acid can be designed based upon the nucleotide sequence of an AGS cDNA disclosed herein, (see, e.g., Cech et al. U.S. Patent No. 4,987,071 and Cech et al. U.S. Patent No. 5,116,742).
  • AGS mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules, (see, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418).
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno- associated viruses) as well as baculoviral vectors, which serve equivalent functions.
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., AGS proteins, mutant forms of AGS proteins, fusion proteins, etc.).
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith,
  • the coding sequence of an AGS protein is cloned into a pGEX expression vector to create a vector encoding a fusion protein comprising, from the N-terminus to the C-terminus, GST-thrombin cleavage site-AGS protein.
  • the fusion protein can be purified by affinity chromatography using glutathione-agarose resin.
  • Recombinant AGS protein unfused to GST can be recovered by cleavage of the fusion protein with thrombin.
  • suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al, (1988) Gene 69:301-315) and pET l id (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89).
  • nucleic acid sequence of the nucleic acid is to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al. (1992) Nucleic Acids Res. 20:2111- 2118).
  • Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to an AGS mRNA.
  • a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to an AGS mRNA.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (e.g., express) an AGS protein.
  • the invention further provides methods for producing AGS proteins using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding an AGS protein has been introduced) in a suitable medium until AGS protein is produced.
  • the method further comprises isolating AGS protein from the medium or the host cell.
  • the host cells of the invention can also be used to produce nonhuman transgenic animals.
  • the nonhuman transgenic animals can be used in screening assays designed to identify agents or compounds, e.g., drugs, pharmaceuticals, etc., which are capable of ameliorating detrimental symptoms of selected disorders such as cardiovascular disorders and proliferative disorders.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which AGS-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous AGS sequences have been introduced into their genome or homologous recombinant animals in which endogenous AGS sequences have been altered. Such animals are useful for studying the function and/or activity of AGS and for identifying and/or evaluating modulators of AGS activity.
  • the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced AGS gene has homologously recombined with the endogenous AGS gene are selected (see e.g., Li, E. et al. (1992) Cell 69:915).
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152).
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene.
  • Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, A.
  • transgenic nonhumans animals can be produced which contain selected systems which allow for regulated expression of the transgene.
  • a system is the cre/loxP recombinase system of bacteriophage PI.
  • cre/loxP recombinase system of bacteriophage PI.
  • a recombinase system is the FLP recombinase system oi Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251 :1351-1355.
  • mice containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones of the nonhuman transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature 385:810-813.
  • Another aspect of the invention pertains to isolated AGS proteins, and biologically active portions thereof, as well as peptide fragments suitable for use as immunogens to raise anti- AGS antibodies.
  • An "isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of AGS protein in which the protein is separated from cellular components of the cells in which it is naturally or recombinantly produced.
  • the language "substantially free of cellular material” includes preparations of AGS protein having less than about 30% (by dry weight) of non- AGS protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non- AGS protein, still more preferably less than about 10% of non- AGS protein, and most preferably less than about 5% non- AGS protein.
  • non- AGS protein also referred to herein as a "contaminating protein”
  • the AGS protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, e.g., culture medium represents less than about 20%), more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of AGS protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of AGS protein having less than about 30%) (by dry weight) of chemical precursors or non- AGS chemicals, more preferably less than about 20% chemical precursors or non- AGS chemicals, still more preferably less than about 10% chemical precursors or non- AGS chemicals, and most preferably less than about 5% chemical precursors or non- AGS chemicals.
  • isolated proteins or biologically active portions thereof lack contaminating proteins from the same animal from which the AGS protein is derived.
  • such proteins are produced by recombinant expression of, for example, a human AGS protein in a nonhuman cell.
  • An isolated AGS protein or a portion thereof of the invention can modulate a G- protein mediated response in a cell.
  • the protein or portion thereof comprises 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.
  • the portion of the protein is preferably a biologically active portion as described herein.
  • the AGS protein has an amino acid sequence shown in SEQ ID NO:2.
  • a preferred AGS protein of the present invention includes an amino acid sequence encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to the nucleotide sequence SEQ ID NO:l or SEQ ID NO: 3 and which can modulate a G-protein mediated response in a cell.
  • the AGS protein is substantially homologous to the amino acid sequence of SEQ ID NO:2 and retains the functional activity of the protein of SEQ ID NO:2 yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above.
  • the AGS protein is a protein which comprises an amino acid sequence which is at least 60%, preferably at least 80%, and more preferably at least 86%o, or 88%, or 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 and which has at least one of the AGS activities described herein.
  • the invention pertains to a full length human protein which is substantially homologous to the entire amino acid sequence of SEQ ID NO:2.
  • Biologically active portions of the AGS protein include peptides comprising amino acid sequences derived from the amino acid sequence of the AGS protein, e.g., the amino acid sequence shown in SEQ ID NO:2 or the amino acid sequence of a protein homologous to the AGS protein, which include less amino acids than the full length AGS protein or the full length protein which is homologous to the AGS protein, and exhibit at least one activity of the AGS protein.
  • the biologically active portion of the protein includes a motif selected from the following: 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-l through G-5, characteristic of GTPases, the C-terminal CAAX (SEQ ID NO: 22) motif, and/or the QAKDKER motif (SEQ ID NO:23) and can modulate the activity of a G-protein.
  • a motif selected from the following: 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 (
  • biologically active portions in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the activities described herein.
  • the biologically active portions of the AGS protein include one or more selected domains/motifs or portions thereof having biological activity.
  • AGS proteins are preferably produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the protein is cloned into an expression vector (as described above), the expression vector is introduced into a host cell (as described above) and the AGS protein is expressed in the host cell. The AGS protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques.
  • an AGS protein, polypeptide, or peptide can be synthesized chemically using standard peptide synthesis techniques.
  • native AGS protein can be isolated from cells, for example using an anti- AGS antibody (described further below).
  • an AGS "chimeric protein” or “fusion protein” comprises an AGS polypeptide operatively linked to a non- AGS polypeptide.
  • An " AGS polypeptide” refers to a polypeptide having an amino acid sequence corresponding to AGS
  • a “non- AGS polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the AGS protein, e.g., a protein which is different from the AGS protein and which is derived from the same or a different organism.
  • the term "operatively linked" is intended to indicate that the AGS polypeptide and the non- AGS polypeptide are fused in- frame to each other.
  • the non- AGS polypeptide can be fused to the N-terminus or C-terminus of the AGS polypeptide.
  • the fusion protein is a GST- AGS fusion protein in which the AGS sequences are fused to the C-terminus of the GST sequences.
  • Such fusion proteins can facilitate the purification of recombinant AGS.
  • the fusion protein is an AGS protein containing a heterologous signal sequence at its N-terminus.
  • an AGS chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • the present invention also pertains to homologues of the AGS proteins which function as either an AGS agonist (mimetic) or an AGS antagonist.
  • the AGS agonists and antagonists stimulate or inhibit, respectively, a subset of the biological activities of the naturally occurring form of the AGS protein.
  • specific biological effects can be elicited by treatment with a homologue of limited function.
  • treatment of a subject with a homologue having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the AGS protein.
  • Homologues of the AGS protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the AGS protein.
  • the term "homologue” refers to a variant form of the AGS protein which acts as an agonist or antagonist of the activity of the AGS protein.
  • An agonist of the AGS protein can retain substantially the same, or a subset, of the biological activities of the AGS protein.
  • An antagonist of the AGS protein can inhibit one or more of the activities of the naturally occurring form of the AGS protein, by, for example, competitively binding to a G-protein or downstream or upstream member of the pheromone response cascade.
  • homologues of the AGS protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the AGS protein for AGS protein agonist or antagonist activity.
  • a variegated library of AGS variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of AGS variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential AGS sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of AGS sequences therein.
  • a degenerate set of potential AGS sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of AGS sequences therein.
  • fusion proteins e.g., for phage display
  • degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential AGS sequences.
  • Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11 :477.
  • libraries of fragments of the AGS protein coding can be used to generate a variegated population of AGS fragments for screening and subsequent selection of homologues of an AGS protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an AGS coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal, C- terminal and internal fragments of various sizes of the AGS protein.
  • REM Recursive ensemble mutagenesis
  • An isolated AGS protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind AGS using standard techniques for polyclonal and monoclonal antibody preparation.
  • the full-length AGS protein can be used or, alternatively, the invention provides antigenic peptide fragments of AGS for use as immunogens.
  • the antigenic peptide of AGS comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:2 and encompasses an epitope of AGS such that an antibody raised against the peptide forms a specific immune complex with AGS.
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of AGS that are located on the surface of the protein, e.g., hydrophilic regions and or regions that are unique to AGS, e.g. not common to all small G proteins.
  • An AGS immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen.
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed AGS protein or a chemically synthesized AGS peptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic AGS preparation induces a polyclonal anti- AGS antibody response.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g. , molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as AGS.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind AGS.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of AGS.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular AGS protein with which it immunoreacts.
  • Polyclonal anti- AGS antibodies can be prepared as described above by immunizing a suitable subject with an AGS immunogen. The anti- AGS antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized AGS.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against AGS can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem .255:4980-83; Yeh et al.
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind AGS, e.g., using a standard ELISA assay.
  • a monoclonal anti- AGS antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with AGS to thereby isolate immunoglobulin library members that bind AGS.
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01 ; and the Stratagene SurfZAPTM Phage Display Kit, Catalog No. 240612).
  • examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S.
  • recombinant anti- AGS antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184, 187; Taniguchi, M., European Patent Application 171 ,496;
  • An anti- AGS antibody e.g., monoclonal antibody
  • An anti- AGS antibody can be used to isolate AGS by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti- AGS antibody can facilitate the purification of natural AGS from cells and of recombinantly produced AGS expressed in host cells. Moreover, an anti- AGS antibody can be used to detect AGS protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance, pattern of expression, and/or subcellular localization of the AGS protein. Anti- AGS antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (e.g., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 I, 35 S or 3 H.
  • compositions suitable for administration to a subject typically comprise the nucleic acid molecule, protein, modulator, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged abso ⁇ tion of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., an AGS protein or anti- AGS antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., an AGS protein or anti- AGS antibody
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the pu ⁇ ose of oral therapeutic administration, the active compound can be inco ⁇ orated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g. , with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g. , Chen et al. (1994) PNAS 91 :3054-3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • An AGS protein of the invention has one or more of the activities described herein and can thus be used, for example, to screen drugs or compounds which modulate AGS protein activity as well as to treat disorders characterized by insufficient or excessive production of AGS protein or production of AGS protein forms which have altered (e.g., increased or decreased) activity compared to wild type AGS.
  • methods are provided that employ AGS molecules and rely on strategies based upon functional readouts using the yeast Saccharomyces cerevisiae, and these methods offer a fast and more reliable way to identify genes whose expression affects key aspects of cellular function.
  • the isolated nucleic acid molecules of the invention can be used to express AGS protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect AGS mRNA (e.g. , in a biological sample) or a genetic lesion in an AGS gene, and to modulate AGS activity, as described further below.
  • AGS protein e.g., via a recombinant expression vector in a host cell in gene therapy applications
  • AGS mRNA e.g. , in a biological sample
  • the anti- AGS antibodies of the invention can be used to detect and isolate AGS protein and modulate AGS protein activity.
  • the invention provides methods for identifying compounds or agents which modulate AGS protein activity and/or AGS nucleic acid expression. These methods are also referred to herein as drug screening assays and typically include the step of screening a candidate/test compound or agent for the ability to interact with (e.g., bind to) an AGS protein, to modulate the interaction of an AGS protein and a target molecule, and/or to modulate AGS nucleic acid expression and/or AGS protein activity, and/or to modulate signal transduction mediated at least in part by an AGS protein.
  • Candidate/test compounds or agents which have one or more of these abilities can be used as drugs to treat disorders characterized by aberrant or abnormal AGS protein activity and/or AGS nucleic acid expression.
  • Candidate/test compounds such as small molecules, e.g., small organic molecules, and other drug candidates can be obtained, for example, from combinatorial and natural product libraries.
  • the invention provides a method for identifying a compound that modulates signal transduction in a cell, comprising the steps of contacting a cell that expresses an AGS protein with a test compound and determining the effect of the test compound on the activity of the AGS protein and 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.
  • the term "identify” as used in the context of "identifying a compound” refers to the identification of compounds for which an activity as an activator of G protein signaling has not been previously recognized or demonstrated.
  • the term “identifying” according to the methods of the present invention is intended to refer to identifying, screening and/or selecting of test compound, for example selecting active compounds not previously recognized as activators of G protein signaling for further analysis and testing.
  • compounds “identified” according to the methods of the present invention can be used as test compounds in a second assay to confirm a G protein activating activity.
  • compounds “identified” according to the methods of the present invention can be tested for other desirable activities or can be tested, for example, at varying doses to determine the efficacy of the compound.
  • Compounds "identified” according to the methods of the present invention can be also tested in cell culture models or in animal models of disease.
  • the AGS protein comprises an amino acid sequence having at least 86%) identity to SEQ ID NO: 2 and stimulates G protein activity in a receptor-independent manner.
  • the AGS protein comprises the amino acid sequence of SEQ ID NO 2.
  • the AGS protein can comprise a structure as described above in the sections discussing AGS proteins and nucleic acids.
  • the effect of the test compound on the 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.
  • the target molecule is a G protein.
  • the invention provides assays for screening candidate/test compounds which interact with (e.g., bind to) AGS protein.
  • the assays are cell-free assays which include the steps of combining an AGS protein or a biologically active portion thereof, and a candidate/test compound, e.g., under conditions which allow for interaction of (e.g., binding of) the candidate/test compound to the AGS protein or portion thereof to form a complex, and detecting the formation of a complex, in which the ability of the candidate compound to interact with (e.g., bind to) the AGS protein or portion thereof is indicated by the presence of the candidate compound in the complex. Formation of complexes between the AGS protein and the candidate compound can be quantitated, for example, using standard immunoassays.
  • a statistically significant change, such as a decrease, in the interaction of the AGS and target molecule (e.g. , in the formation of a complex between the AGS and the target molecule) in the presence of a candidate compound (relative to what is detected in the absence of the candidate compound) is indicative of a modulation (e.g., stimulation or inhibition) of the interaction between the AGS protein and the target molecule.
  • Modulation of the formation of complexes between the AGS protein and the target molecule can be quantitated using, for example, an immunoassay.
  • Biotinylated AGS molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • biotinylation kit Pierce Chemicals, Rockford, IL
  • antibodies reactive with AGS but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and AGS trapped in the wells by antibody conjugation.
  • preparations of an AGS-binding protein and a candidate compound are incubated in the AGS-presenting wells of the plate, and the amount of complex trapped in the well can be quantitated.
  • G proteins interact with a range of ion channels and are able to inhibit certain voltage-sensitive Ca - " transients, as well as stimulating cardiac K + channels.
  • a reduction in the generation of AGS-induced second messengers or mating factor responses e.g., growth arrest or shmoo formation
  • GTP binding or GTPase enzymatic activity of G proteins can be measured in plasma membrane preparations by determining, respectively, the
  • receptors that modulate cAMP are tested, it will be possible to use standard techniques for cAMP detection, such as competitive assays which quantitate [ ⁇ H]cAMP in the presence of unlabelled cAMP.
  • Certain receptors stimulate the activity of phospholipase C which stimulates the breakdown of phosphatidylinositol 4,5, bisphosphate to 1,4,5-IP3 (which mobilizes intracellular Ca++) and diacylglycerol (DAG) (which activates protein kinase C).
  • DAG diacylglycerol
  • cerevisiae pheromone signaling pathway is comprised of a protein kinase cascade composed of the products of the S7E71 , STE7, and FUS3/KSS1 genes (the latter pair are distinct and functionally redundant). Accordingly, phosphorylation and/or activation of members of this kinase cascade can be detected and used to quantitate receptor engagement.
  • Phosphotyrosine specific antibodies are available to measure increases in tyrosine phosphorylation and phospho-specific antibodies are commercially available (New England Biolabs, Beverly, MA). Modified methods for detecting receptor-mediated signal transduction exist and one of skill in the art will recognize suitable methods that may be used to substitute for the example methods listed.
  • an indicator gene can be used for detection.
  • an indicator gene is an unmodified endogenous gene.
  • the instant method can rely on detecting the transcriptional level of such pheromone system pathway responsive endogenous genes as the BARl or FUS1, FUS 2, mating factor, STE3 STE13, KEX1, STE2, STE6, STE7, SST2, or CHS1. (Appletauer and Zchstetter. 1989. ⁇ ur. J. Biochem. 181:243)
  • the present invention teaches engineering the cell such that: 1) growth arrest does not occur as a result of exogenous signal pathway activation (e.g., by inactivating the FAR1 gene); and/or 2) a selective growth advantage is conferred by activating the pathway (e.g., by transforming an auxotrophic mutant with a HIS3 gene under the control of a pheromone- responsive promoter, and applying selective conditions).
  • the promoter may be one which is repressed by the receptor pathway, thereby preventing expression of a product which is deleterious to the cell.
  • a receptor repressed promoter one screens for agonists by linking the promoter to a deleterious gene, and for antagonists, by linking it to a beneficial gene. Repression may be achieved by operably linking a receptor- induced promoter to a gene encoding mRNA which is antisense to at least a portion of the mRNA encoded by the marker gene
  • Repression may also be obtained by linking a receptor-induced promoter to a gene encoding a DNA binding repressor protein, and inco ⁇ orating a suitable operator site into the promoter or other suitable region of the marker gene.
  • the imidazoleglycerol phosphate dehydratase (IGP dehydratase) gene (HIS3) is preferred because it is both quite sensitive and can be selected over a broad range of expression levels.
  • the cell is auxotrophic for histidine (requires histidine for growth) in the absence of activation. Activation leads to synthesis of the enzyme and the cell becomes prototrophic for histidine (does not require histidine). Thus the selection is for growth in the absence of histidine. Since only a few molecules per cell of IGP dehydratase are required for histidine prototrophy, the assay is very sensitive.
  • test polypeptide library may be secreted into the periplasm by any of a number of exemplary mechanisms, depending on the nature of the expression system to which they are linked.
  • the peptide may be structurally linked to a yeast signal sequence, such as that present in the ⁇ -factor precursor, which directs secretion through the endoplasmic reticulum and Golgi apparatus.
  • the compounds to be tested in the subject assays can be derived from libraries. While the use of libraries of peptides is well established in the art, new techniques have been developed which have allowed the production of mixtures of other compounds, such as benzodiazepines (Bunin et al. 1992. J. Am. Chem. Soc. 114:10987; DeWitt et al. 1993. Proc. Nati. Acad. Sci. USA 90:6909) peptoids (Zuckermann. 1994. J. Med. Chem. 37:2678) oligocarbamates (Cho et al. 1993. Science. 261:1303), and hydantoins (DeWitt et al. supra).
  • Exemplary compounds which can be screened for activity include, but are not limited to, peptides, nucleic acids, carbohydrates, small organic molecules, and natural product extract libraries. In such embodiments, both compounds which agonize or antagonize the receptor- or channel-mediated signaling function can be selected and identified.
  • peptide libraries are systems which simultaneously display, in a form which permits interaction with a target, a highly diverse and numerous collection of peptides. These peptides may be presented in
  • the Ladner et al. patent, USSN 5,096,815, describes a method of identifying novel proteins or polypeptides with a desired DNA binding activity.
  • Semi-random 5 (“variegated") DNA encoding a large number of different potential binding proteins is introduced, in expressible form, into suitable yeast cells.
  • the target DNA sequence is inco ⁇ orated into a genetically engineered operon such that the binding of the protein or polypeptide will prevent expression of a gene product that is deleterious to the gene under selective conditions. Cells which survive the selective conditions are thus cells 0 which express a protein which binds the target DNA. While it is taught that yeast cells may be used for testing, bacterial cells are preferred.
  • the compounds tested are in the form of peptides from a peptide library.
  • the peptide library of the present invention takes the form of a cell culture, in which essentially each cell expresses one, and usually only one, peptide of the library. While the diversity of the library is maximized if each cell produces a peptide of a different sequence, it is usually prudent to construct the library so there is some redundancy.
  • the combinatorial peptides of the library can be expressed as is, or can be inco ⁇ orated into larger fusion proteins.
  • the fusion protein can provide, for example, stability against degradation or denaturation, as well as a secretion signal if secreted.
  • the polypeptide library is expressed as thioredoxin fusion proteins (see, for example, U.S. Patents 5,270,181 and 5,292,646; and PCT publication WO94/ 02502).
  • the combinatorial peptide can be attached one the terminus of the thioredoxin protein, or, for short peptide libraries, inserted into the so-called active loop.
  • the peptide library is a combinatorial library of polypeptides which are based at least in part on a known polypeptide sequence or a portion thereof (not a cDNA library). That is, the sequences of the library is semi- random, being derived by combinatorial mutagenesis of a known sequence. See, for example, Ladner et al. PCT publication WO 90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007-16010; Griffths et al. (1993) EMBO J 12:725-734; Clackson et al.
  • polypeptide(s) which are known ligands for a target receptor can be mutagenized by standard techniques to derive a variegated library of polypeptide sequences which can further be screened for agonists and/or antagonists.
  • the cells collectively produce a "peptide library", preferably including at least 10 ⁇ to 10? different peptides, so that diverse peptides may be simultaneously assayed for the ability to interact with the exogenous receptor.
  • a "peptide library” preferably including at least 10 ⁇ to 10? different peptides, so that diverse peptides may be simultaneously assayed for the ability to interact with the exogenous receptor.
  • at least some peptides of the peptide library are secreted into the periplasm, where they may interact with the "extracellular" binding site(s) of an exogenous receptor. They thus mimic more closely the clinical interaction of drugs with cellular receptors.
  • This embodiment optionally may be further improved (in assays not requiring pheromone secretion) by preventing pheromone secretion, and thereby avoiding competition between the peptide and the pheromone for signal peptidase and other components of the secretion system.
  • the peptides of the library are encoded by a mixture of DNA molecules of different sequence.
  • Each peptide-encoding DNA molecule is ligated with a vector DNA molecule and the resulting recombinant DNA molecule is introduced into a yeast cell. Since it is a matter of chance which peptide encoding DNA molecule is introduced into a particular cell, it is not predictable which peptide that cell will produce. However, based on a knowledge of the manner in which the mixture was prepared, one may make certain statistical predictions about the mixture of peptides in the peptide library.
  • the peptides of the library can be composed of constant and variable residues. If the nth residue is the same for all peptides of the library, it is said to be constant. If the nth residue varies, depending on the peptide in question, the residue is a variable one.
  • the peptides of the library will have at least one, and usually more than one, variable residue.
  • a variable residue may vary among any of two to all twenty of the genetically encoded amino acids; the variable residues of the peptide may vary in the same or different manner.
  • the frequency of occurrence of the allowed amino acids at a particular residue position may be the same or different.
  • the peptide may also have one or more constant residues.
  • the DNAs are synthesized a base at a time.
  • a suitable mixture of nucleotides is reacted with the nascent DNA, rather than the pure nucleotide reagent of conventional polynucleotide synthesis.
  • the second method provides more exact control over the amino acid variation.
  • trinucleotide reagents are prepared, each trinucleotide being a codon of one (and only one) of the amino acids to be featured in the peptide library.
  • a mixture is made of the appropriate trinucleotides and reacted with the nascent DNA.
  • test polypeptide library may be secreted into the periplasm by any of a number of exemplary mechanisms, depending on the nature of the expression system to which they are linked.
  • the peptide may be structurally linked to a yeast signal sequence, such as that present in the ⁇ -factor precursor, which directs secretion through the endoplasmic reticulum and Golgi apparatus. Since this is the same route that the receptor protein follows in its journey to the plasma membrane, opportunity exists in cells expressing both the receptor and the peptide library for a specific peptide to interact with the receptor during transit through the secretory pathway. This has been postulated to occur in mammalian cells exhibiting autocrine activation.
  • Such interaction could yield activation of the response pathway during transit, which would still allow identification of those cells expressing a peptide agonist.
  • this system would still be effective, since both the peptide antagonist and receptor would be delivered to the outside of the cell in concert.
  • those cells producing an antagonist would be selectable, since the peptide antagonist would be properly and timely situated to prevent the receptor from being stimulated by the externally applied agonist.
  • An alternative mechanism for delivering peptides to the periplasmic space is to use the ATP-dependent transporters of the STE6/MDR1 class.
  • This transport pathway and the signals that direct a protein or peptide to this pathway are not as well characterized as is the endoplasmic reticulum-based secretory pathway. Nonetheless, these transporters apparently can efficiently export certain peptides directly across the plasma membrane, without the peptides having to transit the ER/Golgi pathway. It is anticipated that at least a subset of peptides can be secreted through this pathway by expressing the library in context of the a-factor prosequence and terminal tetrapeptide.
  • agents identified in the subject assay can be formulated in pharmaceutical preparations for in vivo administration to an animal, preferably a human.
  • the compounds selected in the subject assay, or a pharmaceutically acceptable salt thereof may accordingly be formulated for administration with a biologically acceptable medium, such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.
  • a biologically acceptable medium such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.
  • the optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists.
  • biologically acceptable medium includes any and all solvents, dispersion media, and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation.
  • the use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the activity of the compound, its use in the pharmaceutical preparation of the invention is contemplated. Suitable vehicles and their formulation inclusive of other proteins are described, for example, in the book Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, Pa., US A 1985).
  • the AGS proteins can be used as "bait proteins" in a two-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with AGS (" AGS-binding proteins" or " AGS-bp”) and modulate AGS protein activity.
  • AGS-binding proteins or " AGS-bp”
  • Such AGS- binding proteins are also likely to be involved in the propagation of signals by the AGS proteins as, for example, upstream or downstream elements of the pheromone response pathway.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, (“bait”), the gene that codes for AGS, is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a known transcription factor e.g., GAL-4
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait” and the “prey” proteins are able to interact, in vivo, forming an AGS- dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with AGS.
  • a reporter gene e.g., LacZ
  • Another aspect of the invention is directed to a yeast-based counter-screen for pheromone pathway inhibitors.
  • This screen is based on a yeast cell constitutively expressing a receptor-independent activator (e.g. , AGS 1 ) of the yeast pheromone response pathway such that a lethal marker gene is expressed (e.g., Canl).
  • a cDNA library of interest cloned into an appropriate vector (e.g., pYES2), is introduced into this strain to identify cDNAs that, when expressed, counteract the activators ability to induce the expression of the lethal marker gene.
  • This screen therefore, can rapidly identify proteins that both directly and indirectly regulate activators of the pheromone response pathway.
  • Modulators of AGS protein activity and/or AGS nucleic acid expression identified according to these drug screening assays can be used to treat, for example, diseases or disorders characterized by excessive or insufficient G-protein mediated signal transduction.
  • diseases or disorders which can be treated using modulators of AGS protein activity and/or nucleic acid expression include proliferative disorders and/or diseases.
  • Support for the use of AGS modulators in treating proliferative disorders can be found in the fact that, for example, oncogenic mutations in ras-like G proteins have been implicated in approximately 30%> of human cancers, including 90% of pancreatic and 50% of colon cancers (Bos ( 1988) Mutat. Res. 195:255- 271; Bos (1989) Cancer Res.
  • modulators of AGS function are likely to have pharmaceutical applications.
  • Methods of treatment include the steps of administering the AGS molecules of the present invention or modulators of AGS protein activity and/or nucleic acid expression, e.g., in a pharmaceutical composition as described above, to a subject in need of such treatment.
  • the invention further provides a method for detecting the presence of AGS in a biological sample.
  • the method involves contacting the biological sample with a compound or an agent capable of detecting AGS protein or mRNA such that the presence of AGS is detected in the biological sample.
  • a preferred agent for detecting AGS mRNA is a labeled or labelable nucleic acid probe capable of hybridizing to AGS mRNA.
  • the nucleic acid probe can be, for example, the AGS cDNA of SEQ ID NO: 1, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to AGS mRNA.
  • a preferred agent for detecting AGS protein is a labeled or labelable antibody capable of binding to AGS protein.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used.
  • the term "labeled or labelable", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (e.g., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect AGS mRNA or protein in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of AGS mRNA include Northern hybridizations and in situ hybridizations.
  • AGS protein can be detected in vivo in a subject by introducing into the subject a labeled anti- AGS antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample is a cell sample, a tissue section, for example, a freeze-dried or fresh frozen section of tissue removed from a patient, or a biological fluid obtained from a subject.
  • kits for detecting the presence of AGS in a biological sample can comprise a labeled or labelable compound or agent capable of detecting AGS protein or mRNA in a biological sample; means for determining the amount of AGS in the sample; and means for comparing the amount of AGS in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect AGS mRNA or protein.
  • Additional methods of the invention include methods for treating a subject having a disorder characterized by aberrant AGS activity or expression. These methods include administering to the subject an AGS modulator such that treatment of the subject occurs.
  • the terms "treating” or “treatment”, as used herein, refer to reduction or alleviation of at least one adverse effect or symptom of a disease or disorder, e.g., a disease or disorder characterized by or associated with abnormal or aberrant AGS protein activity or AGS nucleic acid expression.
  • an AGS modulator is a molecule which can modulate AGS nucleic acid expression and/or AGS protein activity.
  • an AGS modulator can modulate, e.g., upregulate (activate) or downregulate (suppress), AGS nucleic acid expression.
  • an AGS modulator can modulate (e.g., stimulate or inhibit) AGS protein activity. If it is desirable to treat a disease or disorder characterized by (or associated with) aberrant or abnormal (non-wild-type) AGS nucleic acid expression and/or AGS protein activity by inhibiting AGS nucleic acid expression, an AGS modulator can be an antisense molecule, e.g., a ribozyme, as described herein.
  • antisense molecules which can be used to inhibit AGS nucleic acid expression include antisense molecules which are complementary to a portion of the 5' untranslated region of SEQ ID NO: 3, which also includes the start codon and antisense molecules which are complementary to a portion of the 3' untranslated region of SEQ ID NO:3.
  • AN AGS modulator which inhibits AGS nucleic acid expression can also be a small molecule or other drug, e.g., a small molecule or drug identified using the screening assays described herein, which inhibits AGS nucleic acid expression.
  • an AGS modulator can be, for example, a nucleic acid molecule encoding AGS (e.g., a nucleic acid molecule comprising a nucleotide sequence homologous to the nucleotide sequence of SEQ ID NO: 1) or a small molecule or other drug, e.g., a small molecule (peptide) or drug identified using the screening assays described herein, which stimulates AGS nucleic acid expression.
  • an AGS modulator can be an anti- AGS antibody or a small molecule or other drug, e.g., a small molecule or drug identified using the screening assays described herein, which inhibits AGS protein activity.
  • an AGS modulator can be an active AGS protein or portion thereof (e.g., an AGS protein or portion thereof having an amino acid sequence which is homologous to the amino acid sequence of SEQ ID NO:2) or a small molecule or other drug, e.g., a small molecule or drug identified using the screening assays described herein, which stimulates AGS protein activity.
  • an AGS modulator is an indirect modulator (e.g., an indirect activator or inhibitor of AGS protein expression and/or activity.
  • an AGS modulator is an indirect modulator which increases the expression and/or activity of an AGS-specific transcription factor.
  • an AGS modulator is an indirect modulator which decreases the expression and/or activity of an AGS-specific transcription factor.
  • Other aspects of the invention pertain to methods for modulating a cell associated activity.
  • a cell associated activity refers to a normal or abnormal activity or function of a cell. Examples of cell associated activities include proliferation, migration, differentiation, production or secretion of molecules, such as proteins, and cell survival.
  • the cell-associated activity is mediated by a G-protein signaling pathway.
  • altered or modulated refers to a change, e.g., an increase or decrease, of a cell associated activity.
  • the agent stimulates AGS protein activity or AGS nucleic acid expression.
  • stimulatory agents include an active AGS protein, a nucleic acid molecule encoding AGS that has been introduced into the cell, and a modulatory agent which stimulates AGS protein activity or AGS nucleic acid expression and which is identified using the drug screening assays described herein.
  • the agent inhibits AGS protein activity or AGS nucleic acid expression.
  • inhibitory agents include an antisense AGS nucleic acid molecule, an anti- AGS antibody, and a modulatory agent which inhibits AGS protein activity or AGS nucleic acid expression and which is identified using the drug screening assays described herein.
  • modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).
  • the modulatory methods are performed in vivo, e.g., the cell is present within a subject, e.g., a mammal, e.g., a human, and the subject has a disorder or disease characterized by or associated with abnormal or aberrant AGS activity or expression.
  • a nucleic acid molecule, a protein, an AGS modulator etc. used in the methods oftreatment can be inco ⁇ orated into an appropriate pharmaceutical composition described herein and administered to the subject through a route which allows the molecule, protein, modulator etc. to perform its intended function. Examples of routes of administration are also described herein. Particular AGS inhibitory agents and AGS stimulatory agents are described further below.
  • AGS activity is inhibited in a cell by contacting the cell with an inhibitory agent.
  • Inhibitory agents of the invention can be, for example, intracellular binding molecules that act to inhibit the expression or activity of AGS.
  • intracellular binding molecule is intended to include molecules that act intracellularly to inhibit the expression or activity of a protein by binding to the protein itself, to a nucleic acid (e.g., an mRNA molecule) that encodes the protein or to a target with which the protein indirectly interacts.
  • intracellular binding molecules examples include polypeptides that directly or indirectly bind an AGS molecule or target molecule, antisense AGS nucleic acid molecules (e.g., to inhibit translation of AGS mRNA), intracellular anti- AGS antibodies (e.g., to inhibit the activity of AGS protein) and chemical inhibitors of the AGS protein.
  • an inhibitor of an AGS or AGS-related molecule is identified by modifying one of the above-mentioned assays to activate a reporter gene that prevents growth (e.g., CAN1).
  • a reporter gene e.g., CAN1
  • candidate inhibitors of an AGS or AGS-related activator that can block the AGS induction of the growth inhibiting gene can be rapidly screened and identified (Fig. 2b).
  • an identified inhibitor of an AGS molecule is the RGS5 polypeptide (SEQ ID NO: 25) which either directly or indirectly down-regulates AGS activity.
  • an inhibitory agent of the invention is an antisense nucleic acid molecule that is complementary to a gene encoding AGS or to a portion of said gene, or a recombinant expression vector encoding said antisense nucleic acid molecule.
  • antisense nucleic acids to downregulate the expression of a particular protein in a cell is well known in the art (see e.g., Weintraub, H. et al, Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986; Askari, F.K. and McDonnell, W.M. (1996) N. Eng. J. Med. 334:316- 318; Bennett, M.R.
  • an antisense nucleic acid is designed so as to be complementary to a region preceding or spanning the initiation codon on the coding strand or in the 3' untranslated region of an mRNA.
  • An antisense nucleic acid for inhibiting the expression of AGS protein in a cell can be designed based upon the nucleotide sequence encoding the AGS protein (e.g., SEQ ID NO: 1), constructed according to the rules of Watson and Crick base pairing (e.g., as described above in subsection I).
  • an antisense nucleic acid can exist in a variety of different forms.
  • the antisense nucleic acid can be an oligonucleotide that is complementary to only a portion of an AGS gene.
  • An antisense oligonucleotides can be constructed using chemical synthesis procedures known in the art.e.g. To inhibit AGS expression in cells in culture, one or more antisense oligonucleotides can be added to cells in culture media, typically at about 200 ⁇ g oligonucleotide/ml.
  • an antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (e.g., nucleic acid transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).e.g., nucleic acid transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).
  • an antisense nucleic acid for use as an inhibitory agent is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region (for reviews on ribozymes see e.g., Ohkawa, J. et al. (1995) J. Biochem. 118:251-258; NASAdsson, S.T. and Eckstein, F. (1995) Trends Biotechnol L3:286-289; Rossi, J.J. (1995) Trends Biotechnol 13:301-306; Kiehntopf, M.
  • a ribozyme having specificity for AGS mRNA can be designed based upon the nucleotide sequence of the AGS cDNA.
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the base sequence of the active site is complementary to the base sequence to be cleaved in an AGS mRNA. See for example U.S. Patent Nos. 4,987,071 and 5,116,742, both by Cech et al.
  • AGS mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See for example Bartel, D. and 5 Szostak, J.W. (1993) Science 261: 1411-1418.
  • inhibitory agent that can be used to inhibit the expression and/or activity of AGS in a cell is an intracellular antibody specific for the AGS protein.
  • intracellular antibodies to inhibit protein function in a cell is known in the art (see e.g., Carlson, J. R. (1988) Mol. Cell. Biol. 8:2638-2646; Biocca, S. et al. (1990) EMBO
  • a recombinant expression vector which encodes the antibody chains in a form such that, upon introduction of the vector into a cell, the antibody chains are expressed as a functional antibody in an intracellular compartment of the cell.
  • an intracellular antibody 5 that specifically binds the AGS protein is expressed in the cytoplasm of the cell.
  • antibody light and heavy chain cDNAs encoding antibody chains specific for the target protein of interest e.g., AGS, are isolated, typically from a hybridoma that secretes a monoclonal antibody specific for the AGS protein.
  • Hybridomas secreting anti-AGS monoclonal antibodies, or 0 recombinant anti-AGS monoclonal antibodies can be prepared as described above.
  • a monoclonal antibody specific for AGS protein e.g., either a hybridoma-derived monoclonal antibody or a recombinant antibody from a combinatorial library
  • DNAs encoding the light and heavy chains of the monoclonal antibody are isolated by standard molecular biology techniques.
  • light and heavy chain cDNAs can be obtained, for example, by PCR amplification or cDNA library screening.
  • cDNA encoding the light and heavy chains can be recovered from the display package (e.g., phage) isolated during the library screening process.
  • Nucleotide sequences of antibody light and heavy chain genes from which PCR primers or cDNA library probes can be prepared are known in the art. For example, many such sequences are disclosed in Kabat, E.A., et al. (1991) Sequences of Proteins of
  • an intracellular antibody expression vector can encode an intracellular antibody in one of several different forms. For example, in one embodiment, the vector encodes full-length antibody light and heavy chains such that a full-length antibody is expressed intracellularly.
  • the vector encodes a full-length light chain but only the VH/CHl region of the heavy chain such that a Fab fragment is expressed intracellularly.
  • the vector encodes a single chain antibody (scFv) wherein the variable regions of the light and heavy chains are linked by a flexible peptide linker (e.g., (Gly4Ser)3) and expressed as a single chain molecule.
  • scFv single chain antibody
  • the expression vector encoding the anti-AGS intracellular antibody is introduced into the cell by standard transfection methods, as discussed hereinbefore.
  • AGS activity is stimulated in a cell by contacting the cell with a stimulatory agent.
  • stimulatory agents include active AGS protein and nucleic acid molecules encoding AGS that are introduced into the cell to increase AGS activity in the cell.
  • a preferred stimulatory agent is a nucleic acid molecule encoding an AGS protein, wherein the nucleic acid molecule is introduced into the cell in a form suitable for expression of the active AGS protein in the cell.
  • an AGS-encoding DNA is first introduced into a recombinant expression vector using standard molecular biology techniques, as described herein.
  • AN AGS-encoding DNA can be obtained, for example, by amplification using the polymerase chain reaction (PCR), using primers based on the AGS nucleotide sequence. Following isolation or amplification of AGS- encoding DNA, the DNA fragment is introduced into an expression vector and transfected into target cells by standard methods, as described herein.
  • Other stimulatory agents that can be used to stimulate the activity of an AGS protein are chemical compounds that stimulate AGS activity in cells, such as compounds that directly stimulate AGS protein and compounds that promote the interaction between AGS and target molecules. Such compounds can be identified using screening assays that select for such compounds, as described in detail above.
  • the modulatory methods of the invention can be performed in vitro (e.g., by culturing the cell with the agent or by introducing the agent into cells in culture) or, alternatively, in vivo (e.g., by administering the agent to a subject or by introducing the agent into cells of a subject, such as by gene therapy).
  • cells can be obtained from a subject by standard methods and incubated (e.g. , cultured) in vitro with a modulatory agent of the invention to modulate AGS activity in the cells. If desired, cells treated in vitro with a modulatory agent of the invention can be readministered to the subject.
  • the modulatory agent can be administered to the subject such that AGS activity in cells of the subject is modulated.
  • the term "subject" is intended to include living organisms in which an AGS-dependent cellular response can be elicited. Preferred subjects are mammals. Examples of subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, goats and sheep.
  • nucleic acids including recombinant expression vectors encoding AGS protein, antisense RNA and intracellular antibodies
  • the agents can be introduced into cells of the subject using methods known in the art for introducing nucleic acid (e.g., DNA) into cells in vivo. Examples of such methods encompass both non-viral and viral methods, including:
  • Naked DNA can be introduced into cells in vivo by directly injecting the DNA into the cells (see e.g., Acsadi et al. (1991) N ⁇ twre 332:815-818; Wolff et al. (1990) Science 247:1465-1468).
  • a delivery apparatus e.g., a "gene gun” for injecting D ⁇ A into cells in vivo can be used.
  • Such an apparatus is commercially available (e.g., from BioRad).
  • Cationic Lipids Naked DNA can be introduced into cells in vivo by complexing the DNA with cationic lipids or encapsulating the DNA in cationic liposomes.
  • suitable cationic lipid formulations include N-[-l-(2,3- dioleoyloxy)propyl]N,N,N-triethylammonium chloride (DOTMA) and a 1 : 1 molar ratio of l,2-dimyristyloxy-propyl-3-dimethylhydroxyethylammonium bromide (DMRIE) and dioleoyl phosphatidylethanolamine (DOPE) (see e.g., Logan, J.J. et al. (1995) Gene Therapy 2:38-49; San, H. et al. (1993) Human Gene Therapy 4:781-788).
  • DOTMA N-[-l-(2,3- dioleoyloxy)propyl]N,N,N-trieth
  • Naked DNA can also be introduced into cells in vivo by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor (see for example Wu, G. and Wu, CH. (1988) J. Biol. Chem. 263:14621; Wilson et al. (1992) J. Biol. Chem. 267:963-967; and U.S. Patent No. 5,166,320). Binding of the DNA-ligand complex to the receptor facilitates uptake of the DNA by receptor-mediated endocytosis.
  • a cation such as polylysine
  • a DNA-ligand complex linked to adenovirus capsids which naturally disrupt endosomes, thereby releasing material into the cytoplasm can be used to avoid degradation of the complex by intracellular lysosomes (see for example Curiel et al. (1991) Proc. Nati Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Nati Acad. Sci. USA 90:2122-2126).
  • Retroviruses Defective retroviruses are well characterized for use in gene transfer for gene therapy pu ⁇ oses (for a review see Miller, A.D. (1990) Blood 76:271).
  • a recombinant retrovirus can be constructed having a nucleotide sequences of interest inco ⁇ orated into the retroviral genome. Additionally, portions of the retroviral genome can be removed to render the retrovirus replication defective. The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M.
  • retroviruses examples include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art.
  • suitable packaging virus lines include ⁇ Crip, ⁇ Cre, ⁇ 2 and ⁇ Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al.
  • Retroviral vectors require target cell division in order for the retroviral genome (and foreign nucleic acid inserted into it) to be integrated into the host genome to stably introduce nucleic acid into the cell. Thus, it may be necessary to stimulate replication of the target cell.
  • Adenoviruses The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for example Berkner et al.
  • adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus are well known to those skilled in the art. Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al.
  • introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA).
  • the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al. cited supra; Haj- Ahmand and Graham (1986) J. Virol. 57:267).
  • Most replication-defective adenoviral vectors currently in use are deleted for all or parts of the viral El and E3 genes but retain as much as 80 % of the adenoviral genetic material.
  • Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a he ⁇ es virus, as a helper virus for efficient replication and a productive life cycle.
  • AAV Adeno-associated virus
  • adenovirus or a he ⁇ es virus
  • helper virus for efficient replication and a productive life cycle.
  • It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol.
  • AAV vector such as that described in Tratschin et al. (1985) Mol Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells.
  • a variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Nati Acad. Sci. USA 8L6466-6470; Tratschin et al. (1985) Mol. Cell. Biol.
  • DNA introduced into a cell can be detected by a filter hybridization technique (e.g., Southern blotting) and RNA produced by transcription of introduced DNA can be detected, for example, by Northern blotting, RNase protection or reverse transcriptase-polymerase chain reaction (RT-PCR).
  • RNA produced by transcription of introduced DNA can be detected, for example, by Northern blotting, RNase protection or reverse transcriptase-polymerase chain reaction (RT-PCR).
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • the gene product can be detected by an appropriate assay, for example by immunological detection of a produced protein, such as with a specific antibody, or by a functional assay to detect a functional activity of the gene product.
  • a retroviral expression vector encoding AGS is used to express AGS protein in cells in vivo, to thereby stimulate AGS protein activity in vivo.
  • retroviral vectors can be prepared according to standard methods known in the art (discussed further above).
  • a modulatory agent such as a chemical compound
  • Such compositions typically comprise the modulatory agent and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents, and the like, compatible with pharmaceutical administration.
  • compositions can be prepared as described above. This invention is further illustrated by the following examples which should not be construed as limiting.
  • Plasmid CP1127 carrying the promoter sequences and first 41 amino acid codons oiGPAl, was prepared by ligation of a sequence encompassing nucleotides -200 to +100 oiGPAl (where translational start is +1) to pRS405 (Sikorski and Hieter (1989) Genetics 122:19).
  • the amplified product was digested with Sacl and Sail., then ligated into SacllSaR digested CP1127.
  • a glycine to alanine alteration at codon 204 of G ⁇ i2 in CP1183 was introduced using Stratagene's QuickChange kit and mutagenic oligos SEQ ID NO: 6 and SEQ ID NO: 7 creating plasmid CP5533. Sequences encoding ⁇ -galactosidase (lacZ) were introduced downstream of the fusl promoter on plasmid pRS424 (Sikorski and Hieter (1989) Genetics 122:19) to create CP1584. Plasmid pSM187, with a 4.3 kb DNA fragment carrying the STE14 gene flanked by BamHI sites, was kindly provided by S. Michaelis.
  • the vector pY ⁇ X4Ti which allows for copper inducible expression in yeast of sequences fused at the N-terminus with GST, was purchased from Amrad Biotech.
  • Vector pGEX-KG which allows for IPTG inducible expression in bacteria of sequences fused at the N-terminus with GST (Guan and Dixon (1991), Anal. Biochem. 192, 262- 267). Similar vectors are also commercially available.
  • the vectors pcDNA3.1HisC and pBlueBacHis2A which allow for expression of N-terminal hexahistidine fusion proteins, were purchased from Invitrogen.
  • the vector pIND which allows for inducible expression from mammalian cells, was purchased from Invitrogen.
  • the cDNA for gene AGS was amplified from pYES2-AGS by PCR using the oligo pair provided in SEQ ID NOS: 8 and 9, cleaved with BamHI and EcoRI , and ligated into if ⁇ /wHI/EeoRI digested pY ⁇ X4Ti, pGEX-KG, pcDNA3.1HisC, pBlueBacHis2A and pIND.
  • the coding sequence for Cdc42 was amplified by PCR from yeast genomic DNA using the oligo pair provided in SEQ ID NOS: 10 and 11, cleaved with BamHI and EcoRI, and ligated into E ⁇ HI/EcoRI digested pG ⁇ X-KG.
  • Single glycine to valine alterations at codons 31 and 36, and a glycine to alanine alteration at codon 81, were constructed using the Stratagene QuickChange kit and protocol and the respective mutagenic oligo pairs provided in S ⁇ Q ID NOS: 12 and 13, 14 and 15, and 16 and 17.
  • the plasmid pcDNA3.1HisC-AGS served as a template for mutagenesis. Following mutagenesis, AGS sequences were excised with Bam HI and Eco RI for subcloning into vectors described above. Bam HI and Eco RI sites were introduced via PCR, for example the Bam HI site was introduced using the primer set forth as S ⁇ Q ID NO: 8 and the Eco RI site, introduced using the primer set forth as S ⁇ Q ID NO:9. Automated dideoxy sequencing was used to verify the correct construction of all plasmids used in the examples.
  • Epistasis tests Epistasis tests - Epistasis analysis on yeast strains was performed by introducing the pYES2 vector alone or AGS carried on the pYES2 vector into the indicated strains. Strains were grown to saturation in liquid sensitive sucrose medium. Approximately 2000 cells from each transformant were spotted onto selective sucrose medium (to determine viability) and selective glucose and galactose medium lacking histidine and containing ImM AT. Plates were incubated at 30° for 2 days prior to photographing. ⁇ -galactosidase assays - Indicated yeast strains carrying a plasmid-borne FUS1- lacZ reporter construct were grown for 16-18 hours in selective glucose or galactose medium. Cells were harvested in log phase.
  • DMEM Dulbecco's minimal essential medium
  • AGS (from the CMV promoter in pcDNA3.1-HisC) was monitored in Neomycin resistant clones by RNA blot analysis using the full length cDNA coding sequence of AGS as probe (labeled with ⁇ -[ J 37 , P]-deoxycytidine triphosphate as described above).
  • Plasmid CP5336 carrying an episomal copy oiSTE14, was also introduced into the yeast strains. For copper induction, cells were grown at 30° to a density of 5 x 10 cells/ml and treated with 0.1 mM 1SO4 for 6 hr. Cells were
  • recombinant phage carrying the pBlueBacHisA-AGS construct were used to infect cultures of Sf9 cells. 2-3 d post- infection, cells were harvested by centrifugation and whole cell extracts prepared by denaturation at 100° for 5 min in denaturation buffer. Stable transfectants of HEK293 cells carrying pcDNA3.1 -HisC-AGS were cultured to -80% confluency in DMEM and harvested by scraping in the presence of PBS.
  • GST proteins were measured by staining membranes with 0.2% amido black. Membranes were blocked in 5% dry milk (w/v) in Tris-buffered saline, 0.1% Tween-20 (TBS-T) and probed with either a 1 :5,000 dilution of anti-Xpress antisera (Invitrogen ® ) or a 1 :2,000 dilution of anti-Ste4 antisera for 2 hr at 25°. Blots were washed extensively with TBS-T and probed with a 1 : 10,000 dilution of horseradish peroxidase-conjugated donkey anti-mouse or anti-rabbit antiserum (Amersham®) for 1 hr at 25°. After washing with TBS-T, blots were developed using the chemiluminescent substrate from Pierce ® according to the manufacturers protocol.
  • GTP ⁇ S Binding Assays A 6 pmol amount of purified His 6 -G ⁇ i2 or myristoylated Gail was incubated for 30 min at 25° either alone or with 170 pmol GST or GST-AGS 1 in the presence of 1.4 nmol GDP in a total volume of 280 ⁇ l assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.6 mM ethylenediaminetetraacetic acid, 5 mM MgCl 2 , 0.01% Thesit).
  • Thawed pellets were lysed by vortexing with beads in 50 mM Tris, pH 7.4, 20 mM MgCl 2 , 50 mM NaCl, 0.5% Triton X-100, 1 mM DTT, protease and phosphatase inhibitors. Samples were centrifuged 10 min at 10000 ⁇ ra and supernatants bound to 150 ⁇ l glutathione sepharose for 2 hr at 4°.
  • GPCR G-protein coupled receptor
  • PAK p-21 activated protein kinase
  • MAP kinase mitogen activated protein kinase
  • RGS regulator of G protein signaling
  • PCR polymerase chain reaction
  • IGP imidazoleglycerolphosphate
  • AT 2-aminotriazole
  • SDS sodium dodecyl sulfate
  • PVDF polyvinylidene difluoride
  • GST glutathione-S-transferase
  • TBS-T Tris-buffered saline containing 0.1% Tween-20
  • DTT dithiothreitol
  • GTP ⁇ S guanosine-5'-o-(3- thiotriphosphate
  • CPRG chlorophenolred- ⁇ -D-galactopyranoside
  • IPTG isopropyl ⁇ -D-thiogalactopyranoside.
  • a his3 strain carrying the fusl-HIS3 construct can be made conditional for growth upon pheromone pathway activation by culturing this strain in medium lacking histidine.
  • f ⁇ sl promoter sequences can be fused to ⁇ -galactosidase gene (lacZ) sequences, and pheromone pathway activation quantitated by colorimetric assays.
  • lacZ ⁇ -galactosidase gene
  • the fits 1 promoter activity of these constructs is often not entirely repressed in the absence of pheromone pathway activation (King et al. ( 1990) Science 250: 121-123).
  • low levels of AT can be added to the culture medium to fully repress HIS3 activity (Manfredi et al. (1996) Mol. Cell. Biol. 16:4700-4709).
  • EXAMPLE 2 Screening of a Human Liver cDNA Library for Pheromone
  • a cDNA library derived from adult human liver was chosen.
  • the human liver library was chosen as a potential general source of regulators.
  • This cDNA library was ligated into the high-copy 2 ⁇ -based yeast vector pYES2. Sequences were ligated downstream of the yeast GAL1 promoter, making expression of cDNAs conditional upon the addition of galactose to the growth medium.
  • the combination of a high-copy number vector and a strong galactose- inducible promoter increases the ability to detect even weak activators of the pheromone response pathway.
  • a schematic of the pathway and yeast screen is shown respectively in Figs. 1A, IB, and 2.
  • Yeast strain CY1316/1183 was transformed with each of the libraries and transformation mixtures plated on a series of agar plates containing sucrose to select for primary transformants.
  • Sucrose was used as a carbon source rather than glucose, as glucose actively represses the GAL1 promoter (Schneider et al. (1991) Meth. Enz.
  • Plasmids were isolated from strains showing galactose-dependent growth and used to re-transform na ⁇ ve CY1316/1183 strains. Transformants were again plated onto sucrose medium, then replica-plated onto both galactose and glucose medium lacking histidine and containing 1 mM AT. Those plasmids that conferred universal galactose- dependent growth on this medium were considered to carry cDNAs encoding pheromone pathway activators. Eight such plasmids were identified from these screens, each containing the same open reading frame downstream of the GALl promoter (designated LI, see Table 2).
  • CY1316/1183 transformants were screened for colony growth on galactose medium lacking histidine and containing aminotriazole (Primary
  • Ijj ORF downstream of the yeast GAI1 promoter are designated LI with their frequency of occurrence in parenthesis. See text for details. c m
  • Strains carrying pYES2-AGSl exhibit galactose-dependent growth as well as galactose-dependent reporter gene activity (in strains also carrying the FUSlp-lacZ construct).
  • the galactose-dependent growth phenotype conferred upon strain C Yl 316/CP 1183 by pYES2-AGS was further characterized by genetic analysis.
  • PYES2-AGS fails to confer galactose-dependent growth on medium lacking histidine and containing 1 mM AT to strains CY4600, CY 12444 and CY12970, containing, respectively genomic disruptions o STE4, STE5, and STE20 (see Table 1). This lack of growth is consistent with AGS activation of signaling occurring through the pheromone response pathway (G ⁇ -» PAK — > MAPK cascade). Neither were the isolates of strains carrying an episomal copy of human RGS4 under control of the constitutive PGKl promoter able to confer galactose-dependent growth.
  • Plasmid pYES2-AGS was also unable to confer galactose-dependent growth on medium lacking histidine and containing 1 mM AT to strain CY1316/CP5533, carrying the G204A allele of GPA1 (f.4 J ⁇ -Gai2.
  • strains CY1316/pYES2 and CY1316/pYES2-AGS carrying plasmid CP5533 (G204A)
  • strains CY1316/pYES2 and CY1316/pYES2-AGS carrying plasmid CPU 83 (WT) were grown on sucrose medium for 2 d.
  • Equal numbers of cells were then resuspended in sterile H2O and spotted onto galactose medium lacking histidine and containing ImM AT. Cells were grown 2 d at 30° prior to photography to visualize growth.
  • G204A Neither the isolates of strains CY1316/pYES2 or CY1316/pYES2-AGS carrying plasmid CP5533 (G204A), exhibited growth above that seen with control strain 5 CY1316/pYES2 carrying plasmid CP1183 (WT). By contrast, positive control strain CY1316/pYES2-AGS carrying plasmid CP1183 (WT) exhibited significant colony growth.
  • the G204A mutation is analogous to the G226A mutation in G s which renders this protein unable to form a high affinity Mg ⁇ # GTP complex (Lee et al. (1992) J. Biol. Chem. 267:1212-1218). The G204A mutation in the chimeric Gpal( ⁇
  • the G ⁇ selectivity profile of AGSl was determined.
  • G ⁇ selectivity profile for AGSl in yeast strains carrying afuslp- l ⁇ cZ reporter construct and expressing different mammalian G ⁇ constructs indicated G ⁇ i specificity.
  • Expression of AGSl resulted in high levels of ⁇ -galactosidase activity in strains expressing GPA i ( 1) -G ⁇ i2 or G/ 7, M1) -G ⁇ i3, but not in strains expressing Gas, GA47 (M1) -Gal6 or GPA1 itself (Table 3).
  • AGSl as a receptor-independent mammalian activator of the yeast pheromone response pathway
  • a novel screen was devised to identify pheromone pathway inhibitors.
  • a strain was created that had its pheromone pathway constitutively activated by AGS 1.
  • a human liver cDNA library in vector pYES2 was introduced into this strain in an attempt to identify cDNAs that, when expressed, counteracted AGS 1 function. This screen, therefore, can identify proteins that both directly and indirectly regulate AGS 1 activity.
  • the marker gene CAN1 was employed which encodes a yeast arginine permease that can transport the arginine analog canavanine (Sikorski (1991) Meth. Enz. 194:302-318). Accordingly, cells expressing Canl and cultured in the absence of arginine can transport canavanine and inco ⁇ orate it into nascent proteins, leading to cell death.
  • CAN1 coding sequences downstream of the FUSl promoter and introducing it into yeast strain CY1141 (Table 1), this strain can be made conditionally non-viable upon pheromone pathway activation.
  • pYES2-RGS4 conferred galactose-dependent growth on medium lacking arginine and containing canavanine.
  • the amount of canavanine used in this test (200 ⁇ g/ml medium) was determined to be optimal in suppressing the growth of pYES2 vector transformants without causing general lethality. Therefore the strain was transformed with the human liver cDNA library and a inhibitor screen analogous to the activator screen was carried out.
  • a schematic of the inhibitor screen is shown in Figure 2 and the screen characteristics are shown in Table 2.
  • AGS The open reading frame of the 8 isolated clones encodes a 281 amino acid protein with homology to small ras-like G proteins. This newly cloned protein was termed AGS.
  • RSR1/BUD1 45%> amino acid identity within amino acids 25- 125 of AGS
  • RAS1 and RAS2 43% amino acid identity within amino acids 25- 125 of AGS.
  • AGS is clearly a member of the ras superfamily, it has several unique structural features, including both N- and C-terminal extensions not seen in most small G proteins. AGS also has alterations in several amino acids that are normally highly conserved in all small G proteins. Indeed, AGS shares alterations at three key amino acid residues with the recently identified G proteins Rndl, Rnd2 and RhoE/Rnd3 (Foster et al. (1996) Mol. Cell. Biol. 16:2689-2699; Nobes et al. (1998) J. Cell Biol. 141:187- 197) (see Figure 5).
  • RhoE was shown to be deficient in GTP hydrolysis activity, and this deficiency could be restored by alteration of these three amino acids to their highly conserved counte ⁇ arts (Foster, R. et al. (1996) Mol. Cell. Biol. .16:2689-2699).
  • AGS shares these three amino acid alterations with Rndl, Rnd2 and RhoE/Rnd3, it does not share the consensus rho effector (amino acids 42-50 in Rndl) or rho insert (amino acids 132-140 in Rndl) domains of these proteins.
  • AGS therefore, does not appear to be a member of the rho family of ras-like proteins.
  • a series of point mutations were created in AGS to analyze their effects on pheromone pathway activation, as measured by galactose-dependent growth in the absence of histidine. Mutations at conserved glycine residues 31 and 36, which are predicted to be in the P site, render AGS unable to confer histidine prototrophy to cells. Su ⁇ risingly, mutation of another conserved glycine in the G' site, glycine-81, does not appear to affect the function of AGS, even with growth on medium containing 20 mM AT. This glycine mutation is analogous to the G(226)A mutation of Gas and the G204A mutation of G ⁇ i2, and would normally be predicted to by an inactivating mutation.
  • mutants listed below in table 4 were tested in the vector pYES2 in CYl 141 on medium lacking histidine and containing 1 mM aminotriazole as described above.
  • the ⁇ -galactosidase activities (relative to wild-type AGSl) of all of the mutants tested are as follows.
  • the G(31 )V mutant carried a glycine to valine alteration at residue 31 (G31 V), an absolutely conserved residue in the P-loop of monomeric G proteins that makes critical contacts with the ⁇ - and ⁇ -phosphates of both GDP and GTP (Valencia et al. (1991) Biochemistry 30:4637-4648).
  • the C(278)S mutant carried a cysteine to serine alteration at residue 278 (C278S) within the C-terminal CAAX box. This cysteine is a major site of lipid modification for most members of the ras superfamily.
  • EXAMPLE 7 Effect of STE14 on AGS Function
  • the yeast strain used in the isolation of AGS contained a genomic disruption of the STE14 locus, which encodes a farnesyl cysteinexarboxyl methyltransferase known to carboxymethylate the yeast G ⁇ , Stel8, Rasl and Ras2 proteins as well as a-factor (Hrycyna and Clarke (1990) Mol. Cell. Biol. 10:5071-5076; Marr et al. (1990) J. Biol. Chem. 265:20057-20060; Hrycyna et al. (1991) EMBO J. K): 1699- 1709).
  • stel4 Disruption of stel4 has been found to reduce background signaling through the pheromone response pathway in strains carrying chimeric G ⁇ constructs.
  • AGS appears to encode a small ras-related G protein with a C-terminal CaaX box sequence consistent with farnesylation, and because small G proteins are often carboxymethylated after farnesylation, we examined the effect of addition of an episomal copy oiSTE14 on AGS function. STE14 gene expression appeared to enhance the fits 1-HIS3 mediated growth of strains expressing AGS, though an increase in background growth on glucose medium was also evident.
  • EXAMPLE 8 Tissue Expression of AGS mRNA The tissue-specific expression of AGS in mRNAs isolated from various human tissues was measured. A 32 P-labeled DNA probe was made from the full length coding sequence of AGS and hybridized under stringent conditions to mRNA Northern blots and an mRNA dot blot generated from human tissues and human cancer cell lines. The
  • AGS probe hybridized to a major transcript at approximately 2 kb and a minor transcript at approximately 5 kb.
  • the major transcript hybridized most strongly with mRNAs derived from liver, skeletal muscle, heart, kidney, brain and placenta, prostate and bone marrow tissues, as well as from a HeLa cell line.
  • the relative ratio of the major and minor transcripts varies with tissue source, and the major transcript is almost entirely absent from mRNA derived from fetal liver.
  • hybridization to the AGS probe is detectable in all mRNA samples, probably indicating low-level of AGS expression in all tissues.
  • EXAMPLE 9 Chromosomal localization of AGSl
  • E. coli or yeast (S. cerevisiae) and insect cells using, respectively, glutathione sepharose and Ni-agarose matrices.
  • a GST-fusion of Cdc42 was also purified from E. coli and yeast. Purification was monitored by SDS-polyacrylamide gel electrophoresis and immunoblotting. Large amounts of purified soluble GST-fusion proteins can be readily obtained from bacterial cultures and yeast cultures.
  • GST-AGS appears to be relatively unstable during purification, even with buffers containing high levels of Mg 2+ and either GDP or GTP.
  • EXAMPLE 12 AGSl Stimulates GTP Binding to G ⁇ Subunits
  • AGSl and a His 6 -tagged version of AGSl have also been constructed in the ecdysone (ponasterone)-inducible mammalian expression vector pIND (Invitrogen). using standard techniques. Upon induction, assays for determining activation of MAP kinase pathways were performed on cells carrying inducible pIND-AGSl constructs. These assays indicated that in HEK293 cells, the ERK1/ERK2 pathway is activated upon induction of expression of AGSl and that this activation is abolished by pretreatment of cells with pertussis toxin (Ptx) (Table 5). Table 5
  • EXAMPLE 15 Cloning and Analysis of a Human AGSl Homolog.
  • AGSl homolog was identified from the GenBank database (Accession No.: CAA18456). Alignment of the AGSl polypeptide sequence to the AGSl homolog indicated that these proteins were 62% identical when comparing residues 5-280 of AGSl (SEQ ID NO: 2) to residues 12-227 of the AGSl homolog (SEQ ID NO: 41). In addition, the cDNAs encoding these proteins were 82%> and 84% identical when comparing AGSl nucleotide bases 102-438 and 510-646 of SEQ ID NO: 1 with, respectively, AGSl homolog nucleotide bases 123- 459 and 531-667 of SEQ ID NO: 40. The overall greatest divergence between these two molecules is found in the C-terminal or 3' region of these molecules.
  • AH for AGSl head and Homolog tail
  • HA for Homolog head and AGSl tail

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Abstract

L'invention se rapporte à une analyse de levure visant à identifier, dans une banque d'ADNc mammalien, des activateurs et des inhibiteurs, indépendants des récepteurs couplés à des protéines G, de la voie de la phéromone. Elle se rapporte notamment à de nouvelles protéines activatrices de la signalisation des protéines G ('AGS' Activator of G protein Signalling), qui sont des protéines associées à Ras et qui stimulent l'activité de la protéine G de manière indépendante du récepteur, ainsi qu'à des molécules d'acides nucléiques codant les protéines AGS. L'invention se rapporte non seulement aux protéines AGS isolées mais également à des protéines de fusion AGS, à des peptides antigéniques et à des anticorps dirigés contre les protéines AGS. L'invention se rapporte également à des molécules d'acides nucléiques isolées AGS, à des vecteurs d'expression recombinés contenant une telle molécule d'acide nucléique, à des cellules hôtes dans lesquelles on a introduit les vecteurs d'expression et à des animaux transgéniques non humains dans lesquels un gène de protéine AGS a été introduit ou rompu. Enfin, l'invention se rapporte à des méthodes diagnostiques, sélectives et thérapeutiques utilisant des compositions contenant les protéines décrites ci-dessus.
PCT/US1999/010151 1998-05-08 1999-05-07 Molecules d'acides nucleiques et proteines ags, et utilisations correspondantes WO1999058670A1 (fr)

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AU39776/99A AU3977699A (en) 1998-05-08 1999-05-07 Ags proteins and nucleic acid molecules and uses therefor
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US09/709,103 US6733991B1 (en) 1998-05-08 2000-11-08 AGS proteins and nucleic acid molecules and uses therefor
US10/804,491 US7144711B2 (en) 1998-05-08 2004-03-19 AGS proteins and nucleic acid molecules and uses therefor

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EP1179006A2 (fr) * 1999-05-07 2002-02-13 MUSC Foundation For Research Development Nouvelles proteines de modulateur de proteine g (gpm), molecules d'acide nucleique, et leurs utilisations
WO2002033082A1 (fr) * 2000-10-19 2002-04-25 Takeda Chemical Industries, Ltd. Nouveau gene associe a des maladies et son utilisation
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WO2002062849A2 (fr) * 2001-02-08 2002-08-15 Pe Corporation (Ny) Proteines humaines isolees du type ras, molecules d'acide nucleique codant pour lesdites proteines et leurs utilisations
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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1179006A2 (fr) * 1999-05-07 2002-02-13 MUSC Foundation For Research Development Nouvelles proteines de modulateur de proteine g (gpm), molecules d'acide nucleique, et leurs utilisations
EP1179006A4 (fr) * 1999-05-07 2003-03-19 Musc Found For Res Dev Nouvelles proteines de modulateur de proteine g (gpm), molecules d'acide nucleique, et leurs utilisations
US7590493B2 (en) 2000-07-31 2009-09-15 Ocimum Biosolutions, Inc. Methods for determining hepatotoxins
WO2002033082A1 (fr) * 2000-10-19 2002-04-25 Takeda Chemical Industries, Ltd. Nouveau gene associe a des maladies et son utilisation
WO2002050104A3 (fr) * 2000-12-01 2003-04-10 Wyeth Corp Methodes et cellules de detection de modulateurs de proteines rgs
WO2002050104A2 (fr) * 2000-12-01 2002-06-27 Wyeth Methodes et cellules de detection de modulateurs de proteines rgs
WO2002062849A2 (fr) * 2001-02-08 2002-08-15 Pe Corporation (Ny) Proteines humaines isolees du type ras, molecules d'acide nucleique codant pour lesdites proteines et leurs utilisations
US6969588B2 (en) 2001-02-08 2005-11-29 Applera Corporation Isolated human ras-like proteins, nucleic acid molecules encoding these human ras-like proteins, and uses thereof
WO2002062849A3 (fr) * 2001-02-08 2003-02-13 Pe Corp Ny Proteines humaines isolees du type ras, molecules d'acide nucleique codant pour lesdites proteines et leurs utilisations
US7415358B2 (en) 2001-05-22 2008-08-19 Ocimum Biosolutions, Inc. Molecular toxicology modeling
US7426441B2 (en) 2001-05-22 2008-09-16 Ocimum Biosolutions, Inc. Methods for determining renal toxins
US7447594B2 (en) 2001-07-10 2008-11-04 Ocimum Biosolutions, Inc. Molecular cardiotoxicology modeling
WO2017187206A1 (fr) * 2016-04-29 2017-11-02 University Of Bradford Peptides et leurs formulations de nanoparticules
US11999770B2 (en) 2016-04-29 2024-06-04 University Of Bradford Peptides and nanoparticle formulations thereof

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