IDENTIFICATION OF FACTORS WHICH MEDIATE THE INTERACTION OF HETEROTRIMERIC G PROTEINS AND MONOMERIC G PROTEINS

Abstract: Monomeric GTPase guanine nucleotide exchange factor (GEF) have been identified which also contain an RGS region analogous to those of GTPase activating proteins (GAP). One of these GEF proteins, a Rho GEF has been demonstrated to contain an RGS sequence that has GAP activity toward a alpha subunit of a heterotrimeric G prote in.

Description: IDENTIFICATION OF FACTORS WHICH MEDIATE THE INTERACTION OF HETEROTRIMERIC G PROTEINS AND MONOMERIC G PROTEINS BACKGROUND OF THE INVENTION Signal transduction pathways linking extracellular factors to the activation of the Rho GTPase have been implicated in cell growth control and cytoskeletal rearrangements. Specifically, heterotrimeric G proteins have been shoαwn to mediate these pathways, although the mechanism of mediation has been unclear. The identification of factors which interact with both heterotrimeric G proteins and Rho GTPase would provide an important tool for investigating and controlling various cell processes, including cell proliferative diseases. SUMMARY OF THE INVENTION The invention relates to a polypeptide, and corresponding nucleic acid, comprising an amino acid sequence of a novel RGS domain, obtainable, e.g., from a guanine nucleotide exchange factor (GEF) protein, where the polypeptide preferably does αot include a dbl homology (DH) domain or a pleckstrin homology (PH) domain. In a preferred embodiment, the polypeptide has GTPase activating activity and binding affinity for an a G protein subunit such as Gα. The polypeptides and nucleic acids can be used as tools for research, therapeutics, and diagnostics as discussed below. The invention also relates to a method of identifying or assaying for a molecule, or mixture of molecules, that regulate the binding of an RGS domain of a GEF protein to a substrate, e.g., a G protein subunit such as Gα . In one embodiment, the method involves incubating, under effective conditions, a polypeptide having an RGS domain of a GEF polypeptide, and optionally having GEF activity, with a Gα subunit, or a fragment thereof, in the presence and/or absence of a test molecule; and determining whether the presence of the test molecule regulates the binding between the polypeptide and the subunit, or fragment thereof. As discussed later, various RGS-GEF polypeptides binding substrates can be utilized. In addition, the invention relates to a method of identifying or assaying for a molecule, or mixture of molecules, that regulates a stimulatory effect of a polypeptide comprising an RGS domain of a GEF protein on a polypeptide having a GTPase activity. In a preferred embodiment, the method comprises incubating a Gα subunit and a GEF protein, under effective conditions, in the presence and absence of a test molecule and determining whether the presence of the test molecule regulates the stimulatory effect of the GEF protein on Gα subunit GTPase activity. The invention also relates to a method of identifying or assaying for a molecule that specifically regulates a stimulatory effect of a first polypeptide, such as an activated Gα subunit, or polypeptide having GTPase activity, on a nucleotide exchange factor activity of a second polypeptide. The second polypeptide preferably comprises a RGS-GEF domain obtainable from a GEF, and more preferably is a guanine nucleotide exchange factor (GEF) for a monomeric G protein. In one embodiment of the method, a first assay is conducted by incubating an activated Gα subunit with a GEF protein and a monomeric G protein in the presence and absence of a test molecule; a second assay is conducted: by incubating a GEF protein and a monomeric G protein in the presence and absence of the test molecule, and a determination is made as to whether the molecule has a different effect when the first assay is compared to the second assay. The invention further relates to a method of identifying or assaying for a molecule, or mixture of molecules, that mimics the stimulatory effect of an activated Gα subunit on GEF mediated nucleotide exchange of a monomeric G protein. In one example, such a method comprises identifying a test compound that exhibits a binding affinity for an RGS domain of GEF proteins, incubating a GEF protein and monomeric G protein in the presence or absence of the test compound, determining whether the test compound exhibits a stimulatory effect on GEF mediated nucleotide exchange of a monomeric G protein. The invention further relates to a method of identifying or assaying for a molecule, or mixture of molecules, that mimics the stimulatory effect of an RGS domain of GEF polypeptide on Gα subunit GTPase activity. In one example, such a method comprises identifying a test compound that exhibits a binding affinity for a Gα subunit and incubating a GTP loaded Gα subunit in the presence or absence of the test compound to determine whether the test compound exhibits a stimulatory effect on GEF mediated nucleotide exchange of a monomeric G protein. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1, Panel A depicts the alignment of the sequences from RGS proteins and the N-terminal region of pi 15 Rho GEF as performed by Clustal W with a secondary structure mask of RGS4 to assign penalties for gaps. The RGS homologous sequences of Lsc, KIAA380, and DrhoGEF2 were further added to this alignment by Clustal W and manual adjustments. The (a) symbols above RGS4 indicate the α helices of the RGS domain of RGS4. Dark shaded boxes indicate conserved residues of the hydrophobic core of the RGS structure. Lightly shaded boxes show other conserved residues. Asterisks mark the residues of RGS4 which contact Gαu. Primary sequences used in the alignment are the following: rat RGS4 (SwissProt accession number P49799), mouse RGS2 (O08849), human GAIP (P49795), rat RGS12 (O08774), rat RGS 14 (O08773), human pi 15 (1654344), mouse Lsc (1389756), human KIAA380 (2224701) and Drosophila DrhoGEF2 (2760368). Figure 1, Panel B depicts constructs of pi 15 Rho GEF that were employed in the studies described herein. Numbers indicate the residues of pi 15 in each construct. The RGS, dbl(DH), and pleckstrin (PH) homology regions are indicated. GST=glutathione-S- transferase. Figure 2, Panel A is a graph showing the hydrolysis of GTP bound to Gαι 3 and Gαj 2 at 15°C either with («o) or without (BΠ) 10 nM pi 15 Rho GEF. Figure 2, Panel B is a graph showing the hydrolysis at 4°C of GTP bound to Gαι (•) and Gαι 2 (o) and in the presence of various concentrations of pi 15 Rho GEF. The initial rates of reaction were plotted as a function of the concentration of pi 15 Rho GEF. Figure 3 is a graph showing the hydrolysis at 15°C of GTP bound to Gαι 3 and Gαι 2 with either full-length pi 15 Rho GEF (•), ΔNpl 15 (■), or RGS-pl 15 (A), or without any pi 15 construct (T). Figure 4 is a graph showing the hydrolysis of GTP bound to Gαπ, Gα z , Gα q , and Gα s with 100 nM pi 15 Rho GEF (Δ), 100 nM RGS4 (α), or buffer control (o). Assays were performed at 4°C for Gαπ and Gα s , at 15°C for Gα z , and at 20°C for Gα q . Figure 5 is a graph showing the selective inhibition of pi 15 GAP activity by the A1F 4 - activated forms of Gα subunits. Panel A: PI 15 (400 nM) was incubated on ice for 15 minutes with various Gα subunits (400 nM) in the presence of 30 μM A1C1 3 , 10 mM NaF, and 10 mM MgS0 4 . The mixture was diluted 20-fold, mixed with 0.3 nM Gα )2 (GTP) and the hydrolysis of bound GTP was measured after incubation at 15°C for 2 minutes. Panel B: PI 15 (400 nM) was incubated with various concentrations of Gαι 2 (GDP-AIF " ) (•) or Gαι 3 (GDP-AIF 4 " ) (■) as described for Panel A. The mixture was diluted 20-fold, mixed with InM Gαπ(GTP) at 4°C and the hydrolysis of bound GTP was assessed over time. The initial rate of GTPase of Gαπ was plotted against the final concentration of α subunit GDP- AIF " . The filled triangle indicates the rate of GTPase of Gαι 3 without pi 15. Figure 6, Panel A is an image of an immunoblot showing the detection of myc- tagged pi 15 Rho GEF expression in COS cells using an anti-myc antibody. Figure 6, Panel B is an image of an immunoblot showing the detection of a coimmunoprecipitate of pi 15 Rho GEF and Gαπ using an anti-myc antibody. Figure 6, Panel C is an image of an immunoblot showing the detection of the coimmunoprecipitate of pi 15 Rho GEF and Gα ) using an anti-Gα i3 antibody. Figure 6, Panel D is an image of an immunoblot showing the detection of pi 15 Rho GEF and Gαπ binding when purified Gαπ is added to immunoprecipitated pi 15 Rho GEF when using an anti Gαπ antibody. Figure 7, Panel A is a graph showing the dissociation of bound GDP from 100 nM RhoA after 10 minutes in the presence or absence of 100 nm Gαπ or Gαπ and in the presence of various concentrations of pi 15 Rho GEF as indicated. Figure 7, Panel B is a graph showing the dissociation of GDP from 100 nM RhoA after 10 minutes in the presence of 25 nm pi 15 Rho GEF and the indicated concentrations of Gαπ or Gαι . Unstimulated dissociation of GDP from RhoA is indicated by the lower dashed line. Figure 7, Panel C is a graph showing the dissociation of GDP from 100 nM RhoA after 10 minutes of incubation with pi 15 Rho GEF and Gαπ that had been treated with AMF, GTPγS or GDPβS as indicated. Figure 7, Panel D is a graph showing the dissociation of of GDP from 100 nM RhoA after 10 minutes of incubation with pi 15 Rho GEF (25 nM) and various Gα subunits (100 nM) as indicated. Figure 8, Panel A is a graph showing the association of 1 nM [ 2 P]GTP to 100 nM RhoA in the presence of the indicated concentrations of truncated for full-length pi 15 Rho GEF as measured by filtration after 30 minutes at 30°C. Figure 8, Panel B is a graph showing the dissociation of [ H]-GDP from 100 nM RhoA after incubation for 10 minutes in the presence or absence of 25 nM pi 15 Rho GEF, 20 nM Gα i3 , and 300 nM GST-RGSpl 15 as indicated. Figure 8, Panel C is a graph showing the dissociation of [ 3 H]-GDP from 100 nM RhoA after incubation for 10 minutes in the presence 25 nM pi 15 Rho GEF and in the presence or absence of 25 nM Gαπ and the indicated concentrations of Gαι 2 . Figure 9, Panel A is an image of an immunoblot showing the detection of myc- tagged KIAA380 (designated FL147) expression in COS cells using an anti-myc antibody. Figure 9, Panel B is an image of an immunoblot showing the detection of a coimmunoprecipitate of KIAA380 (designated FL147) and Gα )2 using an anti-Gαι antibody. Figure 10 is the a listing of the amino acid sequence for pi 15 Rho GEF.. The RGS domain is shown by amino acids 45-170. Figure 11 is a listing of the nucleic acid sequence for pi 15 Rho GEF. The RGS domain is encoded by nucleotides 187-564. Figure 12 is a listing of the amino acid sequence for KIAA380. The RGS domain is shown by amino acids 310-432. Figure 13 is a listing of the nucleic acid sequence for KIAA380. The RGS domain is encoded by nucleotides 1673-2041. Figure 14 is a listing of the amino acid sequence for Lsc. The RGS domain is shown by amino acids 43-168. Figure 15 is a listing of the nucleic acid sequence for Lsc. The RGS domain is encoded by nucleotides 218-595. Figure 16 is a listing of the amino acid sequence for DRhoGEF2. The RGS domain is shown by amino acids 924-1053 Figure 17 is a listing of the nucleic acid sequence for DRhoGEF2. The RGS domain is encoded by nucleotides 3185-3574. Figure 18 is a homology alignment of the RGS region of several proteins, including GEF proteins with RGS domains (e.g. pi 15 Rho GEF, Lsc, KIAA380, DrhoGEF). The alignment was performed using the Clustal method with a PAM250 residue weight table. DETAILED DESCRIPTION OF THE INVENTION G proteins transduce signals from a large number of cell surface heptahelical receptors to various intracellular effectors. Each heterotrimeric G protein is composed of a guanine nucleotide-binding α subunit and a high-affinity dimer of β and γ subunits. Gα subunits are commonly classified into four subfamilies (G s , Gj, G q , and Gι 2 ) based on their amino acid sequence homology and function (A.G. Gilman, Annu. Rev. Biochem, 56, 615 (1987); Y. Kaziro et al., Annu. Rev. Biochem., 60, 349 (1991); Hepler and Gilman, Trends Biochem. Sci, 17, 383, (1992)). The Gι 2 subfamily, consists of two identified members to date, G i2 and G| 3 . In accordance with the present invention, the identification of proteins having activity as both a GTPase activating protein (GAP) for the α subunit of a heterotrimeric G protein and activity as a guanine nucleotide exchange factor (GEF) activity for monomeric G proteins have been described. Also in accordance with the invention, the first identification of a protein having GAP activity for the Gj 2 subfamily of G proteins has been described. Also in accordance with the invention, the ability of an α subunit of a heterotrimeric G protein to stimulate GEF mediated guanine nucleotide exchange activity of a monomeric G protein has been described. GAP and GEF activity, and methods of screening thereof, are described in Berman et al., 1996, Cell 86:445 and Hart et al., 1996, J. Biol Chem., 271:25452. According to the present invention, the GAP activity of GEF proteins has been correlated with a novel RGS domain obtainable from a GEF protein. The present invention relates to all aspects of such an RGS domain, including all aspects of a Rho GEF such as pi 15 Rho-GEF. (U.S. Patent Application No. 08/943,768, herein incorporated by reference). A GEF protein modulates cell signaling pathways, both in in vitro and in vivo, by modulating the guanine nucleotide exchange activity of a GTPase. According to the present invention, a GEF protein which also modulates the GTPase activity of a heterotrimeric Gα subunit is described. By way of illustration, pi 15 Rho-GEF, which modulates the guanine nucleotide exchange activity of a Rho GTPase, as well as the GTPase activity of the G π family of heterotrimeric G protein subunits is described. The present invention particularly relates to polypeptides comprising a RGS domain of a GEF polypeptide, or fragments thereof, and corresponding nucleic acids. The invention also relates to methods of using such polypeptides, nucleic acids, or derivatives thereof, e.g., in therapeutics, diagnostics, and as research tools. Other aspects of the present invention relate to antibodies and other ligands which recognize the RGS domain of GEF polypeptides or nucleic acids, methods for identifying or assaying modulators of the GEF activities and/or the GAP activities of a protein containing a RGS domain, and methods of treating pathological conditions associated with or related to the RGS domain, e.g., a GEF mediated interaction of a Gα subunit and a Rho GTPase. As used herein, an "RGS-GEF polypeptide" means, e.g., a polypeptide containing an RGS domain derived from a GEF protein, such as pi 15 Rho-GEF, Lsc, KIAA0380, or DRhoGEF2, and, which has one or more of the following activities: a specific binding affinity for a polypeptide substrate, e.g., a G protein subunit, preferably an α subunit, such as Gi 2 or G )3 ; a GTPase activating activity (GAP), such as a GAP activity for a G protein α subunit; or, an immunogenic activity. An RGS-GEF polypeptide preferably does not contain a (dbl homology) DH or a (pleckstrin homology) PH domain. DH and PH domains are disclosed in Cerione and Zheng, 1996, Curr. Opin. In Cell Biol, 8:216. For example, the amino acid sequence of pi 15 Rho GEF (Fig.10) contains a novel RGS domain at amino acids 45-170, the DH domain at amino acids 420-637, and the PH domain at amino acids 646-672. By "derived," it is meant that the amino acid sequence is obtainable from a naturally-occurring GEF (such as pi 15, Lsc, KIAA380, and DrrhoGEF2) or a non-naturally- occurring "mutated" sequence which is based upon a naturally-occurring GEF sequence (i.e., different amino acid residues have been substituted for the amino acid residues which occur in the naturally-occurring sequence at a particular position). The polypeptide can be "isolated," i.e., the material is in a form in which it is not found in its original environment, e.g., more concentrated, more purified, or separated from other components,etc. A preferred RGS polypeptide possesses both a GAP and GEF activity, e.g., a mutated pi 15 Rho-GEF. See below. An RGS-GEF nucleic acid codes for an RGS-GEF polypeptide. The nucleic acid refers to both sense and anti-sense nucleic acids. By the term "specific binding affinity," it is meant, e.g., that the RGS-GEF polypeptide has a binding preference for the activated state or transition state of a G protein subunit as compared to a GDP-bound state or the nucleotide depleted state. By "GEF activity," it is meant, e.g., that the polypeptide stimulates or catalyzes the dissociation of GDP from a monomeric G-protein, such as Rho, and subsequent binding of GTP. Monomeric G-proteins include but are not limited to G-proteins in the Ras, Rho/Rac, Sar, Rab, Arf, and Ran families. Of particular interest are the RGS domains of the following GEF proteins: human pi 15 (1654344) (Fig. 10, RGS domain at amino acids 45-170 ), mouse Lsc (1389756) (Fig. 14, RGS domain at amino acids 43-168), KIAA380 (2224701) (Fig. 12, RGS domain at amino acids 310-432) and Drosophila DrhoGEF2 (2760368) (Fig. 16, RGS domain at amino acids 924-1053). Another aspect of the invention relates to novel consensus sequences for RGS domain(s) of a GEF protein, herein referred to as a "sub-RGS consensus sequence." An "RGS domain," as used herein, refers to the amino acid sequence of protein which is able to bind to or physically interact with a G protein and, optionally, stimulates GTPase activity of that protein. A "sub-RGS consensus sequence," as used herein, refers to a consensus sequence which can be used to identify a specific subset of proteins which contain an RGS domain. For example, a homology alignment of the RGS domain from several proteins as shown and described in Fig. 18 and the corresponding legend, shows that several sub-RGS consensus sequences may be defined by the gap of 13 to 14 amino acids that is apparent in the RGS domains of GEF proteins. One of these consensus sequences, herein designated as "RGS-GEF consensus 1," is herein defined to be a consensus sequence of AAι-AA 2 -AA 3 - AA 4 -AA 5 -AA 6 -AA 7 -AA 8 -(gap of 13 amino acids)-AA 22 -AA 2 -AA 24 -AA 25 -AA 26 , wherein: AAi is L; AA 2 is E or V; AA 3 is K or P; AA 4 is T, N, or R; AA 5 is A; AA 6 is V or P AA 7 is L; AA 8 is either S or a gap of one amino acid, contiguous with the gap of 13 amino acids; AA 22 is either R or W; AA 23 is either V or Y; AA 24 is either P,K, or R AA 25 is either V, I, or Q; AA 2 .is either P or D. A second consensus sequence, herein designated as "RGS-GEF consensus 2," is herein defined to be a consensus sequence of AAι-AA 2 -AA -AA -(gap of 13 amino acids)- A A j 8 -A A 1 9ι wherein : AA 2 is V or P; AA 3 is L; AA 4 is either S or a gap of one amino acid, contiguous with the gap of 13 amino acids; AA i8 is either R or W; AA 19 is either V or Y. Other proteins, including other GEF proteins can be aligned with the RGS domain of RGS proteins as shown in Figure 18, and using methods described herein, to determine if they contain a sub-RGS consensus sequence, such as RGS-GEF consensus 1 or RGS-GEF consensus 2, as defined above. In examining Figure 18 it is also apparent that a nucleotide sequence uniques to RGS proteins that are not GEF proteins is shown by the nucleotide sequences which encode the amino acids that correspond to the 13-14 amino acid gap in RGS-GEF proteins. These nucleotide sequences could be used as probes to identify particular types of RGS proteins. RGS-GEF polypeptides are preferably biologically-active. By biologically-active, it is meant that a polypeptide fragment possesses an activity in a living system or with component(s) of a living system. Biological-activities include, but are not limited to a specific binding affinity for a G protein α subunit, as defined above, and GAP activity toward a G protein α subunit. As described in the examples, such polypeptides can be prepared routinely, e.g., by recombinant means or by proteolytic cleavage of isolated polypeptides, and then assayed for a desired activity. A polypeptide of the present invention includes polypeptides which have less than 100% identity to the amino acid sequences of pi 15 Rho-GEF (Fig. 10), Lsc (Fig. 14), KIAA0380 (Fig. 12), or DRhoGEF2 (Fig. 16). For the purposes of the following discussion: Sequence identity means that the same nucleotide or amino acid which is found in the sequences set forth in Fig. 10-17 is found at the corresponding position of the compared sequence(s). A polypeptide having less than 100% sequence identity to the amino acid sequences set forth in Figures 10, 12, 14, and 16 can be substituted in various ways, e.g., by a conservative amino acid. The sum of the identical and conservatively substituted residues divided by the total number of residues in the sequence is equal to the percent sequence similarity. For purposes of calculating sequence identity and similarity, the compared sequences can be aligned and calculated according to any desired method, algorithm, computer program, etc., including, e.g., FASTA, BLASTA. A polypeptide having less than 100% amino acid sequence identity to the amino acid sequences of the GEF proteins shown in Figures 10, 12, 14, and 16 may comprise, for example, about 60, 65 percent sequence similarity and more preferably about 67, 70, 78, 80, 90, 92, 96, 99, etc. percent sequence amino acid sequence similarity. In particular, the present invention relates to polypeptides, and corresponding nucleic acids, of pi 15, Lsc, KIAA380, and DrhoGEF2 which are mutated in the RGS domain of a GEF protein and which possess one or more of the RGS-GEF polypeptide activities mentioned above. By the term "mutated," it is meant herein that such sequences are not naturally-occurring. For example a mutated polypeptide as mentioned can have one or more naturally-occurring positions replaced by a conservative amino acid, e.g., (based on the size of the side chain and degree of polarization) small nonpolar: cysteine, proline, alanine, threonine; small polar: serine, glycine, aspartate, asparagine; large polar: glutamate, glutamine, lysine, arginine; intermediate polarity: tyrosine, histidine, tryptophan; large nonpolar: phenylalanine, methionine, leucine, isoleucine, valine. Such conservative substitutions also include those described by Dayhoff in the Atlas of Protein Sequence and Structure 5 (1978), and by Argos in EMBO J., 8, 779-785 (1989). A polypeptide having an amino acid sequence as set forth in Figures 10, 12, 14, and 16 can be substituted at 1, 5, 10, 15, or 20 positions by conservative amino acids. The mutations can be introduced into the conserved consensus region or the other residues of the RGS domain of a GEF protein. A mutation to an RGS-GEF polypeptide can be selected to have one or more of the activities mentioned above, e.g., a specific binding affinity for a G protein α subunit, a GAP activity toward a G protein α subunit, etc. Assays for such activities can be conducted as described below or as disclosed in Cerione and Zheng, 1996, Curr. Opin. In Cell Biol, 8:216. An RGS-GEF polypeptide can be modified by introducing amino acid substitutions into the hydrophobic core of the RGS domain (See Fig. 1, Panel A). For example, a conservative amino acid substitution would not be expected to affect activity, whereas as non-conservative amino acid substitution, e.g., changing a hydrophobic residue to a hydrophilic residue, would be expected to reduce or eliminate its activity. Hydrophobic resiudes are nonpolar amino acids such phenylalanine, leucine, isoleucine, valine, alanine, methionine, tryptophan, and cysteine. Hydrophilic residues are polar amino acids such as lysine, arginine, histidine, glutamate, and aspartate. Modifications to a RGS-GEF polypeptide of the present invention or corresponding nucleotide sequence, e.g., mutations, can also be prepared based on homology searching from gene data banks, e.g., Genbank, EMBL. Sequence homology searching can be accomplished using various methods, including algorithms described in the BLAST family of computer programs, the Smith-Waterman algorithm, etc. For example, conserved amino acids can be identified between various sequences containing an RGS domain of various GEF proteins. (See Fig. 18) A mutation(s) can then be introduced into such sequences by identifying and aligning amino acids conserved between the polypeptides and then modifying an amino acid in a conserved or non-conserved position. A mutated RGS-GEF sequence can comprise conserved or non-conserved amino acids, e.g., between corresponding regions of homologous nucleic acids. For example, a mutated sequence can comprise conserved or non-conserved residues from any number of homologous sequences as mentioned-above and/or determined from an appropriate searching algorithm. Corresponding mutations can be made in specific regions of an RGS-GEF nucleic acid. For example, mutations may be made wherein amino acids that particpate in the GTPase catalytic function or mutations may be made in amino acids that function as contact points between the RGS-GEF sequence and the Gα subunit. An RGS-GEF polypeptide or fragment thereof, or substituted RGS-GEF polypeptide or fragment thereof, may also comprise various modifications, wherein such modifications include glycosylation, covalent modifications (e.g., of an R-group of an amino acid), amino acid substitution, amino acid deletion, or amino acid addition. Modifications to the polypeptide can be accomplished according to various methods, including recombinant, synthetic, chemical, etc. Polypeptides of the present invention (e.g., RGS-GEF polypeptides, and fragments and mutations thereof) may be used in various ways, e.g., as immunogens for antibodies as described below, as biologically-active agents (e.g., having one or more of the activities associated with an RGS-GEF polypeptide), as inhibitors of the activities of the corresponding full-length polypeptide. For example, upon binding of pi 15 Rho-GEF to the Gα subunit, a cascade of events is initiated in the cell, e.g., promoting cell proliferation and/or cytoskeletal rearrangements. The interaction between pi 15 Rho-GEF and the Gα subunit can be modulated by using a RGS-GEF polypeptide, or fragment thereof, to inhibit the interaction between pi 15 Rho-GEF and the Gα subunit. Such a fragment can be useful for modulating pathological conditions associated with the Rho signaling pathway. A useful fragment may be identified routinely by testing the ability of overlapping fragments of the entire length of the RGS domain of a GEF protein to inhibit the binding of pi 15 Rho-GEF with the Gα subunit or to inhibit the GAP activity of the pi 15 Rho-GEF toward the Gα subunit. The measurement of these activities is described below and in the examples. Peptides can be chemically-modified, etc. A RGS-GEF polypeptide of the present invention can comprise one or more structural domains, functional domains, detectable domains, antigenic domains, and/or other polypeptides of interest, in an arrangement which does not occur in nature, i.e., not naturally-occurring. A polypeptide comprising such features is a chimeric or fusion polypeptide. Such a chimeric polypeptide can be prepared according to various methods, including, chemical, synthetic, quasi-synthetic, and/or recombinant methods. A chimeric nucleic acid coding for a chimeric polypeptide can contain the various domains or desired polypeptides in a continuous or interrupted open reading frame, e.g., containing introns, splice sites, enhancers, etc. The chimeric nucleic acid can be produced according to various methods. See, e.g., U.S. Pat. No. 5,439,819. A domain or desired polypeptide can possess any desired property, including, a biological function such as catalytic, signaling, growth promoting, cellular targeting, etc., a structural function such as hydrophobic, hydrophilic, membrane-spanning, etc., receptor-ligand functions, and/or detectable functions, e.g., combined with enzyme, fluorescent polypeptide, green fluorescent protein GFP (Chalfie et al., 1994, Science, 263:802; Cheng et al., 1996, Nature Biotechnology, 14:606; Levy et al., 1996, Nature Biotechnology, 14:610, etc. In addition, a RGS-GEF nucleic acid, or a fragment thereof, may be used as selectable marker when introduced into a host cell. For example, a nucleic acid coding for an amino acid sequence according to the present invention can be fused in frame to a desired coding sequence and act as a tag for purification, selection, or marking purposes. The region of fusion encodes a cleavage site. A polypeptide according to the present invention can be produced in an expression system, e.g., in vivo, in vitro, cell-free, recombinant, cell fusion, etc., according to the present invention. Modifications to the polypeptide imparted by such system include, glycosylation, amino acid substitution (e.g., by differing codon usage), polypeptide processing such as digestion, cleavage, endopeptidase or exopeptidase activity, attachment of chemical moieties, including lipids, phosphates, etc. For example, some cell lines can remove the terminal methionine from an expressed polypeptide. A polypeptide according to the present invention can be recovered from natural sources, transformed host cells (culture medium or cells) according to the usual methods, including, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography and lectin chromatography. It may be useful to have low concentrations (approximately 0.1-5 mM) of calcium ion present during purification (Price, et al., J. Biol. Chem., 244:917 (1969)). High performance liquid chromatography (HPLC) can be employed for final purification steps. A RGS-GEF nucleic acid of the present invention can comprise the complete coding sequence for an RGS-GEF polypeptide, or fragments thereof. A nucleic acid according to the present invention may also comprise a nucleotide sequence which is 100% complementary, e.g., an anti-sense, to any RGS-GEF nucleotide sequence. A nucleic acid according to the present invention can be obtained from a variety of different sources. It may be obtained from DNA or RNA, such as polyadenylated mRNA, e.g., isolated from tissues, cells, or whole organism. The nucleic acid may be obtained directly from DNA or RNA, or from a cDNA library. The nucleic acid can be obtained from a cell at a particular stage of development, having a desired genotype, phenotype (e.g., an oncogenically transformed cell or a cancerous cell), etc. The nucleic acid may also be chemically synthesized. A nucleic acid according to the present invention may include only coding sequence for an RGS-GEF polypeptide; coding sequence for an RGS-GEF polypeptide and additional functional coding sequences including, for example, leader sequences, secretory sequences, tag sequences (e.g. targeting tags, enzymatic tags, fluorescent tags etc.). A nucleic acid according to the present invention may also include coding sequence for an RGS-GEF polypeptide and non-coding sequences, e.g., untranslated sequences at either a 5' or 3' end, or dispersed in the coding sequence, e.g., introns. A nucleic acid according to the present invention may also comprise an expression control sequence operably linked to a nucleic acid as described above. The phrase "expression control sequence" means a nucleic acid sequence which regulates expression of a polypeptide coded for by a nucleic acid to which it is operably linked. Expression can be regulated at the level of the mRNA or polypeptide. Thus, the expression control sequence includes mRNA-related elements and protein-related elements. Such elements include promoters, enhancers (viral or cellular), ribosome binding sequences, transcriptional terminators, etc. An expression control sequence is operably linked to a nucleotide coding sequence when the expression control sequence is positioned in such a manner to effect or achieve expression of the coding sequence. For example, when a promoter is operably linked 5' to a coding sequence, expression of the coding sequence is driven by the promoter. Expression control sequences can be heterologous or endogenous to the normal gene. A nucleic acid in accordance with the present invention may be selected on the basis of nucleic acid hybridization. The ability of two single-stranded nucleic acid preparations to hybridize together is a measure of their nucleotide sequence complementarity, e.g., base- pairing between nucleotides, such as A-T, G-C, etc. The invention thus also relates to nucleic acids which hybridize to a nucleic acids comprising a nucleotide sequence as set forth in Figures 11, 13, 15, and 17. The present invention includes both strands of nucleic acid, e.g., a sense strand and an anti-sense strand. According to the present invention, a nucleic acid or polypeptide can comprise one or more differences in the nucleotide or amino acid sequence set forth in Figures 10-17. Changes or modifications to the nucleotide and/or amino acid sequence can be accomplished by any method available, including directed or random mutagenesis. A nucleic acid coding for an RGS-GEF polypeptide according to the invention may comprise nucleotides which occur in a naturally-occurring GEF gene e.g., naturally- occurring polymorphisms, normal or mutant alleles (nucleotide or amino acid), mutations which are discovered in a natural population of mammals, such as humans, monkeys, pigs, mice, rats, or rabbits. By the term naturally-occurring, it is meant that the nucleic acid is obtained from a natural source, e.g., animal tissues and cells, body fluids, tissue culture cells, forensic samples. Naturally-occurring mutations include deletions, substitutions, or additions of nucleotide sequence. These genes can be detected and isolated by nucleic acid hybridization according to methods well known to one skilled in the art. It is recognized that, by analogy to other oncogenes, naturally-occurring variants of GEF proteins will include variants with deletions, substitutions, and additions in the RGS domain of a GEF protein, which produce pathological conditions in the host cell and organism. A nucleotide sequence coding for an RGS-GEF polypeptide of the invention may contain codons found in a naturally-occurring gene, transcript, or cDNA, for example, or it may contain degenerate codons coding for the same amino acid sequences. In addition, a nucleic acid or polypeptide of the present invention may be obtained from any desired mammalian organism, but also non-mammalian organisms. Homologs from mammalian and non-mammalian organisms can be obtained according to various methods. For example, hybridization with an oligonucleotide (see below) selective for an RGS domain of a GEF, or a RGS-GEF, of the present invention can be employed to select such homologs, e.g., as described in Sambrook et al., Molecular Cloning, 1989, Chapter 11. Such homologs may have varying amounts of nucleotide and amino acid sequence identity and similarity to previously identified RGS domain or RGS-GEF nucleotide or polypeptide sequence. Non-mammalian organisms include, e.g., vertebrates, invertebrates, zebra fish, chicken, Drosophila, yeasts (such as Saccharomyces cerevisiae), C. elegans, roundworms, prokaryotes, plants, Arabidopsis, viruses, etc. A nucleic acid according to the present invention may comprise, for example, DNA, RNA, synthetic nucleic acid, peptide nucleic acid, modified nucleotides, or mixtures thereof. A DNA can be double- or single-stranded. Nucleotides comprising a nucleic acid can be joined via various known linkages such as, for example, ester, sulfamate, sulfamide, phosphorothioate, phosphoramidate, methylphosphonate, carbamate, etc., depending on the desired purpose. Linkages may be modified for purposes such as, for example, resistance to nucleases such as RNase H and improved in vivo stability. See, e.g., U.S. Pat. Nos. 5,378,825. Various modifications can be made to the nucleic acids, such as attaching detectable markers (avidin, biotin, radioactive elements), moieties which improve hybridization, detection, or stability. The nucleic acids can also be attached to solid supports, e.g., nitrocellulose, nylon, agarose, diazotized cellulose, latex solid microspheres, polyacrylamides, etc., according to a desired method. See, e.g., U.S. Pat. Nos. 5,470,967, 5,476,925, 5,478,893. Another aspect of the present invention relates to oligonucleotides and nucleic acid probes. Such oligonucleotides or nucleic acid probes can be used, e.g., to detect, quantify, or isolate an RGS-GEF nucleic acid in a test sample. Detection can be desirable for a variety of different purposes, including research, diagnostic, and forensic. For diagnostic purposes, it may be desirable to identify the presence or quantity of a specific RGS-GEF nucleic acid sequence in a sample obtained from tissues, cells, body fluids, etc. In a preferred method, the present invention relates to a method of detecting a target RGS-GEF nucleic acid in a test sample comprising contacting the test sample with an oligonucleotide under conditions effective to achieve hybridization between the target and oligonucleotide; and detecting hybridization. An oligonucleotide in accordance with the invention can also be used in synthetic nucleic acid amplification such as PCR, e.g., Saiki et al, 1988, Science, 241:53; U.S. Pat. No. 4,683,20, or or differential display (See, e.g., Liang et al., 9{ cl. Acid. %es., 21:3269-3275, 1993; USP 5,599,672; WO97/18454). Oligonucleotides can be identified routinely, e.g., to the DH, PH, and RGS-GEF domains to differentially display and/or amplify gene products containing such sequences. Both sense and antisense nucleotide sequences are intended as part of the invention. A unique nucleic acid according to the present invention may be determined routinely. An RGS-GEF nucleic acid may be used as a hybridization probe to identify the presence of RGS-GEF nucleotide sequence in a sample comprising a mixture of nucleic acids, e.g., on a Northern blot. Hybridization can be performed under stringent conditions to select nucleic acids having at least 95% identity (i.e., complementarity) to the probe, but less stringent conditions can also be used. A unique RGS-GEF nucleotide sequence can also be fused in- frame, at either its 5' or 3' end, to various nucleotide sequences, including, for example, coding sequences for enzymes or expression control sequences, etc. Hybridization can be performed under different conditions, depending on the desired selectivity, e.g., as described in Sambrook et al., Molecular Cloning, 1989. For example, to specifically detect RGS-GEF sequences, an oligonucleotide can be hybridized to a target nucleic acid under conditions in which the oligonucleotide only hybridizes to the GEF sequence from which the RGS -GEF sequence was derived, e.g., where the oligonucleotide is 100% complementary to the target. Different conditions can be used if it is desired to select target nucleic acids which have less than 100% nucleotide complementarity, at least about, e.g., 99%, 97%, 95%, 90%, 70%, 67%. Since a mutation in GEF genes can cause diseases or pathological conditions, e.g., cancer, benign tumors, an oligonucleotide according to the present invention can be used diagnostically. For example, a patient having symptoms of a cancer or other condition associated with the Rho signaling pathway (see below) can be diagnosed with the disease by using an oligonucleotide according to the present invention, in polymerase chain reaction followed by DNA sequencing to identify whether the sequence is normal, in combination with other oncogene oligonucleotides, etc., e.g., p53, Rb, p21, Dbl, MTS1, Wtl, Bcl-1, Bcl-2, MDM2, etc. Oligonucleotides according to the present invention can be of any desired size, preferably 14-16 oligonucleotides in length, or more. Such oligonucleotides can have non- naturally-occurring nucleotides, e.g., inosine. In accordance with the present invention, the oligonucleotide can comprise a kit, where the kit includes a desired buffer (e.g., phosphate, tris, etc.), detection compositions, etc. The oligonucleotide can be labeled or unlabeled, with radioactive or non-radioactive labels as known in the art. Anti-sense nucleic acid can also be prepared from a nucleic acid according to the present, preferably an anti-sense RGS-GEF nucleotide sequence corresponding to an RGS- GEF nucleotide sequence of Figures 11, 13, 15, and 17. Anti-sense RGS-GEF nucleic acid can be used in various ways, such as to regulate or modulate expression of GEF proteins containing RGS domains or to detect expression of RGS-GEF proteins, including by in situ hybridization. For the purposes of regulating or modulating expression, an anti-sense oligonucleotide may be operably linked to an expression control sequence. The RGS-GEF nucleic acids according to the present invention can be labelled according to any desired method. The nucleic acid can be labeled using radioactive tracers such as 32 P, 35 S, l25 1, 3 H, or 14 C, to mention only the most commonly used tracers. The radioactive labeling can be carried out according to any method such as, for example, terminal labeling at the 3' or 5' end using a radiolabeled nucleotide, polynucleotide kinase (with or without dephosphorylation with a phosphatase) or a ligase (depending on the end to be labeled). A non-radioactive labeling can also be used, combining a nucleic acid of the present invention with residues having immunological properties (antigens, haptens), a specific affinity for certain reagents (ligands), properties enabling detectable enzyme reactions to be completed (enzymes or coenzymes, enzyme substrates, or other substances involved in an enzymatic reaction), or characteristic physical properties, such as fluorescence or the emission or absorption of light at a desired wavelength, etc. An RGS-GEF nucleic acid according to the present invention, including oligonucleotides, anti-sense nucleic acid, etc., can be used to detect expression of RGS-GEF nucleic acids in whole organs, tissues, cells, etc., by various techniques, including Northern blot, PCR, in situ hybridization, etc. Such nucleic acids can be particularly useful to detect disturbed expression, e.g., cell-specific and/or subcelfular alterations of RGS-GEF expression. The levels of RGS-GEF proteins can be determined alone or in combination with other genes products (oncogenes such as p53, Rb, Wtl, etc.), transcripts, etc. A nucleic acid according to the present invention can be expressed in a variety of different systems, in vitro and in vivo, according to the desired purpose. For example, a nucleic acid can be inserted into an expression vector, introduced into a desired host, and cultured under conditions effective to achieve expression of a polypeptide coded for the nucleic acid. Effective conditions includes any culture conditions which are suitable for achieving production of the polypeptide by the host cell, including effective temperatures, pH, medias, additives to the media in which the host cell is cultured (e.g., additives which amplify or induce expression such as butyrate, or methotrexate if the coding nucleic acid is adjacent to a dhfr gene), cyclohexamide, cell densities, culture dishes, etc. A nucleic acid can be introduced into the cell by any effective method including, e.g., calcium phosphate precipitation, electroporation, injection, DEAE-Dextran mediated transfection, fusion with liposomes, and viral transfection. A cell into which a nucleic acid of the present invention has been introduced is a transformed host cell. The nucleic acid can be extrachromosomal or integrated into a chromosome(s) of the host cell. It can be stable or transient. An expression vector is selected for its compatibility with the host cell. Host cells include, mammalian cells, e.g., COS-7, CHO, HeLa, LTK, NIH 3T3, yeast, insect cells, such as Sf9 (S. frugipeda) and Drosophila, bacteria, such as E. coli, Streptococcus sp., Bacillus sp., yeast, fungal cells, plants, embryonic stem cells (e.g., mammalian, such as mouse or human), cancer or tumor cells Sf9 expression can be accomplished in analogy to Graziani et al., Oncogene, 7:229-235, 1992. Expression control sequences are similarly selected for host compatibility and a desired purpose, e.g., high copy number, high amounts, induction, amplification, controlled expression. Other sequences which can be employed include enhancers such as from SV40, CMV, inducible promoters, cell-type specific elements, or sequences which allow selective or specific cell expression. A labelled polypeptide can be used, e.g., in binding assays, such as to identify substances that bind or attach to pi 15 Rho-GEF, to track the movement of pi 15 Rho-GEF in a cell, in an in vitro, in vivo, or in situ system, etc. A nucleic acid or polypeptide of the present invention can also be substantially purified. By substantially purified, it is meant that nucleic acid or polypeptide is separated and is essentially free from other nucleic acids or polypeptides, i.e., the nucleic acid or polypeptide is the primary and active constituent. Another aspect of the present invention relates to the regulation of biological pathways in which a RGS-GEF polypeptide is involved, particularly pathological conditions, e.g., cell proliferation (e.g., cancer), growth control, morphogenesis, stress fiber formation, and integrin-mediated interactions, such as embryonic development, tumor cell growth and metastasis, programmed cell death, hemostasis, leucocyte homing and activation, bone resorption, clot retraction, and the response of cells to mechanical stress. See, e.g., Clark and Brugge, Science, 268:233- 239, 1995; Bussey, Science, 272:225-226, 1996. Thus, the invention relates to all aspects of a method of modulating an activity of a RGS-GEF polypeptide comprising, administering an effective amount of an RGS-GEF polypeptide or a biologically-active fragment thereof, an effective amount of a compound which modulates the activity of a RGS-GEF polypeptide, or an effective amount of a nucleic acid which codes for a RGS-GEF polypeptide or a biologically-active fragment thereof. The activity of the RGS-GEF which is modulated may include binding to a Gα subunit or GAP activity toward a Gα subunit. The activity can be modulated by increasing, reducing, antagonizing, or promoting expression or activity of the RGS-GEF. The present invention also relates to antibodies which specifically recognize a RGS- GEF polypeptide. Antibodies, e.g., polyclonal, monoclonal, recombinant, chimeric, can be prepared according to any desired method. For example, for the production of monoclonal antibodies, an RGS-GEF polypeptide according to Figures 10, 12,14, or 16 can be administered to mice, goats, or rabbit subcutaneously and/or intraperitoneally, with or without adjuvant, in an amount effective to elicit an immune response. The antibodies can also be single chain or FAb. The antibodies can be IgG, subtypes, IgG2a, IgGl, etc. An antibody specific for RGS-GEF means that the antibody recognizes a defined sequence of amino acids within or including the amino acid sequence of the RGS domain of a GEF polypeptide. Thus, a specific antibody will bind with higher affinity to an amino acid sequence, i.e., an epitope, found in the RGS domain of a GEF polypeptdie than to a different epitope(s), e.g., as detected and/or measured by an immunoblot assay. Thus, an antibody which is specific for an epitope within or including the RGS domain of pi 15 Rho-GEF is useful to detect the presence of the epitope in a sample, e.g., a sample of tissue containing pi 15 Rho-GEF gene product, distinguishing it from samples in which the epitope is absent. Additionally, in accordance with the present invention, ligands which bind to an RGS domain of a GEF polypeptide can also be prepared, e.g., using synthetic peptide libraries or aptamers (e.g., Pitrung et al., U.S. Pat. No. 5,143,854; Geysen et al., 1987, J. Immunol. Methods, 102:259-274; Scott et al., 1990, Science, 249:386; Blackwell et al., 1990, Science, 250:1104; Tuerk et al., 1990, Science, 249: 505. Antibodies and other ligands which bind the RGS domain of a GEF polypeptide, and specifically antibodies and other ligands which bind the RGS domain of pi 15 Rho GEF, can be used in various ways. These include, but are not limited to, uses therapeutic, diagnostic, and commercial research tools, e.g, to quantitate the levels of pi 15 Rho-GEF polypeptide in animals, tissues, cells, etc., to identify the cellular localization and/or distribution of pi 15 Rho-GEF, to purify pi 15 Rho-GEF or a polypeptide comprising a part of pi 15 Rho-GEF, to modulate the function of pi 15 Rho-GEF, etc. Antibodies can be used in Western blots, ELIZA, immunoprecipitation, RIA, etc. The present invention relates to such assays, compositions and kits for performing them, etc. An antibody according to the present invention can be used to detect polypeptides or fragments containing an RGS domain of a GEF polypeptide in various samples, including tissue, cells, body fluid, blood, urine, cerebrospinal fluid. A method of the present invention comprises contacting a ligand which binds to an RGS-GEF polypeptide of Figure 10, 12, 14, or 16 under conditions effective, as known in the art, to achieve binding, detecting specific binding between the ligand and peptide. By specific binding, it is meant that the ligand attaches to a defined sequence of amino acids, e.g., within or including the amino acid sequence of the RGS domain as shown in Figures 10, 12, 14, and 16, or derivatives thereof. The antibodies or derivatives thereof can also be used to inhibit expression of GEF proteins containing an RGS domain. The levels of a GEF polypeptide containing an RGS domain may be determined alone or in combination with other gene products. In particular, the amount (e.g., its expression level) of the GEF polypeptide containing an RGS domain can be compared (e.g., as a ratio) to the amounts of other polypeptides in the same or different sample, e.g., p21, p53, Rb, WT1, etc. A ligand for the RGS domain of GEF polypeptides can be used in combination with other antibodies, e.g., antibodies that recognize oncological markers of cancer, including, Rb, p53, c-erbB-2, oncogene products, etc. In general, reagents which are specific for the RGS domain of GEF polypeptides can be used in diagnostic and/or forensic studies according to any desired method, e.g., as U.S. Pat. Nos. 5,397,712; 5,434,050; 5,429,947. The present invention also relates to a transgenic animal, e.g., a non-human- mammal, such as a mouse, comprising an RGS-GEF polypeptide. Transgenic animals can be prepared according to known methods, including, e.g., by pronuclear injection of recombinant genes into pronuclei of 1-cell embryos, incorporating an artificial yeast chromosome into embryonic stem cells, gene targeting methods, embryonic stem cell methodology. See, e.g., U.S. Patent Nos. 4,736,866; 4,873,191; 4,873,316; 5,082,779; 5,304,489; 5,174,986; 5,175,384; 5,175,385; 5,221,778; Gordon et al., Proc. Natl Acad. Sci., 77:7380-7384 (1980); Palmiter et al., Cell, 41 :343-345 (1985); Palmiter et al., Ann. Rev. Genet., 20:465-499 (1986); Askew et al., Mol. Cell. Bio., 13:4115-4124, 1993; Games et al. Nature, 373:523-527, 1995; Valancius and Smithies, Mol Cell Bio., 11: 1402-1408, 1991; Stacey et al., Mol Cell Bio., 14: 1009-1016, 1994; Hasty et al., Nature, 350:243-246, 1995; Rubinstein et al., Nucl Acid Res., 21 :2613-2617,1993. A nucleic acid according to the present invention can be introduced into any non-human mammal, including a mouse (Hogan et al., 1986, in Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York), pig (Hammer et al., Nature, 315:343-345, 1985), sheep (Hammer et al., Nature, 315:343-345, 1985), cattle, rat, or primate. See also, e.g., Church, 1987, Trends in Biotech. 5: 13-19; Clark et al., 1987, Trends in Bio tech. 5:20-24; and DePamphilis et al., 1988, BioTechniques, 6:662-680. Additionally, custom transgenic rat and mouse production is commercially available. These transgenic animals are useful, for example, as a cancer model or as a model to evaluate the effects of overexpression of the RGS-GEF polypeptide. Generally, the nucleic acids, polypeptides, antibodies, etc. of the present invention can be prepared and used as described in, U.S. Pat. Nos. 5,501,969, 5,506,133, 5,441,870; WO 90/00607; WO 91/15582; Other aspects of this invention relate to methods to assay for, or identify, molecules that modulate the following interactions and effects: the interaction between an RGS domain of a GEFand its cognate binding substrate; the interaction between an RGS domain of a GEF and a Gα subunit; the effect of G protein subunit stimulation on a guanine nucleotide exchange activity of a GEF protein containing an RGS domain; the effect of a GEF protein having an RGS domain as a GTPase activating protein for a G protein subunit. Activity can be modulated in various ways, e.g., enhancing, activating, stimulating, suppressing, preventing, inhibiting, etc. A modulatory molecule can be an agonist, antagonist, or have partial activities thereof. Modulating molecules can be any type of molecule, including but not limited to small molecules, proteins, peptides, antibodies, nucleic acids, etc. In general, a compound having an in vitro activity will be useful in vivo to modulate a biological pathway associated with a GEF protein containing an RGS domain, e.g., to treat a pathological condition associated with the biological and cellular activities mentioned above. The modulatory molecules can comprise a mixture of the same or different molecules. A binding substrate for the RGS domain of a GEF protein can be any material to which the RGS domain binds specifically, including members of the Gαl2 family. See, e.g., Strathman and Simon, Proc. Natl. Acad. Sci., 88:5582, 1991. For example, a method of identifying or assaying for a molecule that modulates or regulates the binding of a G protein α subunit to a GEF protein containing an RGS domain, such as pi 15 Rho-GEF, can be conducted in accordance with this invention. In one embodiment, a GTP bound α subunit, or derivative thereof, is incubated with a GEF protein, or fragment thereof, containing the RGS domain, in the presence and absence of a test molecule to determine whether the presence of the test compound modulates the binding between the GEF protein and the G protein α subunit. The incubation is accomplished under effective conditions, i.e., conditions under which binding or attachment occurs. Binding can be detected in one or more ways. For example, the GEF protein or the binding substrate is labeled detectably; the labelled bound component is separated from the labelled free component; and the amount of bound-detectably labeled GEF protein or binding substrate determined. The detectable label can be of any desired composition, e.g., radioactive, fluorescent, etc. Such an assay can be performed in either solid or liquid phase. In one aspect of the invention, it is desirable to identify molecules that regulate the binding of the Gαπ family of subunits, eg. Gαπ and Gαπ, with a GEF, e.g., pi 15 Rho GEF, Lsc, KIAA380, or DrhoGEF2. The assay can be conducted using a complete GEF protein, or any subfragments thereof which contain the RGS domain, or biologically active subfragments of the RGS domain. The assay is typically conducted with stable analogs of the GTP bound state of the Gα subunit, including α subunits bound to either GDP-AIF 4 " or GTPγS. For example, a binding assay may be conducted by the procedure described in Example 5 below wherein a COS cell is transfected with a nucleic acid construct for a myc- tagged polypeptide, such as pi 15 Rho GEF, or a fragment thereof and complexes of the polypetide and a Gα subunit are detected by precipitation of any bound complex with a first antibody to one of components and detection of the amount of a second bound component with a second antibody. Binding assays could also be performed using techniques that are well known in the art such as by binding one of the components to a column and then determining the amount of a second labelled compoent that binds to the column. Relevant assay methods are also disclosed, for example, in Berman et al, 1996, J. Biol. Chem. 271:27209. A method of isolating or assaying for a molecule that modules or regulates the stimulatory effect of a RGS-GEF polypeptide on GTPase activity, such as a GTPase activity of a Gα subunit, can also be conducted in accordance with the invention. For example, a Gα subunit is incubated under effective conditions with an RGS-GEF polypeptide having GTPase stimulatory effect in the presence and absence of a test inhibitor to determine whether the presence of the test inhibitor modulates its stimulatory effect. The assay can conducted using a complete RGS-GEF polypeptide, a GEF protein, or any subfragments thereof which contain the RGS domain, or biologically active subfragments of the RGS domain. An RGS-GEF polypeptide can be pi 15 Rho GEF, Lsc, KIAA380, DrhoGEF2, or biologically-active fragments thereof. For example, an assay can be conducted using a pi 15 Rho GEF in conjuction with an αl2 or an αl3 subunit, as described in the examples discussed herein, as well as using other variations or assay methods which are well known in the art. For example, the assay may be conducted in accordance with Example 5 below, in which Gα subunits were loaded with [γ- 32 P]GTP and the amount of hydrolysis under various conditions, including the presence of an RGS-GEF polypeptide, was determined by measuring the amount of 32 Pi in the supernatant after centrifugation of the assay mixture. Relevant assay methods are also disclosed, for example, in Berman et al, 1996, J. Biol. Chem. 271:27209. A method of identifying or assaying for a molecule that modulates the stimulatory effect of an activated Gα subunit on a RGS-GEF polypeptide having GEF mediated nucleotide exchange for a monomeric G protein can also be conducted in accordance with this invention. For instance, a first assay can be conducted by incubating an activated Gα alpha subunit with a GEF protein (e.g., pi 15 Rho GEF, Lsc, KIAA380, DrhoGEF2, or biologically-active fragments thereof, which retain GEF activity) and a monomeric G protein in the presence and absence of a test modulator to determine whether the test modulator has an inhibitory, enhancing, etc. effect on the ability of an activated Gα subunit to stimulate GEF mediated nucleotide exchange of a monomeric protein. See e.g. Hart et al., 1996, J. Biol. Chem. 221:25452. The test modulator can be further evaluated by conducting a second assay in which said GEF protein and a monomeric G protein, without the G protein subunit, are incubated in the presence or absence of the test modulator to determine whether the test modulator had any effect on GEF mediated nucleotide exchange of the monomeric protein, and then comparing the modulation effect in the first and second assays to determine whether the modulating effect in the first assay is different from the modulating effect in the second assay, thereby indicating that the test modulator modulates the interaction of an activated Gα subunit with the GEF protein rather than the interaction of the GEF protein with the monomeric G protein. For example, the stimulatory effect on GEF mediated guanine nucleotide exchange may be measured according to Example 6 below, wherein RhoA was loaded with [ 3 H]GDP and the dissociation of GDP from RhoA was measured under various conditions by the determination of bound GDP by filtration, prior to an after incubation. (See e.g. Northrup et al., J. Biol Chem., 257, 11416-11423 (1982)). A method of identifying a molecule that mimics the stimulatory effect of an activated Gα subunit on GEF mediated nucleotide exchange of a monomeric G protein may also be conducted in accordance with the invention. The method comprises identifying a test compound that exhibits a binding affinity for the RGS domain of GEF proteins and then incubating a GEF protein and monomeric G protein in the presence or absence of the test compound to determine whether the test compounds exhibits a stimulatory effect on GEF mediated nucleotide exchange of a monomeric G protein. The identification of test compounds that exhibit a binding affinity for the RGS domain of GEF proteins may be accomplished using techniques well known in the art. For example, an RGS polypeptide may be bound to a column and cocktails of test compounds may be passed over the column to determine if any were selectively bound by the column. A method of identifying a molecule, or mixture of molecules, that mimics the stimulatory effect of an RGS domain of GEF polypeptide on Gα subunit GTPase activity may also be conducted in accordance with the invention. The method comprises identifying a test compound that exhibits a binding affinity for a Gα subunit and incubating a GTP loaded Gα subunit in the presence or absence of the test compound to determine whether the test compound exhibits a stimulatory effect GTPase activity of the Gα subunit. The identification of test compounds that exhibit a binding affinity for the G α subunit may be accomplished using techniques well known in the art. For example, a Gαπ may be bound to a substrate and incubated with both a GEF polypeptide containing an RGS domain and the test compound to determine whether the test compound competes with the RGS domain for binding to the Gα subunit. The modulation of oncogenic transforming activity by an RGS-GEF component, or derivatives thereof, can be measured according to various known procedures, e.g., Eva and Aaronson, Nature, 316:273-275, 1985; Hart et al., J. Biol. Chem., 269:62-65, 1994. A compound can be added at any time during the method (e.g., pretreatment of cells; after addition of the RGS-GEF, etc.) to determine its effect on the oncogenic transforming activity of the RGS-GEF component. Various cell lines can also be used. Other assays for monomeric GTPase-mediated signal transduction can be accomplished according to the invention by analogy to procedures known in the art, e.g., as described in U.S. Pat. Nos. 5,141,851 ; 5,420,334; 5,436,128; and 5,482,954; W094/16069; WO93/16179; WO91/15582; WO90/00607. The present invention thus also relates to the treatment and prevention of diseases and pathological conditions associated with signal transduction mediated by GEF proteins that contain an RGS domain, e.g., cancer, diseases associated with abnormal cell proliferation. For example, the invention relates to a method of treating cancer comprising administering, to a subject in need of treatment, an amount of a compound effective to treat the disease, where the compound is a regulator of the stimulatory effect of GEF protein containing an RGS on Gα subunit GTPase activity or where the compound is a regulator of the stimulatory effect of a Gα subnit on GEF mediated nucleotide exchange by a monomeric GTPase. Treating the disease can mean, delaying its onset, delaying the progression of the disease, improving or delaying clinical and pathological signs of disease. A regulator compound, or mixture of compounds, can be synthetic, naturally-occurring, or a combination. A regulator compound can comprise amino acids, nucleotides, hydrocarbons, lipids, polysaccharides, etc. A regulator compound is preferably a compound that regulates expression of a GEF protein containing an RGS domain, e.g., inhibiting or increasing its mRNA, protein expression, or processing, or a compound that regulates the interaction of the RGS domain of the GEF protein with a Gα subunit. To treat the disease, the compound, or mixture, can be formulated into pharmaceutical composition comprising a pharmaceutically acceptable carrier and other excipients as apparent to the skilled worker. See, e.g., Remington's Pharmaceutical Sciences, Eighteenth Edition, Mack Publishing Company, 1990. Such composition can additionally contain effective amounts of other compounds, especially for treatment of cancer. EXAMPLES Example 1. Identification of homology between a Rho GEF and proteins which regulate G- protein signaling. The RGS family of proteins act as negative regulators of G protein signalling. Nineteen mammalian members of the family have been identified, all of which encode proteins that contain a homologous core domain called the RGS box. Examination of the sequence of pi 15-GEF, a GEF specific for Rho, revealed an N- terminal region with specific homology to the conserved domain of RGS proteins, including RGS4, RGS2, GAIP, RGS 12, and RGS 14 (Fig. 1). Analysis of three other Rho GEF proteins, Lsc, KIAA380, and DrhoGEF also showed that they contained regions of specific homology to the conserved domain of RGS proteins (Fig. 1). The crystal structure of a complex between RGS4 and AlF 4 -activated Gα,ι revealed that the functional core of RGS4 (the RGS box) contains nine α-helixes that fold into two small subdomains (Tesmer et al., Cell, 89, 251 (1997)). The RGS box has been shown to contain the GAP activity towards Gα subunits (Popov et al., Proc. Natl Acad. Sci. USA, 94, 7216 (1997)). The hydrophobic core residues of the box, which are conserved in members of the RGS family, are important for stability of structure and GAP activity (Tesmer et al., Cell, 89, 251 (1997) and Srinivasan et al., J. Biol. Chem., 273, 1529 (1998). RGS4 stimulates the GTPase activity of Gα^ by interacting with its three switch regions, primarily by stabilization of the transition state of GTP hydrolysis (Tesmer et al., Cell, 89, 251 (1997)). Most of the hydrophobic residues that form the core of the RGS domain are conserved in pi 15 Rho GEF (17 out of 23) (Fig. 1). The position of gaps in the alignment correspond to the loops between alpha helixes of RGS domain structure. This homology suggested that the N-terminal region of pi 15-GEF may have a similar structure to the RGS4 box domain and possess GAP activity. In contrast, the residues of RGS4 that make contact with the switch regions of Gα ϋ (GDP-AlF 4 -) are not well conserved, and any GAP activity of pi 15 Rho GEF will have a unique mechanism or a significantly different specificity than those previously identified. A search of the gene bank revealed three other Rho-GEF members that have regions homologous to the RGS region of pi 15. These include Lsc, KIAA380, and DrhoGEF2 (Fig. 1). Lsc appears to be the mouse homolgue of pi 15 RhO GEF and KIAA380 appears to be the human homolgue of Drosophila DrhoGEF2 (Whitehead et al., J. Biol. Chem., 271, 18643 (1996); Barrett et al., Cell, 91, 905 (1997)). These four Rho-GEF's define a new RGS related family of proteins which also possess guanine nucleotide exchange activtity for Rho. An alignment of RGS domains of the four GEF proteins known to contain RGS domains (pi 15 Rho GEF, Lsc, KIAA380, DRhoGEF2) with the RGS domains of RGS proteins RET-RGS1, RGS1, RGS2, RGS3, RGS4, RGS7, RGS 10, RGS 12, RGS 14, Rapl/2B.P., and GAIP shows that a novel sub-RGS consensus sequence is defined by the RGS sequence of the four GEF proteins (Fig. 18). As shown in the bottom set of sequences shown in Fig. 18, a novel sub-RGS consensus sequence is shown by the large gap of 13 to 14 amino acids in the homology alignment, along with the conservation of amino acids on either side of the gap. Example 2. The RHO GEF protein, pi 15 RHO-GEF. stimulates the GTPase activity of Gαπ and Gap subunits. PI 15 Rho GEF was tested to determine it's capability in stimulating the intrinsic GTPase activity (GAP activity) of Gαπ and G π. Gαπ was expressed in Sf9 cells and purified as described in Kozasa and Gilman, J. Biol. Chem., 270, 1734 (1995). Gαπ was prepared by a similar procedure using the previously described baculovirus method (Singer and Miller, J. Biol. Chem., 269, 19796 (1994)) and octylglucoside during washing and elution of the α subunit after immobilization of the heterotrimer on Ni-NTA resin (Qiagen). The eluted Gαπ was further purified by absorption to and elution from hydroxyapatite. Gαπ or Gαπ (20-30 pmol) was loaded at 30°C for 30 or 40 minutes, respectively with 5 μM[γ- 32 P]GTP (50-100 cpm/fmol) and in the presence of 5 mM EDTA. Samples were then rapidly filtered by centrifugation at 4°C through Sephadex G50 which had been equilibrated with buffer A (50 mM NaHepes (pH 8.0), 1 mM dithiolthreitol, 5 mM EDTA, and 0.05% polyoxyethylene 10-laurylether) to remove free [γ- 32 P]GTP and [ 32 Pi]. Hydrolysis of GTP was initiated by adding Gα loaded with [γ- 32 P]GTP in buffer A containing 8 mM MgSO 4 , 1 mM GTP and the indicated amount of pi 15. The reaction mixture was incubated at 4°C or 15°C. Aliquots (50 μl) were removed at the indicated times and mixed with 750 μl of 5%(w/v) NoritA in 50 mM NaH 2 PO 4 . The mixture was centrifuged at 2000 rpm for 5 minutes and 400 μl of supernatant containing 32 Pi were counted by liquid scintillation spectrometry. The hydrolysis of GTP bound to Gαπ and G π was performed at 15°C either with or without lOnM full-length pi 15 (Fig. 2, Panel A). The hydrolysis of GTP bound Gα !3 and Gαπ was measured at 4°C in the presence of various concentrations of pi 15 (Fig. 2, Panel B). Full-length pi 15 was able to stimulate a single round of hydrolysis of [γ- 32 P]GTP which had been prebound to the G π subunit. The intrinsic GTPase activity of Gαπ, the closest homologue of Gαπ, was also stimulated by full-length pi 15. At 15°C, the k cat for hydrolysis of GTP by Gαπ (0.07 min "1 ) and Gαπ (0.24 min "1 ) were respectively increased 5-fold and 10-fold by 10 nM pi 15 (Fig. 2, Panel A). Similar results were obtained with several preparations of Gαπ and Gαπ. Treatment of pi 15 at 90°C inactivated this GAP activity. Due to the rapid hydrolytic rates of Gαπ, assays were performed at 4°C to better estimate the effect of pi 15 on the initial rate of GTPase activity by the G protein (Fig. 2, Panel B). Under these conditions, 100 nM pi 15 caused and 80-fold increase in the GTPase activity of Gαπ. In contrast, the hydrolytic rate of Gαπ was increased only 6-fold. Although stimulation of both proteins was observed at concentrations of pi 15 as low as 1 nM, measurements at both temperatures indicate that pi 15 is a more efficacious GAP for Gαπ than Gαπ. In the absence of a receptor, the rate limiting step in the binding of GTPγS to Gα and the steady state hydrolysis of GTP is the release of GDP. PI 15 did not affect either the rate of GTPγS binding to G π and Gαπ or the steady state of GTPase activity of either subunit. Therefore, pi 15 stimulates only the intrinsic GTPase activity of Gαπ and Gαπ without effecting their rates of nucleotide exchange. The conserved RGS box region of RGS proteins is sufficient to show GAP activity in vitro (Popov et al., Proc. Natl Acad. Sci. USA, 94, 7216 (1997)). Therefore, a fusion protein (Fig. 1 , Panel B) of glutathione-S-transferase and the N-terminal region of pi 15, GST-RGS, was tested for GAP activity. This region retains RGS homology domain but not the Dbl or PH domains of pi 15. This "RGS domain" of pi 15 (10 nM) was almost as active as full-length pi 15 when tested for GAP activity for G π and Gαπ (Fig. 3). In contrast, a construct of pi 15 missing this N-terminal region was ineffective. Thus, the data indicates that the RGS homology region is responsible for the GAP activity of p 115. Example 3. The pi 15 RHO-GEF, does not stimulate the GTPase activity of Gα,. Gα z Gα α and G ; subunits. The specificity of the GAP activity of pi 15 for various G protein α subunits was examined as follows. Gαs was expressed in and purified from Escherichia coli as described in Lee et al., Meth. Enzymol, 237, 146 (1994). Gα^ Gα 2 , and Gα q R183C were expressed in Sf9 cells and purified as described in Kozasa and Gilman, J. Biol Chem., 270, 1734 (1995) and Biddlecome et al., J. Biol. Chem., Ill, 7999 (1996). Gα„ Gα z , and Gα z were loaded with 5- 10 μM [γ- 32 P]GTP at 20°C (for Gα s ) or 30°C (for Gα, and Gα z ) for 20 minutes in the presence of 5 mM EDTA and GAP assays were performed as described above for Gαπ and Gαπ. Gap activity on Gα q was assessed with a mutant Gα q R183C. An analogous mutation in Gα, Rl 178C, causes markedly reduced GTPase activity but response to RGS proteins was retained (Berman et al., Cell, 86, 445 (1996)). The slow GTPase activity of Gα q R183C enables loading of [γ- 32 P]GTP on Gα q without using receptor to accelerate nucleotide exchange. Gα q R183C was loaded with 10 μM [γ- 32 P]GTP in the presence of 50 mM Hepes (pH7.4), 0.1 mg/ml BSA, 1 mM DTT, 1 mM EDTA, 0.9 mM MgSO 4 , 30 mM (NH 4 ) 2 SO 4 , 4% glycerol, and 5.5 mM CHAPS at 20°C for 2 hours. The reaction mixture was rapidly filtered through Sephadex G50 which had been equilibrated with 50 mM Hepes (pH 7.4), 1 mM DTT, 1 mM EDTA, 0.9 mM SO 4 , 0.1 mg/ml BSA, and 1 mM CHAPS. The results of this study showed that pi 15 (100 nM) did not stimulate the GTPase activity of Gαi, Gα z, or Gα q under conditions where RGS4 acts as a GAP for these Gα subunits (Figure 4). Similarly, pi 15 did not accelerate the GTPase activity of Gα s , nor did pi 15 Rho GEF have any GAP activity towards RhoA or racl. Thus, pi 15 is a GAP with specificity for Gαπ and Gαπ. Example 4. Selective inhibition of pi 15 GAP activity by AffV activated forms of Gα subunits. RGS proteins have been shown to have high affinity for the GDP-AIF4 " bound form of α subunits, a configuration similar to the transition state of GTP hydrolysis (Tesmer et al., Cell, 89, 251 (1997), Berman et al., J. Biol. Chem., 211, 27209 (1996)). Therefore, the GDP-AHY forms of Gα should compete with GαGTP for interaction with pi 15 and block the observed GAP activity. As shown in Fig. 5, Panel A, GDP-AEEV bound Gαπ and Gαπ effectively inhibited the GAP activity of pi 15 for Gαπ, while similar forms of Gα s , Gα;, and Gα q were without effect. Additionally, a tritration of GDP-AIF 4 " bound forms of Gαπ and Gαπ demonstrated that the subunits are equipotent in inhibiting the GAP activity of Gαπ (Fig. 5, Panel B). These competition assays suggest that the two G protein subunits have a similar affinity for pi 15 and supports the apparent differential efficacy of pi 15 towards the subunits as shown in Fig. 2. Example 5. Binding of Gαn to pi 15 Rho GEF in vivo. The following experiments demonstrated that Gαπ and pi 15 Rho GEF interact in a GTP-dependent manner. EXV-myc tagged (for COS cell transfections) and pAc-Glu tagged (for baculovirus expression) proteins with deletions of the RGS or DH domains were constructed as previously described in Hart et al., J. Biol Chem., 211, 25452-25458 (1996). Full-length versions were constructed in the same vectors. A fusion of GST to the first 246 amino acids of pi 15 Rho GEF was constructed in pGEX4T-2 (Pharmacia). Transfections, immunoprecipitations, and purifications were performed as previously described in Hart et al., J. Biol Chem., 271, 25452-25458 (1996). In COS cells transfected with myc -tagged pi 15 Rho GEF, Gαπ can be specifically immunoprecipitated using the anti-myc antibody (Fig. 6, Panels A and B). This interaction is dependent on the presence of aluminum fluoride which is added to mimic the activated GTP-bound state of the Gα J . Additionally, a truncated mutant of pi 15 Rho GEF which lacks the amino-terminal RGS domain is incapable of mediating co-immunoprecipitation, while full-length protein with a deletion in the DH domain does mediate co- immunoprecipitation. The differential binding of full-length and truncated Rho GEF proteins could also be detected using antibodies to Gαπ to immunoprecipitate the complex (Fig. 6, Panel C). A very weak interaction with Gαπ was detectable, while antibodies to Gα s , Gas, Gα q and Gα z do not detect immunoreactive bands in the anti-myc immunoprecipitates, in spite of the fact that their respective antigens are detectable in the whole cell lysates. The co-immunoprecipitation of pi 15 Rho GEF and Gαπ can be reproduced in a semi-purified system in which purified Gαπ is added to immunoprecipitated pi 15 Rho GEF (Fig. 6, Panel D), suggesting a direct interaction. This direct interaction is consistent with the observation that pi 15 Rho GEF stimulates Gαπ GTPase activity, but also indicates that pi 15 Rho may be an effector of Gαπ- Binding could also be detected between the Rho GEF protein, KIAA380 and the απ G protein subunit (Fig. 9, KIAA380 is referred to as FL147). In COS cells transfected with myc -tagged KIAA380, Gαπ can be specifically immunoprecipitated using the anti-myc antibody (Fig. 9. Panels A and B, KIAA380 is referred to as FL147). This interaction is dependent on the presence of aluminum fluoride which is added to mimic the activated GTP-bound state of the Gαι 3 . Example 6. Stimulation of pi 15 Rho GEF activity by Gαπ. The ability of Gαπ to affect the exchange activity of pi 15 Rho GEF was examined by incubating RhoA and pi 15 Rho GEF with or without Gαπ to determine the effect on guanine nucleotide exchange. RhoA (2.5 μM) was loaded with [ 3 H]GDP by incubation at 30°C for 1 hour with 25 μM GDP (10,000 cpm pmol) in 50 mM NaHepes, pH 7.5, 50 mM NaCl, 4 mM EDTA, ImM dithiolthreitol and 0.1%Triton X-100. After addition of MgCl 2 to 9 mM and octylglucoside to 1%, the Rho was incubated for an additional 5 minutes and separated from free GDP by rapid filtration through Sephadex-G50 that had been equilibrated with 50 mM NaHEPES, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM dithiolthreitol, 5 mM MgCl 2 , and 1% octylglucoside. Dissociation of GDP from RhoA was measured at 30°C in 20 μl of 50 mM NaHEPES, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM dithiolthreitol, 5 mM MgCl 2 , 30 mM A1C1 3 , 5 mM NaF, and 5 μM GTPγS. Unless specified, G protein alpha subunits were preincubated with AMF (30 μM A1C1 3 , 5 mM MgCl 2 and 5 mM NaF) prior to mixing with other proteins. Where indicated, alpha subunits were treated with 25 μM GTPγS or GDPβS rather than AMF and reactions were incubated without AMF but with 5 μM of the respective nucleotide. Reactions were started with the addition of [ 3 H]-GDP-RhoA and bound GDP was determined by filtration (Northup et al., J. Biol. Chem., 257, 11416-11423 (1982)) prior to and after incubation. The Gα s and Gαj alpha subunits were purified after expression in Escherichia coli (Lee et al., Meth. Enzymol, 237, 146-164 (1994)). The Gα q and Gα z alpha subunits were coexpressed in Sf9 cells with hexahistidine-tagged beta and gamma subunits and isolated as described (Kozasa and Gilman, J. Biol. Chem., 270, 1734-1741 (1995)). Gα ] 3 was prepared by a similar procedure to Gαπ using baculovirus (Singer et al., J. Biol. Chem., 269, 19796- 19802 (1994)) and octylglucoside during washing and elution of the α subunit after immobilization of the heterotrimer on Ni-NTA resin (Qiagen). The eluted Gαπ was further purified by absorption to and elution from hydroxyapatite. About 500 ug of purified Gαπ can be obtained from 3 liters of cells. The expression of GST-RhoA in Sf9 cells, cleavage of the GST tag and isolation of the free RhoA were as described in Singer et al., J. Biol. Chem., Ill, 4505-4510, (1996). These studies demonstrated that the Gαπ is capable of stimulating the activity of full-length pi 15 Rho GEF in a manner which depends on the concentrations of both pi 15 Rho GEF (Fig. 7, Panel A) and Gαπ (Fig- 7, Panel B). The closely related alpha subunit Gαπ was ineffective in stimulating the activity of pi 15 Rho GEF in these experiments (Fig. 7, Panel A). Stimulation of Rho exchange was also monitored as a function of the activation state of Gan- The data graphed in Fig. 7, Panel C confirm that the stimulation of exchange activity is dependent on either aluminum fluoride (AMF) or GTPγS, but is not stimulated by the deactivated nucleotide state mimicked by GDPβS. Additionally, a series of other alpha subunits including Gα q , Gα z , Gα s , and Gαj also did not affect the activity of pi 15 Rho GEF (Fig. 7, Panel D). These results are consistent with with the activated Gαπ -dependent binding shown in Figure 6, and suggest that the productive binding of Gαπ to pi 15 Rho GEF may be sufficient for activation. Example 7. Effects of domains of pi 15 and G π on the pi 15 nucleotide exchange activity. The theory that the RGS domain of pi 15 Rho GEF is normally autoinhibitory and that binding to G π relieves this inhibition was examined by comparing the effects of full- length Rho-GEF versus truncated Rho-GEF on Rho exchange activity. Preparation of pi 15 proteins was as described in Example 1 above and as described in Hart et al., J. Biol. Chem., 271, 25452-25458 (1996). The assays shown in Figure 8, Panels B and C were performed as described in Example 5 above. AMF was the activating agent. The results of these experiments showed that truncated pi 15 Rho GEF lacking the RGS domain demonstrates consistently elevated Rho exchange activity when compared with equal concentrations of the full-length protein (Fig. 8, Panel A). Additionally, addition of the isolated RGS domain (as a GST fusion protein) resulted in abrogation of Gαπ- stimulated pi 15 Rho GEF activity (Fig. 8, Panel B). These data do not preclude additional Gαπ-binding sites on pi 15 Rho GEF, although they do suggest a primary mode of action via the RGS domain. The inability of the Gαπ subunit to activate pi 15 Rho GEF was puzzling in light of the fact that pi 15 Rho GEF is capable of activating the GTPase of both Gαπ and Gαπ- Therefore, an experiment was conducted in which Gαπ was added to a Gαπ-stimulated pi 15 Rho GEF assay (Fig. 8, Panel C). The results showed that Gαπ was able to inhibit the coupling of Gαπ with pi 15 Rho GEF. This data is consistent with a model in which G π competes with Gαπ for binding to the RGS domain of pi 15 Rho GEF. However, binding of Gαπ to pi 15 Rho GEF is clearly not sufficient to stimulate Rho exchange activity. These results suggest that either the interaction of Gαπ with the RGS domain of pi 15 Rho GEF is quite different from that of Gαπ or that there may be an additional site of interaction between Gα ]3 and pi 15 Rho GEF. For other aspects of the nucleic acids, polypeptides, antibodies, etc., reference is made to standard textbooks of molecular biology, protein science, and immunology. See, e.g., Davis et al. (1986), Basic Methods in Molecular Biology, Elsevir Sciences Publishing, Inc., New York; Hames et al. (1985), Nucleic Acid Hybridization, IL Press, Molecular Cloning, Sambrook et al.; Current Protocols in Molecular Biology, Edited by F.M. Ausubel et al., John Wiley & Sons, Inc; Current Protocols in Human Genetics, Edited by Nicholas C. Dracopoli et al., John Wiley & Sons, Inc.; Current Protocols in Protein Science; Edited by John E. Coligan et al., John Wiley & Sons, Inc.; Current Protocols in Immunology; Edited by John E. Coligan et al., John Wiley & Sons, Inc. The entire disclosure of all patent applications, patents, and publications cited herein are hereby incorporated by reference. From the foregoing description, on skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Claims: CLAIMS What is claimed is: 1. An isolated RGS-GEF polypeptide, or a biologically active fragment thereof, consisting essentially of an RGS domain of a GEF protein. 2. An isolated RGS-GEF polypeptide, or a biologically active fragment thereof, comprising an RGS domain of a GEF protein, with the proviso that the polypeptide does not comprise a DH domain or a PH domain. 3. An isolated RGS-GEF polypeptide, or a biologically active fragment thereof, wherein the polypeptide is selected from the group consisting of pi 15 Rho-GEF, Lsc, KIAA380, and wherein the polypeptide is mutated in the RGS domain, and wherein the polypeptide has a specific binding affinity for a G protein α subunit or a GTPase activating activity for a G protein α subunit. 4. An isolated RGS-GEF polypeptide, or a biologically active fragment thereof, according to claim 1 or 2, wherein the GEF protein is a Rho GEF protein. 5. An isolated RGS-GEF polypetide, or a biologically active fragment thereof, according to claim 4, wherein the Rho GEF protein is pi 15 Rho-GEF. 6. An isolated RGS-GEF polypeptide, or biologically active fragment thereof, according to claim 4 wherein the Rho GEF protein is selected from the group consisting of Lsc, KIAA380, and DrhoGEF2. 7. An isolated RGS-GEF polypeptide, or a biologically active fragment thereof , according to claim 1 or 2, wherein the polypeptide has a specific binding affinity for a G protein α subunit or a GTPase activating activity for G protein α subunits. 8. An isolated RGS-GEF polypeptide, or a biologically active fragment thereof, according to claim 4, wherein the polypeptide has a specific binding affinity for a G protein α subunit or a GTPase activating activity for G protein α subunits. 9. An isolated RGS-GEF polypeptide, or a biologically active fragment thereof, according to claim 5, wherein the polypeptide has a specific binding affinity for a G protein α subunit or a GTPase activating activity for G protein α subunits. 10. An isolated RGS-GEF nucleic acid consisting essentially of a nucleotide sequence encoding a polypeptide comprising an RGS domain of a GEF protein. 11. An isolated RGS-GEF nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising an RGS domain of a GEF protein, wherein the polypeptide does not include a DH domain or a PH domain. 12. An isolated RGS-GEF nucleic acid according to claim 10 or 11, wherein the GEF protein is a Rho GEF protein. 13. An isolated RGS-GEF nucleic acid according to claim 12, wherein the Rho GEF protein is p 115 Rho GEF. 14. An isolated RGS-GEF nucleic acid according to claim 12 wherein the Rho GEF protein is selected from the group consisting of Lsc, KIAA380, and DrhoGEF2. 15. An isolated RGS-GEF nucleic acid according to claim 10 or 11, wherein the polypeptide has a specific binding affinity for a G protein α subunit or a GTPase activating activity for a G protein α subunit. 16. An isolated RGS-GEF nucleic acid according to claim 12, wherein the polypeptide has a specific binding affinity for a G protein α subunit or a GTPase activating activity for a G protein α subunit. 17. An isolated RGS-GEF nucleic acid according to claim 13, wherein the polypeptide has a specific binding affinity for a G protein α subunit or a GTPase activating activity for a G protein α subunit. 18. A method of modulating an activity of a G protein α subunit comprising, administering to a mammal an effective amount of a polypeptide according to claim l or 4. 19. A method of identifying or assaying a molecule that inhibits or enhances binding of a monomeric G protein guanine nucleotide exchange factor to a G protein α subunit comprising incubating the G protein α alpha subunit, or fragments thereof, with the monomeric G protein nucleotide exchange factor, or fragments thereof, in the presence and absence of a test molecule and determining whether the presence of the test molecule inhibits or enhances binding between the monomeric G-protein guanine nucleotide exchange factor and the G protein α subunit. 20. A method of identifying or assaying a molecule that inhibits or enhances a stimulatory effect of a GEF on a Gα subunit GTPase activity comprising incubating a Gα alpha subunit, or fragments thereof, with a GEF protein, or fragments thereof, in the presence and absence of a test molecule and determining whether the presence of the test molecule inhibits or enhances the stimulatory effect of the GEF protein on Gα subunit GTPase activity. 21. A method of identifying or assaying a molecule that specifically inhibits the stimulatory effect of an activated Gα subunit on GEF mediated nucleotide exchange of a monomeric G protein, compising conducting a first assay by incubating an activated Gα alpha subunit, or fragments thereof, with a GEF protein, or fragments thereof, and a monomeric G protein, or fragments thereof, in the presence and absence of a test inhibitor, conducting a second assay by incubating a GEF protein, or fragments thereof, and a monomeric G protein, or fragments thereof, in the presence and absence of the test inhibitor, and determining whether any inhibitory effect of the test inhibitor in the first assay is greater than any inhibitory effect of the test inhibitor in the second assay. 22. A method of identifying or assaying a molecule that specifically enhances the stimulatory effect of an activated Gα subunit on GEF mediated nucleotide exchange of a monomeric G protein, compising conducting a first assay by incubating an activated Gα alpha subunit, or fragments thereof, with a GEF protein, and fragments thereof, and a monomeric G protein, or fragments thereof, in the presence and absence of a test enhancer, conducting a second assay by incubating a GEF protein, or fragments thereof, and a monomeric G protein, or fragments thereof, in the presence and absence of the test enhancer, and determining whether any enhancing effect of the test enhancer in the first assay is greater than any enhancing effect of the test enhancer in the second assay. 23. A method of identifying or assaying a molecule that mimics the stimulatory effect of an activated Gα subunit on GEF mediated nucleotide exchange of a monomeric G protein comprising identifying a test compound that exhibits a binding affinity for the RGS domain of GEF proteins, or fragments thereof, incubating a GEF protein,or fragments thereof, and monomeric G protein, or fragments thereof, in the presence or absence of the test compound, determining whether the test compound exhibits a stimulatory effect on GEF mediated nucleotide exchange of a monomeric G protein. 24. A method of identifying or assaying a molecule that mimics the stimulatory effect of an RGS domain of a GEF protein on GTPase activity of a Gα subunit comprising identifying a test compound that exhibits a binding affinity for a Gα subunit and incubating a GTP loaded Gα subunit in the presence or absence of the test compound to determine whether the test compound has a stimulatory effet on Gα subunit GTPase activity. 25. A method according to claim 19, 20, 21, 22, 23, or 24 wherein the GEF protein is selected from the group consisting of pi 15 Rho GEF, Lsc, KIAA380, and DrhoGEF2. 26. A method of expressing in transformed host cells, a polypeptide coded for by a nucleic acid, comprising culturing transformed host cells containing a nucleic acid according to claim 11. 27. A transformed cell containing a nucleic acid according to claim 11. 28. A vector comprising a nucleic acid according to claim 11.

Inventors: BOLLAG GIDEON, HART MATTHEW J, JIANG XUEJUN, KOZASA TOHRU, POLAKIS PAUL, ROSCOE WILLIAM, STERNWEIS PAUL, BOLLAG, GIDEON, HART, MATTHEW, J., JIANG, XUEJUN, KOZASA, TOHRU, POLAKIS, PAUL, ROSCOE, WILLIAM, STERNWEIS, PAUL
Applicants: ONYX PHARMA INC, ONYX PHARMACEUTICALS, INC.
Application year: 1999
Main classification: C07K14
IPC codes: C07K14, C12N5, C12N9, C12N15, C12Q1