WO1996029089A1 - A novel ras p21-interacting protein and uses thereof - Google Patents

Abstract

A novel protein which interacts with ras p21, has been identified. This protein, RGL, shares 69 % amino acid homology with ral guanine nucleotide dissociation stimulator (ralGDS). ralGDS was also found to bind ras p21. The ras p21-interacting domain of RGL (RID) bound to ras p21 through the effector loop of ras p21. Polypeptide and polynucleotide compositions of the RGL protein are provided as well as methods for implementing the diagnostic and therapeutic uses of these compositions. The uses include isolating effector proteins of ras p21 and modulating ras activity.

Description

A NOVEL RAS P21 -INTERACTING PROTEIN AND USES THEREOF

This invention was made with government support under Grant No. HL07731, awarded by the National Institutes of Health. The government has certain rights in this invention.

BACKGROUND OF THE INVENTION ras p21 is a member of the small GTP-binding protein (G-protein) superfa ily. ras proteins are thought to regulate key signalling pathways involved in cell growth and development. ras proteins reside on the inner surface of the plasma membrane where they participate in transmitting signals from tyrosine kinase receptors and some receptors coupled to heterotrimeric G proteins.

Because mutated ras proteins have been found in lung, bladder, colon, and many other human carcinomas, and are associated with human carcinogenesis, it is of value to study the processes by which ras mediates cell proliferation and differentiation. One approach to dissecting ras function is to identify and study the molecules that interact with ras , specifically the regulatory and effector molecules of ras p21. Knowledge of the interacting proteins in the ras signaling pathway will allow the screening of drugs for agonists and antagonists of ras-dependent cell proliferation and differentiation. Until recently, the hunt for direct downstream targets through which ras acts has not been fruitful. The discoveries of the present invention provide tools useful to elucidate the mechanism of ras signaling and new approaches to modulate ras activity and to alleviate disease conditions due to ras dysfunction. SUMMARY OF THE INVENTION ras p21 is a member of the small GTP-binding protein (G-protein) superfamily. ras proteins regulate key signalling pathways involved in cell growth and development. A novel protein, RGL, which interacts with ras p21, has been identified. RGL was found to share 69% amino acid homology with ral guanine nucleotide dissociation stimulator (ralGDS) , a GDP/GTP exchange protein for ral p24, ralGDS was similarly tested and was found to bind ras p21. It was found that the ras p21-interacting domain of RGL (RID) binds to ras p21 through the effector loop of ras p21.

The present invention provides the nucleotide and amino acid sequence of RGL as well as polypeptide and polynucleotide compositions based on RGL, including the RID of RGL. The uses and methods of use of the RGL polynucleotide and polypeptide compositions are disclosed.

One aspect of this invention is to provide an isolated polynucleotide comprising at least 80% sequence identity with the nucleotide sequence of SEQ ID N0:1, an allelic or species variation thereof, or a fragment thereof. A polynucleotide comprising the nucleotide sequence of SEQ ID N0:1 or the sequence of SEQ ID NO:3 is provided.

Another aspect of the invention is to provide an isolated polypeptide comprising the sequence of SEQ ID NO:2, an allelic or species variation thereof, or a fragment thereof. An isolated polypeptide comprising the sequence of SEQ ID NO:4, or at least 80% sequence identity to SEQ ID NO:4, is also provided. In a specific embodiment, the polypeptide is one capable of binding the effector loop of ras p21. Another aspect of the invention is to provide an isolated polypeptide comprising the sequence of SEQ ID NO:2, an allelic or species variation thereof, or a fragment thereof, wherein the isolated polypeptide is a fusion protein. A RID-GAL4 fusion protein is specifically provided. In one aspect, the fusion protein comprises a tag, or a product of a second gene or fragment of that second gene product. A polypeptide is provided wherein the tag is GST, an epitope tag or an enzyme or wherein the second gene is lacZ. A further aspect of the invention is to provide a recombinant DNA molecule comprising the nucleotide sequence of SEQ ID NO:l or a fragment thereof. In one embodiment, the recombinant DNA molecule is pGAD/RID encoding a RID-GAL4 transactivation domain fusion protein. A recombination DNA molecule, pGAD/ralGDS, encoding a ralGDS-GAL4 transactivation domain fusion protein is also provided. A cell is provide which contains the recombinant DNA molecule comprising the nucleotide sequence of SEQ ID NO:l or a fragment thereof. Yet another aspect of the invention is the provision of antibodies that specifically bind a polypeptide comprising the sequence of SEQ ID NO:2, an allelic or species variation thereof, or a fragment thereof. In one embodiment, an antibody specifically binds to the polypeptide comprising the sequence of SEQ ID NO:4. These antibodies can be polyclonal or monoclonal. A hybridoma capable of producing a monoclonal antibody to any one of these described polypeptides is provided. Also provided is a kit comprising any antibody preparation to the above-mentioned polypeptides. In a different aspect of the invention, a method is provided for isolating a RGL gene or fragment thereof, comprising screening a DNA library using a RGL probe to identify a hybridizing clone and isolating said RGL gene or gene fragment from said hybridizing clone. An RGL probe suitable for use in this method is one which comprises the nucleotide sequence of SEQ ID NO:l or a fragment thereof. The method is useful to isolate a human RGL gene as well as RGL genes from other species.

A further aspect of the invention is to provide a method of identifying a gene encoding a ras p21-binding protein. The method comprises screening a DNA library with a RID probe to identify a hybridizing clone containing a RID sequence, the presence of a RID sequence being indicative of a gene encoding a ras p21-binding protein. Provided for use in this method is a RID probe comprising the sequence of SEQ ID NO:3 or a fragment thereof. The method is useful to identify genes encoding ras p21-binding proteins which are regulators or an effector proteins of ras p21. The invention also provides a method of identifying a ras effector loop-binding protein, comprising screening a gene library with an RID probe for a gene that is substantially homologous to the RGL gene, isolating the substantially homologous gene, producing a polypeptide encoded by the substantially homologous gene and finally determining if the polypeptide binds an effector loop of a ras protein, binding indicating that the polypeptide is a ras effector loop-binding protein. Another aspect of the invention is to provide a method of modulating or blocking ras p21 activity comprising providing a RID polypeptide in a cell expressing ras p21 protein wherein said RID polypeptide binds to said ras p21 protein to block ras p21 activity. In one embodiment, the RID polypeptide is provided by introducing an expression vector encoding a RID polypeptide into the ras p21 expressing cell. The RID polypeptide for use in this method will be derived from the RGL or ralGDS proteins.

It is yet another aspect of the invention to provide a method of blocking a ras p21 protein binding to a ras effector protein in a cell whereby the method comprises expressing a RID polypeptide from a RGL or a ralGDS protein in a cell. The ras effector protein can be Raf, GAP, NF1 or PI (3)K. Another aspect of the invention is a method of isolating a RGL interacting protein, comprising contacting a cell lysate suspected of containing a RGL interacting protein with a RGL polypeptide and isolating any protein bound to the RGL polypeptide, as a RGL interacting protein. If the RGL interacting protein is ras p21, a RID polypeptide can be used to bind and isolate ras .

The invention further provides a pharmaceutical composition useful in the treatment of a cell proliferative condition, the composition comprising a RID polypeptide and a pharmaceutically acceptable carrier. A second pharmaceutical composition useful for the same purpose includes an expression vector capable of expressing a RID polypeptide in an affected cell and a pharmaceutically acceptable carrier. It is another aspect of the invention to provide a method of alleviating a patient suffering from a cell proliferative condition, comprising administering to the patient, a therapeutically effective amount of the pharmaceutical composition described above. This method is useful to treat such cell proliferative conditions as cancer or restenosis caused by ras dysfunction.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. IA shows the nucleotide (SEQ ID NO: 1) and predicted amino acid sequences (SEQ ID NO: 2) of a 2.7 kb cDNA encoding RGL. The single-letter amino acid code is shown below the DNA sequence.

Fig. IB shows alignment of the amino acid sequences of RGL (SEQ ID NO:2) and ralGDS (SEQ ID NO:5). Sequences were aligned by using the Best Fit program. Amino acid identity is denoted by a black background. Dots indicate gaps. ralGDS refers to mouse ralGDSa.

Fig. 1C is a schematic representation of RGL and ralGDS sequence homology. Fig. 2A shows coexpression of ralGDS with ras p21 in

Sf9 cells. Aliquots (5 μl each) of lysates expressing no protein (lane 1), ralGDS alone (lane 2), both ralGDS and v-ras p21 (lane 3), or both ralGDS and ras p21^sl7N (lane 4) were probed with the anti-ra GDS and ras p21 antibodies. Fig. 2B shows the interaction of ralGDS with ras p21 in Sf9 cells. Sf9 cells expressing both ralGDS and -v-ras p21 (lanes 1 and 3) and ralGDS alone (lane 2) were lysed, and the proteins of the lysates were immunoprecipitated (IP) with the anti-ras p21 antibody (Ab; lanes 1 and 2) or nonimmune rat immunoglobulin (Ig; lane 3) . The precipitates were probed with the anti-ralGDS and ras p21 antibodies.

Fig. 2C shows the inability of ralGDS to interact with ras p21^sl7N. Sf9 cells coexpressing ralGDS with v-ras p21 (lane 1) or ralGDS with ras p21^sl7N (lane 2) were lysed, and the proteins of the lysates were immunoprecipitated with the anti-ras p21 antibody. The precipitates were probed with the anti-ralGDS and ras p21 antibodies. Fig. 2D shows the inability of Y13-259 to immunoprecipitate a ras p21-ralGDS complex. Sf9 cells expressing v-ras p21 alone (lanes 1 and 2) or both ralGDS and v-ras p21 (lanes 3 and 4) were lysed, and the proteins of the lysates were immunoprecipitated with Y13-238 (lanes 1 and 3) or Y13-259 (lanes 2 and 4) . The precipitates were probed with the anti-ralGDS and ras p21 antibodies. An arrowhead and an arrow indicate the positions of ralGDS and ras p21, respectively. The results shown are representative of three independent experiments.

Fig. 3A shows the protein staining of ralGDS and c- ras p21. The purified ralGDS and c-ras p21 (0.5 μg of protein each) were subjected to SDS-PAGE (12% polyacrylamide gel) and stained with Coomassie brilliant blue. Fig. 3B shows the interaction of ralGDS with the

GTP-bound form of ras p21. ralGDS (20 pmol) was incubated without (lane 1) or with the indicated amounts of the GTP7S- bound form (lanes 2 to 5) or GDP-bound form (lanes 6 to 9) of ras p21, and the mixtures were immunoprecipitated with the anti-ras p21 antibody. The precipitates were probed with the anti-ralGDS antibody. An arrowhead and an arrow indicate the positions of ralGDS and ras p21, respectively. The results shown are representative of three independent experiments.

Fig. 4A shows the time course for the GAP inhibition activity of ralGDS. The [γ-³²P]GTP-bound form of ras p21 was incubated for the indicated periods of time with or without 300 nM ralGDS in the presence or absence of 10 nM GST-NF1. The mixtures were then collected on filters, washed, and counted. __, without ralGDS or GST-NF1; A, with ralGDS; D, with GST-NF1, •, with ralGDS and GST-NF1.

Fig. 4B shows the dose-dependent effect of ralGDS and Raf on GAP inhibition activity. The [γ-³²P]GTP-bound form of ras p21 was incubated for 6 min with the indicated amounts of ralGDS or GST-N-Raf. •, with ralGDS; O, with GST-N-Raf. Fig. 5 shows the inhibition of the interaction of

Raf-1 with ras p21 by ralGDS. The [Qf-³²P]GTP-bound form of ras p2l was incubated for 30 min with 20 nM GST-N-Raf in the presence of the indicated amounts of ralGOS . GST-N-Raf was precipitated by using glutathione-Sepharose 4B, the precipitates were washed, and the remaining radioactivity was counted.

DESCRIPTION OF THE PREFERRED EMBODIMENT ras p21 is a member of the small GTP-binding protein

(G-protein) superfamily and plays an important role in cell growth and differentiation.

Mammalian ras genes are expressed in all cell lineages and organs, ras is synthesized in the cytosol and mature ras becomes associated with the inner side of the plasma membrane after posttranslational modifications where it participates in transmitting signals from tyrosine kinase receptors and some receptors coupled to heterotrimeric G proteins. ras p21 has GDP/GTP-binding and GTPase activities and cycles between the GDP-bound inactive and GTP-bound active forms. The GDP-bound inactive form can be activated by guanine nucleotide exchange proteins (Lowy et al., Annu. Rev. Biochem.. 62:851-891 (1993)) which promote the exchange of GDP for GTP, thereby converting ras p21 to the GTP-bound active form, ras proteins are then deactivated by interaction with GTPase-activating proteins (GAPs) that promote GTP hydrolysis by ras. Without being bound by a particular theory, it is presumed that the GTP-bound active form of ras p21 interacts with effector proteins that can mediate ras p21-dependent processes such as growth factor-stimulated cell proliferation.

Identification of effector proteins of the active form of ras has been difficult. One candidate effector protein of ras p21 is Raf, a cytoplasmic serine/threonine protein kinase that has been previously shown to act downstream of ras p21 (Dickson et al., Nature (London), 360:600-603 (1992) and Han et al., Nature (London), 363:133- 140 (1993)). It has been recently demonstrated that Raf interacts with the GTP-bound but not with the GDP-bound form of ras p21, that Raf binds to the effector loop of ras p21 (Van Aelst et al., Proc. Natl. Acad. Sci. USA, 90:6213-6217 (1993); Vojtek et al. , Cell. 74:205-214 (1993); and Warne et al., Nature (London), 364:352-355 (1993)), and that Raf inhibits the GTPase-activating activity of GTPase-activating protein (GAP) , which is known to interact with the effector loop of ras p21 (Warne, supra and Zhang et al., Nature

(London), 364:308-313 (1993)). These results have indicated that Raf is an effector protein of ras p21, consistent with previous observations that Raf acts downstream of ras p21 in signaling pathways that mediate both the differentiation and mitogenic responses to receptor tyrosine kinases (Dickson et al. , supra and Han et al., supra) . However, it is possible that ras p21 has effector proteins other than Raf, since ras p21 has multiple functions (Lowy, supra) .

The present invention identifies a novel protein that interacts with ras p21 in the yeast two-hybrid system. This protein, termed RGL (ra GDS-like) , is highly homologous with ral guanine nucleotide dissociation stimulation (ralGDS) , a GDP/GTP exchange protein for ral p24, a member of small G- protein superfamily and a 115-kD protein (Albright et al., EMBO J.. 12:339-347 (1993)). According to the present invention, the ras p21-binding domain of RGL binds to ras p21 through the effector loop of ras p21 and that this domain is highly conserved in ralGDS. Therefore, RGL and ralGDS were examined as to whether they could be effector proteins of ras p21. Since ralGDS has been well characterized, it was tested in this invention. Three characteristics that could be considered to be criteria for identification of an effector protein of ras p21 were tested: (i) the protein must interact with the GTP-bound active form of ras p21 but not with the GDP-bound inactive form, (ii) it must interact with ras p21 through the effector loop of ras p21, and (iii) ideally the protein should inhibit the interaction of ras p21 with other effector proteins such as Raf. By both in vitro and in vivo studies, it was demonstrated that ralGDS fulfills these three characteristics.

The yeast two-hybrid system (Chien et al. , PNAS. 88:9578-9582 (1991)) allows detection of proteins capable of interacting with a known protein that results in the immediate availability of the cloned genes for these interacting proteins. Briefly, the method is as follows. Plasmids are constructed to encode two hybrid proteins which are coexpressed in Saccharo yces cerevisiae. One hybrid consists of the DNA-binding domain of the yeast transcriptional activator protein GAL4 fused to the known protein; the other hybrid consists of the GAL4 activation domain fused to protein sequences encoded by a library of yeast genomic DNA fragments. Interaction between the known protein and a protein encoded by one of the library plasmids lead to transcriptional activation of a reporter gene containing a binding site for GAL4. A suitable reporter gene is the Saccharomyces cerevisiae HIS3 gene and the E. coli lacZ gene (encoding /3-galactosidase (3- gal)). Yeast cells are tested for growth in media lacking histidine and for expression of jS-gal activity which can be assayed by detecting blue colonies on a plate containing the substrate 5-bromo-4-chloro-3-indolyl /3-D-galactoside.

In the present invention, the yeast reporter strain is cotransformed with PC62/ras p21 which encodes a GAL4 DNA binding domain fused to c-H-ras, and P51/mouse embryonic cDNA library linked to the GAL4 transactivation domain.

A clone was identified that encodes a 164-amino-acid domain (RID) which interacts with H-ras p21. However, RID has no primary sequence homology with Raf and GAP, which interact with ras p21. Using this RID clone as a probe, cDNA of RGL was isolated and sequenced. The nucleotide and amino acid sequence of RGL is shown in Figure IA. The GenBank¹" Accession number for the RGL sequence is U14103. It was found that RGL shares 69% amino acid homology with ralGDS. It was also found that the RIDs of RGL and ralGDS are located on the C-terminal side of the CDC25-like domains of these molecules. The high degree of homology between the RIDs of RGL and ralGDS indicates that these domains are functionally important. The findings of the present invention indicate that ralGDS and RGL are in a family of proteins that contain a domain that binds to ras p21. Definitions

An "RGL interacting protein" or associated protein is one which has an affinity for RGL and binds or physically interacts with RGL. The term "RGL interacting molecule" does not imply any particular molecular size or other structural or compositional feature other than that the molecule or compound in question is capable of binding or otherwise interacting with RGL. This interaction can be transient, lasting only a fraction of a second or it can be stable so as to enable the detection of the complex of RGL-interacting molecule. Preferably, this interaction persists for at least ten seconds, ideally several minutes. The interacting molecule may be a substrate of RGL, an enzyme that acts on RGL, a protein that RGL is involved in localizing, an effector molecule of ras p21 or a molecule that alters the conformation of RGL upon interaction. Interacting or associating proteins that can be investigated by this invention include but are not restricted to agonists and antagonists for ras proteins, cellular proteins encoded by oncogenes or proto-oncogenes, lipids, toxins, hormones, sugars, cofactors, peptides, proteins, enzyme substrates, drugs and compounds from plant or animal sources.

The "effector loop of raε is a region in ras defined by amino acids 30-38 known to bind to effector proteins of ras such as Raf, GAP or NF1.

A "ras effector protein" is a protein capable of transmitting signals from ras to various intracellular signalling cascades. Ras effector proteins include phosphatidylinositol-3-OH kinase (PI(3)K) and the kinases Raf, Mek and Erk. However, the identification of ras effector proteins as described in the present invention encompasses only those effector proteins that directly physically interact or associate with ras p21.

Unless stated otherwise, "RGL protein or polypeptide" as used herein refers to the full length RGL protein, the RID polypeptide, fusion proteins of both and mutant derivatives of these proteins or polypeptides. The full-length RGL polypeptide and naturally occurring mutants can be the isolated, naturally produced form or recombinantly synthesized. Preferably, all other derivatives will be produced recombinantly.

"RID" which stands for ras p21-interacting domain, is the carboxy-ter inal 164 amino acid domain constituting amino acids 605-768 of the RGL protein. As the name suggests, RID interacts with ras p21, at the effector loop of ras. When the RID of ralGDS is mentioned, it refers to the region in the ralGDS protein that shares homology with the RID of RGL, the region encompassing amino acids 698-852 in ralGDS.

"CDC25-like domain" describes the region of RGL (amino acids 210 to 499) which is similar to a comparable region of CDC25, a GDP/GTP exchange protein for ras p21.

An "isolated polynucleotide" is a polynucleotide, e.g., an RNA, DNA, or a mixed polymer, which is substantially separated from other DNA sequences which naturally accompany a native human sequence, e.g. , ribosomes, polymerases, and many other human genome sequences. The term embraces a polynucleotide sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. A substantially pure molecule includes isolated forms of the molecule. An "isolated polypeptide" or protein carries a similar meaning with the polypeptide or protein being substantially separated from any cellular contaminants and components naturally associated with the protein in vivo.

An "allelic variation" in the context of a polynucleotide or a gene is an alternative form (allele) of a gene that exists in more than one form in the population. At the polypeptide level, "allelic variants" generally differ from one another by only one, or at most, a few amino acid substitutions. A "species variation" of a polynucleotide or a polypeptide is one in which the variation is naturally occurring among different species of an organism.

A "fragment" of a polynucleotide is a stretch of at least about 18 nucleotides, more typically at least about 40 nucleotides. A polypeptide "fragment" or "segment" is a stretch of amino acid residues of at least about 6 contiguous amino acids from a particular sequence, more typically at least about 12 amino acids.

The term "recombinant" or "recombinant DNA molecule" refers to a nucleic acid sequence which is not naturally occurring, or is made by the artificial combination of two otherwise separated segments of sequence. By "recombinantly produced" is meant artificial combination often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g. , by genetic engineering techniques. Such is usually done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a single genetic entity comprising a desired combination of functions not found in the common natural forms. Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site specific targets, e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incorporated by design. "Recombinant DNA molecules" include cloning and expression vectors.

Two nucleic acids or polynucleotides share sequence "homology" or "identity" if the two polynucleotides or designated segments thereof, when optimally aligned with appropriate nucleotide insertions or deletions, are identical in at least about 50% of the nucleotides. "Substantial homology" in the nucleic acid context means either that the segments, or their complementary strands, when compared, are identical when optimally aligned, with appropriate nucleotide insertions or deletions, in at least about 60% of the nucleotides, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95 to 98% of the nucleotides.

Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to a strand, or its complement, typically using a sequence derived from the RID or other regions of the RGL polynucleotide. Selectivity of hybridization exists when hybridization occurs with a certain degree of specificity rather than being random. Typically, selective hybridization will occur when there is at least about 55% homology over a stretch of at least about 14 nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90%. See. Kanehisa, Nuc. Acids Res.. 12:203-213 (1984). The length of homology comparison, as described, may be over longer stretches, and in certain embodiments will often be over a stretch of at least about 17 nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides. Selective hybridization conditions will be stringent combined conditions of salt, temperature, organic solvents, and other parameters typically controlled in hybridization reactions. Stringent temperature conditions will generally include temperatures in excess of 30°C, typically in excess of 37°C, and preferably in excess of 45°C. Stringent salt conditions will ordinarily be less than 1M, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is much more important than the measure of any single parameter. See. e.g., Wetmur et al., J. Mol. Biol.. 31:349-370 (1968).

"Oligonucleotides" are short-length, single- or double-stranded polydeoxynucleotides that are chemically synthesized by known methods (such as phosphotriester, phosphite, or phosphoramidite chemistry, using solid phase techniques such as described in EP 266,032 published 4 May 1988, or via deoxynucleoside H-phosphonate intermediates as described by Froehler et al., Nucl. Acids Res. , 14:5399-5407 (1986)). They are then purified on polyacrylamide gels.

The technique of "polymerase chain reaction," or "PCR," as used herein generally refers to a procedure wherein minute amounts of a specific piece of nucleic acid, RNA and/or DNA, are amplified as described in U.S. Pat. No. 4,683,195 issued 28 July 1987. Generally, sequence information from the ends of the region of interest or beyond needs to be available, such that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands on the template to be amplified. The 5' terminal nucleotides of the two primers may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, etc. See, generally, Mullis et al., Cold Spring Harbor Svmp. Quant. Biol.. 51:263 (1987) ; Erlich, ed. , PCR Technology. (Stockton Press, NY, 1989) . As used herein, PCR is considered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, comprising the use of a known nucleic acid (DNA or RNA) as a primer.

Generally, the nomenclature used hereafter and the laboratory procedures in cell culture, molecular genetics, and nucleic acid chemistry described below are those well known and commonly employed in the art. Standard techniques such as described in Sambrook et al., Molecular Cloning. A Laboratory Manual. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989) , are used for recombinant nucleic acid methods, polynucleotide synthesis, cell culture, and transgene incorporation, e.g., electroporation, injection, lipofection. Generally enzymatic reactions, oligonucleotide synthesis, and purification steps are performed according to the specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references which are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader.

Specific Embodiments

Polynucleotide and Polypeptide Compositions The present invention provides an "isolated" polynucleotide encoding a novel, ras p21-interacting protein, defined herein as RGL. The nucleotide and amino acid sequences of RGL, SEQ ID NO:l and SEQ ID NO:2, respectively, are shown in Figure IA. The RID nucleotide sequence, SEQ ID NO:3, corresponding to nucleotide positions 1972-2463 shown in Figure IA is also provided. SEQ ID NO:4 refers to the RID amino acid sequence encompassing amino acids 605-768. RID is a 164 amino acid domain of the RGL protein that interacts with ras p21. These sequences are also shown in Kikuchi et al. Mol. Cell. Biol.. 14:7483-7491 (1994).

Specifically provided by the invention is an isolated polynucleotide comprising at least 80% sequence identity with the nucleotide sequence of SEQ ID NO:l, an allelic or species variation thereof, or a fragment thereof. A polynucleotide comprising the RID sequence of SEQ ID NO:3 is also provided. The invention also provides certain recombinant DNA molecules comprising the nucleotide sequence of SEQ ID NO:l or a fragment thereof, such as the pGAD/RID plasmid encoding a RID-GAL4 transactivation domain fusion protein. Another recombination DNA molecule is pGAD/ralGDS, encoding a ralGDS- GAL4 transactivation domain fusion protein. These plasmids/expression vectors are described in the experimental examples below.

Compositions of the RGL polypeptide and derivatives thereof are also provided. These compositions will be full length natural forms, the natural forms including allelic and species variations of the polypeptide encoded by SEQ ID NO:2, fragments of the natural forms, fusion proteins with those fragments and modified forms of each. The compositions include an isolated polypeptide of less than about 200 amino acids, usually about 164 amino acids comprising a ras interacting-domain (RID) , this polypeptide referred to as a RID polypeptide and encoded by the sequence of SEQ ID NO:4. In a preferred embodiment, the RGL polypeptide or fragment thereof is capable of binding the effector loop of ras p21. Also provided are particular fusion proteins based on the RGL sequence, such as the RID-GAL4 fusion protein used in the yeast two-hybrid system and described in the experimental examples. In addition, pharmaceutical compositions are provided that include the RGL polypeptide and its derivatives or the RID polypeptide and its derivatives with a pharmaceutically acceptable carrier.

Uses of RGL Polynucleotide The RGL polynucleotide and fragments thereof have various uses. In one embodiment, the RGL polynucleotide or fragments thereof will be used to prepare expression constructs for RGL. Some of the expression constructs are described in detail under Experimental Examples. The expression vectors will contain the necessary elements for transcription and translation of the DNA fragments into polypeptide if these elements are not already present in the DNA fragments themselves. These necessary elements include a promoter 5¹ of the DNA insert to be expressed, a transcription and translation initiation site, stop codons, poly-A signal sequence, splice signals. DNA sequences encoding the protein will be operably linked to a promoter appropriate for expression in a particular cell type. Usually a strong promoter will be employed to provide for high level transcription and expression. Examples of strong promoters include human cytomegalovirus promoter. An enhancer may be necessary to function in conjunction with the promoter. The expression construct normally comprises one or more DNA sequences encoding RGL under the transcriptional control of a native or other promoter. Usually the promoter will be a eukaryotic promoter for expression in a mammalian cell, where the mammalian cell may or may not result in the expression of RGL. The selection of an appropriate promoter will depend upon the host, but promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters are known. Non-fungal promoters will be preferred where expression occurs in nonfungal cells. Occasionally, it might be useful to express the sequences in other types of cells and appropriate promoters may be selected. In some circumstances, an inducible promoter may be preferred. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Guide. Vols. 1-3 (1989) , Cold Spring Harbor Press. Plasmid, viral or YAC vectors are contemplated. Conveniently available expression vectors which include the replication system and transcriptional and translational regulatory sequences together with a polylinker restriction site for insertion of the protein encoding sequence, may be employed. Examples of workable combinations of cell lines and expression vectors are described in Sambrook et al. (1989) ; see also. Metzger et al. (1988), Nature 334:31-36.

It may be desirable to produce the RGL protein or fragments thereof in a prokaryotic host, in which case a prokaryotic promoter is preferred. Examples of prokaryotic promoters are trp, lac, and lambda. See Sambrook et al. (1989) for other useful prokaryotic promoters. Usually a strong promoter will be employed to provide for high level transcription and expression.

The expression construct will often be contained in a vector capable of stable extrachromosomal maintenance in an appropriate cellular host or may be integrated into the host genome. The expression construct may be bordered by sequences which allow for insertion into a host, such as transposon sequences, lysogenic viral sequences, or the like. Normally, markers are provided with the expression construct which allow for selection of host cells containing the construct. The marker is preferably on the same DNA molecule but can be on a different DNA molecule that is cointroduced into the host cell. In prokaryotic cells, markers such as a resistance to a cytotoxic agent, complementation of an auxotrophic host to prototrophy, production of a detectable product, etc., serve the purpose. The expression construct can be joined to a replication system recognized by the intended host cell. Various replication systems include viral replication systems such as retroviruses, simian virus, bovine papilloma virus, or the like. While the wild-type sequences of RGL or RID will generally be employed, in some situations one or more mutations or minor modifications may be introduced, such as deletions, substitutions or insertions resulting in changes in the amino acid sequence, providing silent mutations or modifying amino acid residues or amino or carboxyl terminal groups. Conservative amino acid substitutions can be introduced. These amino acid changes can be made using techniques such as PCR or site-directed mutagenesis. There will be circumstances where gene fusions between RGL and another protein can be useful. The fusion proteins will be recombinantly produced. The recombinant nucleic acid sequences used to produce fusion proteins of the present invention will often be derived from natural or synthetic sequences.

The nucleic acid constructs will be useful to introduce into cells, providing an efficient and economical means to produce commercially useful quantities of the protein compositions. Transfected cells producing varying quantities of full length RGL or only the RID fragment will also be useful in evaluating the effect of overexpression of RGL on ras function and transformation. Nucleic acid constructs expressing various lengths and mutant forms of RGL can be used to determine the minimum region involved in the RGL/ ras interaction to the specific amino acid contacts.

The means of introduction of the expression construct into a host cell will vary depending upon the particular vector and the target host. Introduction can be achieved by any convenient means, including fusion, conjugation, transfection, transduction, electroporation, injection, or the like. See, e.g., Sambrook, et al. (1989), supra. The DNA expression vectors encoding the active fusion kinase polypeptide are introduced into the appropriate cellular host under conditions which favor expression of the polypeptide and isolation of the resultant expressed polypeptide. This implies using an expression vector compatible with the host cell, the vector containing the necessary elements described above for expression of the polypeptide. The tranfected cells are then provided with the optimum nutrient, gas and temperature conditions for optimal protein production. These conditions will depend on the cell type. Transient or stable transfection procedures can be used.

The host cells will normally be immortalized cells, i.e., cells that can be continuously passaged in culture. For the most part, these cells will be convenient mammalian cell lines which are able to express a RGL protein and, where desirable, process the polypeptide so as to provide an appropriate mature polypeptide. By processing is intended glycosylation, ubiquitination, disulfide bond formation, general post-translational modification, or the like.

A wide variety of both prokaryotic and eukaryotic hosts will be employed for expression of the proteins and peptides. Useful hosts include bacteria, such as E. coli . , yeast, filamentous fungi, insect cells such as Sf9, mammalian cells, typically immortalized, e.g., various mouse cell lines, monkey cell lines, Chinese hamster ovary cell lines, human cell lines, derivatives of them, or the like. In some cases, the cells will be derived from a neoplastic host cell or wild- type cells will be transformed with oncogenes, tumor causing viruses or the like. Cells carrying the RGL polynucleotide compositions are covered by this invention.

Cells transformed with the polynucleotide compositions can be used to create transgenic mice. Such transgenic mice are useful e.g. to study the effect of overexpression of the RID polypeptide on growth and development of the animal. The procedure for producing transgenic mice is known in the art and are described e.g. in detail in Hogan et al., Manipulating the Mouse Embryo. Cold Spring Harbor laboratory, Cold Spring Harbor, NY (1986) . In another aspect of the invention, the mouse full length RGL polynucleotide according to the sequence of SEQ ID NO:i, or fragments thereof will be used to prepare probes to screen DNA libraries to isolate RGL genes or gene fragments encoded by other species, particularly human. The methods of screening DNA libraries are generally well known, see eg.

Sambrook et al. (1989) . The probes can be from about 50 bp to several kb in length. Preferably, the probe should be free of vector sequences. The probes are typically prepared labeled. Radiolabels such as ³²P are normally used although non- radioactive labels are also suitable.

Genomic and cDNA libraries prepared from mammalian, insect or yeast cells are included for screening purposes. The DNA libraries may be constructed in phage, bacteria or yeast. Clones that hybridize to the probe are identified such as by autoradiography if radiolabeled probes are used. DNA is isolated from hybridizing clones and analyzed for the presence of RGL gene sequence as verification that the hybridizing clone carries all or part of the RGL gene. The DNA sequence carried by the clone is compared with that of SEQ ID NO:l. The RGL gene or fragment thereof will then be isolated from the vector by restriction endonucleaseε. It may be necessary to isolate several overlapping DNA sequences from different hybridizing clones to recombinantly reproduce the full length gene in one contiguous DNA fragment.

Alternatively, with the availability of the mouse RGL sequence (Fig. IA) , RGL genes from other species can be isolated by Polymerase Chain Reaction (PCR) by selecting appropriate pairs of primers based on the known sequence and using genomic DNA or cDNA prepared from cells as the template. Primers can be chemically synthesized and will be at least 10 nucleotides in length, more usually 14 nucleotides, preferably 17 nucleotides but can be as long as 100 bp nucleotides. Pairs of primers corresponding to the 5* and 3' ends of the gene or to the internal regions of the gene can be used. Several rounds of PCR may be required to prepare overlapping clones that can then be linked by recombinant methods to produce the entire gene in one DNA fragment. The invention also provides a method of determining if the RGL gene from a cell of interest is mutated. The cells can be from cultured cell lines or from tissue isolated from an animal or human. For example, cells can be prepared from a human tumor biopsy. PCR can be used to amplify all or part of the RGL gene using selected primers and the amplified DNA fragment sequenced or analyzed for restriction enzyme cleavage patterns. The nucleotide sequence or restriction analysis is compared to the wild type sequence of RGL from the appropriate species. Therefore, the wild type sequence acts as a standard or positive control.

In another aspect, the RGL polynucleotide or oligonucleotides derived from it find use to isolate a gene encoding other members of the RGL/ralGDS family of proteins that binds to ras p21 and that share "substantial homology" with RGL and ralGDS genes. Such ras binding proteins could potentially be effector or regulator proteins of ras. Thus, the invention provides a method of identifying a gene encoding a ras p21-binding protein, by screening a DNA library with a RID probe to identify a hybridizing clone containing a RID sequence, the presence of a RID sequence being indicative of a gene encoding a ras p21-binding protein.

One specific embodiment of the invention is a method of identifying a ras effector loop-binding protein other than RGL. Probes corresponding to the RGL sequence are used to screen a gene library. Library screening has been described above. Preferably, the probes for screening the gene library will comprise oligonucleotides corresponding to the RID sequence of RGL or ralGDS. The entire RID sequence can be used as probe. The probes will be oligonucleotides or DNA fragments having at least about 25 nucleotides, more usually at least about 100 nucleotides, and fewer than about 5 knt (kilonucleotides) , usually fewer than about 0.5 knt. The screening of mammalian cDNA or genomic DNA libraries, especially human DNA libraries will be targeted although eukaryotes such as yeast and insects are also of interest for evolutionary comparisons.

A gene that hybridizes with the probe and is determined to be substantially homologous to the RGL gene in nucleotide sequence will be isolated. The homologous gene will be inserted into an appropriate expression vector and introduced into a suitable host for expression to produce the encoded polypeptide. The encoded polypeptide will then be assayed to determine if it binds the effector loop of ras using the same procedure for analyzing the interaction of ralGDS with ras p21, described below in the Experimental Examples. If binding is observed, the polypeptide is determined to be a newly discovered, ras effector loop-binding protein.

The RGL or RID probes can also be used to determine whether RNA encoding RGL or an RGL homolog is present in a cell. This can be done by the procedure of Northern Blotting. In situ hybridization can also be performed on tissue sections of the organism to determine developmental regulation and compare expression levels in various tissues. The probes can be labeled using any suitable labels or tags eg. radiolabel, biotin-avidin. The procedures of preparing probes, Southern blotting, Northern blotting and in si tu hybridizations are well known in the art. See, for example, Sambrook et al., 1989.

Conditions for hybridization can be varied. Initially, less stringent conditions can be used. However, if a high background of non-specific hybridization is observed, more stringent conditions will be employed.

The RGL polynucleotide or fragments thereof also find use in the construction of vectors encoding fusion proteins. Fusion proteins encoding RGL fused to the GAL4 DNA binding domain can be used in the yeast two hybrid system to isolate proteins that interact with the RGL protein. This method allows the isolation of the cloned genes for the interacting proteins and eventually the identification of the interacting proteins. Knowledge of the interacting proteins in the ras signaling pathway will allow the screening of drugs for agonists and antagonists of ras dependent cell proliferation and differentiation. The yeast two-hybrid system is described above and in the experimental examples. The RID sequence in particular is suitable for fusing to the GAL4 DNA binding domain. The second fusion protein will be encoded by a cDNA library linked to the GAL4 transactivation domain. A yeast reporter strain is then cotransformed with plasmids encoding both fusion proteins.

Isolation of RGL and RID Polypeptide

The RGL polypeptide can be isolated from a normally expressing cell or a transfected cell by immunoprecipitation or affinity chromatography of cell lysates using RGL-specific antibody. The antibody can be in solution or affixed on a solid substrate. It may be more efficient to isolate the protein from transfected cells that may produce larger quantities of the protein due to particular characteristics of the expression construct, such as a strong promoter. The Experimental Examples describe purification of ralGDS from the cytosolic fraction of transfected Sf9 insect cells. The RGL protein can be similarly expressed and purified. Instead of or in addition to immunological methods, the peptide will generally be isolated by techniques employing FPLC, HPLC, electrophoresis, gradient centrifugation and other methods routinely used in protein purification to provide a substantially pure product, i.e., particularly free of cellular contaminants. For protein purification methods, see, e.g., Jacoby, Methods in Enzvmologv. Vol. 104 (1984), Academic Press, New York; Scopes, Protein Purification: Principles and Practice. (2nd Ed.) (1987) Springer-Verlag, New York; Deutscher (ed.), Guide to Protein Purification. Methods in Enzvmologv. Vol. 182 (1990).

Uses of RGL Polypeptide Compositions

The RGL polypeptide compositions find several uses. The polypeptide compositions of RGL and RID are useful for raising antibodies, both polyclonal and monoclonal. Such antibodies are powerful tools that can be employed in various assays and diagnostic situations particularly where immunoprecipitation, immunoblotting and affinity purification procedures are necessary. Thus, one object of the invention is to provide antibodies that specifically binds to the RGL polypeptide comprising the sequence of SEQ ID NO:2, an allelic or species variation thereof, or a fragment thereof. Antibodies that bind just the RID encoded by SEQ ID NO:4 are included. The invention also provides hybridoma lines that produce monoclonal antibodies to the RGL polypeptide. These antibodies find use in isolating the RGL protein and any structurally related proteins expressing an epitope recognized by the anti-RGL antibody. Isolation of RGL and structurally related proteins can be accomplished by simply immunoprecipitating the proteins from lysates of normally expressing or transfected cells. Alternatively, affinity purification of eg. cell lysates can be performed using the RGL antibody fixed on a solid matrix such as a column of beads or a filter paper.

RGL antibodies are also useful to study the interaction of RGL with ras in vivo in normal and growth disregulated or cancerous cells. The same protocol described in the experimental examples for studying the interaction of ralGDS with ras p21 in intact cells can be followed. Antibodies capable of specifically binding RID or otherwise blocking the binding of RGL to ras are desirable reagents. Thirdly, the RGL specific antibodies find use in isolating any RGL-associating protein that co- immunoprecipitate with RGL. RGL-associating proteins are useful to study the downstream effectors of ras and the regulation of ras and RGL function.

In a different aspect, RGL specific antibodies can serve as a diagnostic reagents to detect deficiencies in RGL such as expression levels in tumor cells from cancer patients, particularly in cases of bladder and colon cancers.

Polyclonal and/or monoclonal antibodies with specificity to RGL can be prepared by in vi tro or in vivo techniques following standard procedures as described in, e.g., Harlow, et al., Antibodies: A Laboratory Manual (1988), Cold Spring Harbor Press, New York. Antibodies are produced by immunizing an appropriate vertebrate host, e.g. , rabbit or rodents, with the entire RGL protein or peptides derived thereof, or in conjunction with an adjuvant. Usually two or more immunizations will be involved, and the blood or spleen will be harvested a few days after the last injection.

For immunization, preferably peptides corresponding to regions of the protein comprising hydrophilic residues or residues exposed to the aqueous phase are selected.

Immunogens comprising the entire RID polypeptide (SEQ ID NO:4) or peptide derivatives thereof are also desirable. Synthetic peptide fragments may be prepared in a peptide synthesizer and coupled to a carrier molecule (e.g. , keyhole limpet hemocyanin) and the conjugate injected into rabbits at selected times over several months.

For production of polyclonal antibodies, an appropriate target immune system is selected, typically a rabbit or a mouse. The substantially purified antigen is presented to the immune system in a fashion determined by methods appropriate for the animal and other parameters well known to immunologists. Typical sites for injection are in the footpads, intramuscularly, intraperitoneally, or intradermally. Of course, another species will sometimes be substituted for a mouse or rabbit, including goats, sheep, cows, guinea pigs, and rats.

The rabbit sera is tested for immunoreactivity to the RGL protein or peptide immunogen by an immunoassay, typically with preimmune sera as one of the negative controls. The immunoassay can be a radioimmunoassay, an enzyme-linked assay (ELISA) , a fluorescent assay, or any of many other choices, most of which are functionally equivalent but may exhibit advantages under specific conditions. The polyclonal antibodies can be provided commercially in the form of antisera or in purified form. From the polyclonal antisera, the immunoglobulins may be precipitated, isolated and purified, such as by affinity purification. Preferably, the purified form is substantially free of non-specific antibodies and cellular contaminants.

Monoclonal antibodies with affinities of 10⁸ M^"1 preferably 10⁹ to 10¹⁰, or stronger will typically be made by standard procedures as described, e.g., in Harlow et al., Antibodies: A Laboratory Manual. CSH Laboratory (1988) ; or

Goding, Monoclonal Antibodies: Principles and Practice (2d ed) (1986) , Academic Press, New York. Normally, mice are used to produce monoclonal antibodies although rats, guinea pigs and other animals can also be used. After the appropriate period of time from the immunization schedule, the spleens of such animals are excised and individual spleen cells fused, typically, to immortalized myeloma cells under appropriate selection conditions. Thereafter the cells are clonally separated and the supernatants of each clone are tested for their production of an appropriate antibody specific for the desired region of the antigen.

Other suitable techniques involve in vi tro exposure of lymphocytes to the antigenic polypeptides or alternatively to selection of libraries of antibodies in phage or similar vectors. See, Huse et al., "Generation of a Large Combinatorial Library of the I munoglobulin Repertoire in Phase Lambda," Science 246:1275-1281 (1989). The polypeptides and antibodies of the present invention may be used with or without modification. Frequently, the polypeptides and antibodies will be labeled by conjugating, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are-known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents, teaching the use of such labels include U.S. Patent NOS. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulins may be produced, see Cabilly, U.S. Patent No. 4,816,567. The RGL antibodies of the invention can also be provided in a kit for use in any of the various applications described above. The contents of the kit will vary depending on the intended application of the antibody and may include other reagents and instructions for the use of the antibody preparation and the reagents. At least one aliquot of the antibody will be provided. Different RGL antibodies with specificities for different regions of the protein can be provided. Different antibodies may be desirable for verification of an assay result. The aliquots can be contained in any suitable container such as a vial or a tube. The polyclonal antibody can be in the form of antisera or affinity purified. Monoclonal antibodies can be provided in the form of ascites, culture media or a buffer such as phosphate buffered saline solution. The antibody preparation can be provided in solution or in lyophilized form, and may even be immobilized on a substrate such as a column matrix. The antibody preparation may also contain in it preservatives such as sodium azide or protease inhibitors such as EDTA. A carrier protein such as BSA or ovalbumin, usually between 0.5- 5%, may be included for stability. The solution form of the antibody, especially the purified form, may contain up to 50% glycerol if the kit is to be stored frozen at -20°C to -70°C. if the antibody is provided in lyophilized form, the kit can include a reconstitution buffer to reconstitute the antibody.

If the antibody is to be used in western blotting, reagents for use in the blotting procedure can be included in the kit. A secondary, labeled antibody capable of binding to the RGL antibody allowing detection of the binding, can be included. The labeled antibody may be conjugated to an enzyme such as alkaline phosphatase or horse radish peroxidase.

Since the RID of RGL binds ras, the RGL polypeptide or the RID polypeptide is useful in the isolation of any RGL interacting protein including ras p21.

Thus, the invention also provides a method of isolating a RGL interacting protein. The method comprises contacting a cell lysate suspected of containing a RGL interacting protein with a RGL polypeptide and isolating any protein bound to said RGL polypeptide as a RGL interacting protein.

One embodiment of the method, an RGL polypeptide is immobilized on a solid matrix. If the isolation of ras p21 is desired, it is preferable that an immobilized RID polypeptide be used. The solid matrix can comprise various materials such as is commonly used in column chromatography, including sepharose, sephadex, agarose, polystyrene and latex beads. The solid matrix can also be filter paper or membrane, such as nitrocellulose and polyvinylidene fluoride (PVDF) membrane. The RGL polypeptide can be coupled to the solid matrix directly or indirectly. Direct methods include covalent coupling to sepharose beads using cyanogen bromide. Indirect coupling can take advantage of an RGL antibody or some other moiety suitable for linking the two components such as biotin- avidin binding pairs. RGL or RID fusion proteins may be more conveniently immobilized if the fusion protein can bind a ligand provided on the matrix. Lysates are produced from cells that normally express RGL. Cell lysates will be contacted with the immobilized RGL polypeptide such as by running the lysates over an RGL affinity column to allow any RGL interacting protein to bind the immobilized RGL polypeptide under optimum conditions. Preferably the binding reaction is carried out betwen 4°C and normal physiological temperature. Buffer conditions can be modified to favor capture of this binding. For example, the pH and salt conditions can be varied. The matrix is then washed with buffer to remove any unbound or nonspecifically bound cellular components. Any protein that bound to the RGL polypeptide will be isolated by elution off the solid matrix such as by using a salt gradient or using soluble RGL polypeptide and fragments thereof to compete for binding. RGL fusion proteins find particular use in the yeast two hybrid system to isolate RGL interacting proteins as described earlier. Peptide expression libraries such as by phage display methodology, can be screened for ligand binding to the RGL polypeptide or fragments thereof or fusion proteins thereof.

RGL or RGL fusion proteins also find use to detect RGL-interacting proteins by Western blotting, using the RGL protein in solution to bind protein bands on the blot and detecting the bound RGL. In this circumstance, the RGL protein can be labeled directly or indirectly. Indirect labeling will include eg. a labeled antibody binding to RGL. RGL or RID can be fused, e.g., with glutathione-S- transferase (GST) , to produce GST-RGL or GST-RID fusion proteins. Expression vectors carrying GST sequence and specifically constructed to facilitate recombinantly producing GST fusion proteins are commercially available from most sources that supply cloning vectors. The fusion proteins may also comprise RGL or fragments thereof fused to the product or polypeptide encoded by a second gene. A product encoded by only a portion or a fragment of the second gene instead of the entire gene, may be sufficient. For example, RGL or a fragment thereof may be fused to a second gene such as the E. coli lacZ gene that will allow detection of expression. Other convenient fusion proteins will comprise RGL or RID sequence or portions thereof linked to a tag.

In constructing fusion proteins, it will be understood that the amino or carboxy terminus of the RGL protein, or wherever the fusion junction is, may be modified to facilitate cloning or for other reasons eg. to allow cleavage of the fusion protein and release of the separate portions.

The tag can be a label or some means that allows identification of the fusion protein. The tag is introduced into a site in the polypeptide that will not interfere with the folding and the function of the protein, generally at the N- or the C-terminus. The tag can be an epitope tag recognizable by an antibody, a member of a binding pair, an enzyme or any other suitable entity. The tag can be a cleavable sequence such as the phosphatidylinositol-glycan (PIG) signal sequence present in proteins such as alkaline phosphatase, DAF and acetylcholinesterase. The PIG sequence is cleavable by the enzyme phosphatidylinositol phospholipase C (PI-PLC) (Ferguson, Ann. Rev. Biochem.. 57:285-320 (1988)). The influenza virus hemagglutinin (HA) and the yc (10 amino acid - EQKLISEEDL) epitopes are particularly useful tags. Examples of binding pairs are ligand-receptor, antigen- antibody and small molecules like avidin-biotin. Enzyme tags include horse radish peroxidase, alkaline phosphatase and β- galactosidase which can act on a substrate to produce a color signal. For example, the protein can be fused to an epitope tag recognizable by an available antibody. The antibody to the tag is useful eg. to immobilize the RGL protein on an affinity column or to detect the protein such as when the fusion protein is used in Western blotting.

The invention also provides a method to block or modulate ras p21 activity in vitro and in vivo using the RID polypeptide. The binding of RID to the effector loop of ras p21 prevents the GTP-bound active form of ras p21 from interacting with effector proteins that bind at the same site of ras . These effector proteins mediate ras p21-dependent processes such as growth factor-stimulated or oncogene-induced cell proliferation. Various medical conditions are attributed to a disregulation in ras activity, i.e., ras dysfunction. Mutated ras can result in uncontrolled proliferation leading to cancer, e.g. , lung, bladder, and colon carcinoma. The RID polypeptide derived from either the RGL protein or the ralGDS protein will generally be effective in modifying the extent to which ras disregulates cell proliferation, differentiation and other ras dependent processes, this modification being implied when the term "modulating ras p21 activity" is used herein. By competing for binding to the ras effector loop, the RID polypeptide will block or reduce the ability of active GTP-bound ras p21 to interact with downstream effector proteins such as PI(3)K, Raf, neurofibromatosis gene product (NF1) and GAP proteins including rasGAP and neurofibromin. This, in turn, will affect the cellular signalling events downstream of ras p2l. For example, blocking the binding of GAP to ras could increase the amount of ras bound in the GTP-bound active state upon cell stimulation. The method of blocking ras p21 activity comprises providing a RID polypeptide in a cell expressing ras p21 protein wherein the RID polypeptide will bind to said ras p2l protein to block ras p21 activity. The RID polypeptide itself can be directly introduced into the cell under study where arrest or modulation of ras function is desired. Methods of introducing the RID polypeptide into the cell include microinjection of the isolated polypeptide (expressed in other cells) or the use of appropriate drug delivery vehicles such as liposomes to deliver the polypeptide. Alternatively, the RID polypeptide can be provided by introducing an expression construct encoding the RID polypeptide into an affected ras expressing cell wherein the RID polypeptide will be expressed in an amount effective to interfere with ras p21 activity and thus inhibit proliferation. Expression constructs can be targeted to a particular cells by using nucleic acid delivery vehicles that contain targeting moieties on the surface of the vehicle. Examples of such vehicles include liposomes or recombinant viruses expressing receptors for cell surface markers. In some circumstances, complete blockage of ras activity may require high level expression i.e. overexpression of the RID polypeptide for effective competition. In that circumstance, the expression construct can be designed to contain the necessary elements such as strong promoters, inducible promoters and enhancers to achieve high level expression of the RID polypeptide. The RID polypeptide expressed intracellularly will be contacted with and bind to the ras p21 protein.

The method of modifying ras p21 activity in vivo can be applied to alleviating a patient suffering from a cell proliferative condition such as cancer. The method comprises administering to the patient, a therapeutically effective amount of a pharmaceutical composition comprising a RID polypeptide, and a pharmaceutically acceptable carrier. The RID polypeptide will be specifically targeted to the affected cells, such cells being tumor cells in the case of cancer, and cells of the cardiac valve in restenosis. Another pharmaceutical composition for use in the treatment method will comprise an expression vector suitable for introduction into and expression of a therapeutically effective amount of a RID polypeptide in, cancer and other affected cells.

Drug delivery vehicles such as liposomes, can be used to deliver and provide sustained release of the formulations in the body. The liposomes can have targeting moieties exposed on the surface such as antibodies, ligands or receptors to specific cell surface molecules. For example, it may be desirable to limit the delivery of the formulation to only tumor cells. Such cells can be targeted to receive the therapeutic formulation by incorporating into the liposome carrier, a targeting moiety that recognizes and binds a specific tumor surface marker. Liposome drug delivery is known in the art (see, e.g., Biochimica et Biophvsica Acta 113:201-227 (1992)).

The quantities of reagents determined to be an effective amount for treatment will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vi tro may provide useful guidance in the amounts useful for in situ administration of these reagents. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Various considerations are described, e.g., in Gilman et al. (eds) , Goodman and Gilman's: The Pharmacological Bases of Therapeutics. 8th Ed. (1990), Pergamon Press; and Remington's Pharmaceutical Sciences. 17th Ed. (1990), Mack Publishing Co., Easton, Pennsylvania. Methods for administration are discussed therein, e.g., for oral, intravenous, intraperitoneal, or intramuscular administration, transdermal diffusion, and others.

The pharmaceutical compositions will be administered by intravenous, parenteral, intraperitoneal, intramuscular, oral, or local administration, such as by aerosol or transdermally, for therapeutic treatment. The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include powder, tablets, pills, capsules and dragees.

The pharmaceutical compositions will often be administered intravenously. Thus, this invention provides compositions for intravenous administration which comprise a solution of the polypeptide dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. Slow release formulations, or slow release delivery vehicles will often be utilized for continuous administration.

"Pharmaceutically acceptable carriers" will include water, saline, buffers, and other compounds described, e.g., in the Merck Index, Merck & Co. , Rahway, New Jersey. These compositions will sometimes be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, preferably about 20% (see. Remington's, supra..

The compositions containing the compounds can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, compositions are administered to a patient already suffering from a disease, as described above, in an amount sufficient to cure or at least alleviate the symptoms of the disease and its complications. An amount adequate to accomplish this is defined as "therapeutically effective dose." Amounts effective for this use will depend on the severity of the disease and the weight and general state of the patient.

In prophylactic applications, compositions containing the compounds of the invention are administered to a patient susceptible to or otherwise at risk of a particular disease. Such an amount is defined to be a "prophylactically effective dose." In this use, the precise amounts again depend on the patienfs state of health and weight. Experimental Examples

The following examples are by way of illustration and are not meant to be construed as a limitation on the scope of the invention.

Materials and Methods

Materials and chemicals.

PC62 and the PC51/mouse embryonic cDNA library were provided by P.M. Chevray and D. Nathans (Johns Hopkins University, Baltimore, Maryland) (Chevray et al., Proc. Natl. Acad. Sci.. USA. 89:5789-5793 (1992)). PC62 contains an ADH promoter expressing the GAL4 DNA-binding domain (amino acids 1 to 147) , and PC51 contains the ADH promoter expressing the GAL4 transactivation domain (amino acids 768 to 881) . The ralGDSb and ra B p24 cDNAs and the anti-ralGDS antibody were provided by B. w. Giddings, C. F. Albright, and R. A. Weinberg

(Whitehead Institute for Biomedical Research, Cambridge, Massachusetts) (Albright et al., EMBO J.. 12:339-347 (1993)). The c-H-ras p21 cDNA, dominant negative ras p21 cDNA (ras p21^sl7N [a form of ras p21 in which Ser-17 is changed to Asn]), and the hybridoma cells producing anti-ras p21 antibody (Y13-259) were provided by J. Downward (Imperial Cancer Research Institute, London, England). pGBT9, pGBT/ ras p21, and pGBT/rapl p21 were provided by L. Van Aelst and M. Wigler (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) (Van Aelst et al. , Proc. Natl. Acad. Sci. USA. 90:6213-6217 (1993)). S. cerevisiae YPB2 and pGAD were provided by G. Hannon and D. Beach (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) . S. cerevisiae Y153 was provided by S. Field (University of California, Berkeley) (Durfee et al., Genes Dev.. 7:555-569 (1993). racl p21^G12v (a form of racl p21 in which Gly-12 is changed to Val) was provided by Alan Hall (Institute of Cancer Research, London, England) . The neurofibro atosis 1 (NF1) cDNA was provided by G. Xu (University of Utah, Salt Lake City) (Xu et al., Cell. 63:835- 841 (1990)). Spodoptera frugiperda (Sf9) cells, pVL1393, and BaculoGold linearized baculovirus DNA were purchased from Pharmingen (San Diego, California) . High-five cells were from Invitrogen (San Diego, California) . The anti-ras p21 antibodies (Y13-238 for immunoprecipitation assay and F235 for immunoblot analysis) were from Oncogene Science Inc. (New York, New York). [γ-³²P]GTP and [α-³²P]GTP were from DuPont NEN Research Product (Boston, Massachusetts) . All procedures of passage, infection, and transfection of Sf9 cells and the isolation of recombinant baculoviruses were carried out as described previously (Summers et al., 1987, A manual of methods for baculovirus vectors and insect cell culture procedures. Texas Agricultural Experiment Station, College Station) . c-ras p21 and ralGDS were purified from the cytosolic fraction of Sf9 cells and High-five cells, respectively, as described previously (Albright, supra; Mizuno et al., Proc. Natl. Acad. Sci. USA. 88:6442-6446 (1991)). Glutathione S-transferase

(GST) fused to the N-terminal region of Raf (amino acids 1 to 322) (GST-N-Raf) and GST fused to the NF1 catalytic domain (GST-NF1) were purified from Sf9 cells expressing GST-N-Raf and Escherichia coli expressing GST-NF1, respectively, as described previously (Kikuchi et al., J. Biol. Chem.. 269:20054-20059 (1994) ; Xu, suorai .

Plasmid descriptions and construction.

PC51/mouse embryonic cDNA library contains the ADH promoter expressing the GAL4 transactivation domain (amino acids 768 to 881) .

pGAD contains the ADH promoter expressing the GAL4 transactivation domain (amino acids 768 to 881) .

pGAD/RID

To make pGAD encoding the ras p21-interacting domain (RID) of RGL (amino acids 605 to 768), 0.5-kb fragment containing RID with BamHl and Sa l sites was synthesized by PCR. This fragment was digested with flamHI and Sail and inserted into BeuπHI- and Sa l-cut pGAD to generate pGAD/RID. pGAD/πz/GDS

To construct pGAD containing ralGDS, pBluescript KS/ralGDS was digested with Ncol . The 1.8-kb fragment which represents the Ν-terminal two-thirds of ralGDS (Ν-ra GDS) was blunted with Klenow enzyme and inserted into pGAD which was digested with Sjnal to generate pGAD/Ν-ra GDS. Then pEV55/ralGDS was digested with Avrll and EcoRI, and the 1.1-kb fragment, which represents the C terminus of ralGDS, was inserted into Avrll- and EcoRI-cut pGAD/Ν-ralGDS to generated pGAD/ralGDS.

PC62 contains an ADH promoter expressing the GAL4 DΝA-binding domain (amino acids 1 to 147) .

PC62/rαs p21

To construct PC62 encoding c-H-ras p21, the 0.6-kb fragment containing c-H-ras p21 with the Sail site upstream from the initiator methionine codon and the Ba-flHI site downstream from the termination codon was synthesized by PCR. This fragment was digested with Sa l and BaiSHI and inserted into Sail- and BamHI-cut PC62 to generate PC62/ras p21.

ras P21^C186S is a form of ras p21 in which Cys-186 is changed to Ser. This mutant is not posttranslationally modified (Hancock et al. , Cell 57:1167-1177 (1989)).

ras p21^T35A is a form of ras p21 in which Thr-35 is changed to

Ala resultinngg iin an effector loop mutant. Both ras P21^C186S and ras p21^τ35A were made by PCR.

pGBT9 contains an ADH promoter expressing the GAL4 DΝA-binding domain (amino acids 1 to 147) .

pGBT/TOs^* p2l^cl86s and pGBT/res- p2l ^T35A

To construct pGBT9 encoding ras p21^cl86S and ras p21^T35A, the 0.6-kb fragments containing ras P21^C186S and ras p21^τ35A with Sinai and BamHI sites were synthesized by PCR. These fragments were digested with Smal and BainHI and inserted into Smal- and BamHI-cut pGBT9 to generate pGBT/ras p21^cl86S and pGBT/ ras p21 ^T35A. To construct pGBT9/ras p21^cl86S and pGBT/ras p21^T35A.

pGBT/r /B p24 and pGBT/racl p21^αl2v

To construct pGBT9 encoding ra B p24 and racl p21^G12v, the 0.6-kb fragments containing ralB p24 and racl p21^G12V with

BamHI and Sa l sites were synthesized by PCR. These fragments were digested with BamHI and Sa l and inserted into BairtHI- and Sail-cut pGBT9 to generate pGBT/ra B p24 and pGBT/racl _P21^G12V.

pVLl393/c-nw p21 and pVL1393//Y» p21^S17N

To construct pVL1393 encoding c-ras p21 and ras p21^sl7N, the 0.6-kb fragments containing c-ras p21 and ras p21^sl7N with BamHI and PstI sites were synthesized by PCR. These fragments were digested with BaiΛHI and PstI and inserted into BamHI- and Pstl-cut pVL1393.

Constructions of v-ras p21 (a form of ras p21 in which Gly-12 is changed to Val) and the N-terminal region of Raf in pVL1393 and pV-IKS were carried out as described previously (Kikuchi et al. , supra..

Two-hybrid screening.

The yeast reporter strain YPB2 was cotransformed with PC62/ras p21 and the PC51/mouse embryonic cDNA library and plated at a density of 2.5 x 10⁴ colonies per plate on synthetic minimal media lacking histidine, leucine, and tryptophan and supplemented with 30 mM 3-aminotriazole. The plates were incubated for 4 days at 30°C. Of the 0.5 million colonies that were plated, approximately 200 grew in the absence of histidine. These colonies were patched to selective plates and assayed for 3-galactosidase activity by a filter assay (Breeden et al., Cold Spring Harbor Symo. Quant. Biol.. 50:643-650 (1985)).

Eight colonies were positive. Of these, five were specific for PC62/ras p21 when tested with PC62/SH3 domain of mouse p85 (amino acids number 1 to 100) . Library inserts from these five colonies were sequenced by using a Promega Sequenase kit (Promega Corporation, Madison, Wisconsin) after subcloning the inserts as Sa l-NotI fragments into pBluescript KS. cDΝAs from five colonies had the same orientation, and both strands of these cDΝAs were determined. To identify the full-length cDΝA of RGL, a probe was made by using the Sall-NotI fragment with a Quick Prime kit (Pharmacia Biotechnology, Piscataway, New Jersey) and used to screen a BALB/C3T3 fibroblast cDNA library as described previously (Klippel et al., Mol. Cell. Biol.. 14:2675-2685 (1994)).

Interaction assay of RID or ralGDS with ras p21 and other small G proteins in the yeast two-hybrid system. The yeast reporter strain Y153 (Durfee et al., supra) was cotransformed with pGAD/RID or pGAD/ra GDS and the indicated plasmids and plated on synthetic minimal medium lacking leucine and tryptophan. Plates were incubated for 3 days at 30°C and assayed for 0-galactosidase activity by filter assay (Breeden et al. , supra ) .

Interaction assay of ralGDS and ras p21 in vivo. Monolayers of Sf9 cells (2 x 10⁷ cells) were infected singly or doubly with high-titer recombinant baculoviruses (10⁸ PFU/ml) at a multiplicity of infection of 5 per cell. At 72 h postinfection, the cells were washed with cold phosphate- buffered saline and lysed in 1 ml of lysis buffer (20 mM Tris- HCl [pH 7.5], 1% Nonidet P-40, 137 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 20 μg of aprotinin per ml, 10 μg of leupeptin per ml) at 4°C for 1 h. Insoluble material was removed by centrifugation at 4^βC for 30 min at 13,000 x g, and 0.2 ml of lysate (0.24 mg of protein) was used for each assay. The lysates expressing ralGDS and v-ras p21 or ras p2l^sl7N were prepared, and the proteins of the lysates were immunoprecipitated with the anti-ras p21 antibody. Y13-238 was used in the immunoprecipitation experiments except that Y13-259 was used for Fig. 2D. The immunoprecipitates were washed once with lysis buffer, twice with 100 mM Tris-HCl (pH 7.5) and 0.5 M LiCl, and once with 10 m Tris-HCl (pH 7.5). The precipitates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12% polyacrylamide gel) (Laemmli, Nature (London), 227:680-685 (1970)), transferred to nitrocellulose filters, and probed with the anti-ralGDS or anti-ras p21 antibody.

Interaction assay of ralGDS and ras p21 in vitro. To make the guanosine 5'-(3-0-thio)triphosphate (GTPγS)- or GDP-bound form of ras p21, c-ras p21 (20 pmol) was incubated for 10 min at 30°c with 25 μM GTPγS or GDP in 40 μl of reaction mixture (20 mM Tris-HCl [pH 7.5], 10 mM EDTA, 5 mM MgCl₂, l mM dithiothreitol [DTT] . After the incubation, 600 mM MgCl₂ was added at a final concentration of 15 mM. The GTPγS- or GDP-bound form of ras p21 was incubated for 30 min at 4°C with ralGDS (20 pmol) in 80 μl of reaction mixture (20 mM Tris-HCl [pH 7.5], 5 mM EDTA, 10 mM MgCl₂, 0.5 mM DTT, 25 μM GTPγS or GDP) . Then, the anti-ras p21 antibody (Y13- 238) was added to this mixture, and the mixture was subjected to immunoprecipitation. The precipitate was subjected to SDS- PAGE, transferred to nitrocellulose filters, and probed with the anti-ralGDS antibody.

GAP assay of NF1. The GAP assay for c-ras p21 was performed as described previously (Gibbs et al., Proc. Natl. Acad. Sci. USA. 85:5026- 5030 (1988)). Briefly, c-H-ras p21 (2.5 pmol) was preincubated for 5 min at 30°C in 5 μl of preincubation mixture (100 mM sodium phosphate [pH 6.8], 0.5 mM EDTA, 0.5 mg of bovine serum albumin per ml, 0.5 mM DTT, 0.5 μM [γ-³²P]GTP [20,000 to 30,000 cpm/pmol]). To this preincubation mixture, 45 μl of reaction mixture (500 μM GTP, 22.2 mM sodium N- 2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES; pH 7.5], 1.1 M MgCl₂, 1.1 mg of bovine serum albumin per ml, 0.11 mM DTT, 2.2 mM Tris-HCl [pH 7.5]) containing 10 nM

GST-ΝF1 and the indicated amounts of ralGDS or GST-Ν-Raf was added, and a second incubation was performed at 24°C. Assays were quantified by rapid filtration on nitrocellulose filters. GAP activity was calculated from the decrease of the radioactivity of [γ-³²P]GTP compared with a reaction performed in the absence of GST-ΝF1, and GAP inhibition activity was expressed as percent decrease of GAP activity of GST-ΝF1. Interaction assay of Raf and ras p21.

To make the [α-³²P]GTP-bound form of ras p21, c-H-ras p21 (2.5 pmol) was preincubated for 15 min at 30°C in 5 μl of the preincubation mixture described above except that [cr-³²P]GTP was used instead of [γ-³ P]GTP. To the preincubation mixture, 45 μl of the reaction mixture described above containing 20 mM GST-N-Raf and the indicated amounts of ralGDS was added, and a second incubation was performed for 30 min at 4°C. GST-N-Raf was precipitated with glutathione-Sepharose 4B, the precipitates were washed, and the remaining radioactivity was counted.

Other assays. Protein concentrations were determined with bovine serum albumin as a standard using the Bradford assay (Bradford, Anal. Biochem.. 72:248-254 (1976)).

Nucleotide sequence accession number. The GenBank accession number for the mouse RGL cDNA sequence is U14103.

Results Isolation of a protein which interacts with rαs p21 in the yeast two-hybrid system.

To identify proteins that physically interact with H-ras p21, plasmid PC62/ras p21 encoding a fusion protein of ras p21 and the GAL4 DNA-binding domain was cotransformed with a mouse embryonic cDNA library expressed as a fusion with the GAL4 transactivation domain (PC51/mouse embryonic cDNA) into a yeast reporter strain YPB2 carrying the GAL4 binding sites upstream of both the S. cerevisiae HIS3 gene and the E. coli lacZ gene. Of the 0.5 million yeast transformants, five grew in the absence of histidine and expressed /3-galactosidase activity in a ras p21-dependent manner. The cDNA inserts from these five all encoded a single sequence containing an open reading frame of 164 amino acids and the consensus sequence for a stop codon and polyadenylation. This ras p21-interac ing domain was termed RID. Characterization of RID.

To characterize RID, the association of RID with ras p21 mutants and other small G proteins was examined in the yeast two-hybrid system (Table 1) . As assessed by filter assays of /3-galactosidase, RID interacted with ras p21. To examine the effect of posttranslational modification of ras p21 on its interaction with RID, ras p21^cι86s was used. It is known that this mutant is not posttranslationally modified (Hancock et al., Cell. 57:1167-1177 (1989)). Coexpression of RID with ras p21^cι86s reconstituted

/3-galactosidase activity. However, RID did not interact with the effector loop mutant of ras p21, ras p21^T35A. These findings indicate that the posttranslational modification of ras p21 is not necessary for its binding to RID and that the effector loop of ras p21 is required. Also examined was the specificity of small G proteins which interact with RID. RID interacted with rapl p21 as well as with ras p21 but not with ralB p24 or with racl p21^G12v. rapl p21 is known to have the same effector loop as ras p21 and to associate with the same effector-loop binding protein as ras p21.

TABLE 1. Interaction of RID with ras p21 in the yeast two-hybrid system³

GAL4 DNA-binding GAL4 transactivation /3-Galactosidase domain fusion domain fusion activity

Vector RID ras p21 Vector ras p21 RID +

rapl p21 RID ra B p24 RID

^aY153 was cotransformed with RID and ras p21 mutants or other small G proteins and assayed for 3-galactosidase activity. A blue signal, representing /3-galactosidase activity, is indicated by a +, and a white signal, indicating a lack of /3-galactosidase activity, is shown as .

In each case, _/8-galactosidase expression was not detected when cells were transformed with the DNA-binding domain (amino acids 1 to 147) or the transactivation domain (amino acids 768 to 881) fusion alone.

Molecular cloning of RGL.

Using RID to probe a BALB/C3T3 fibroblast cDNA library, a 2.7-kb cDNA containing an open reading frame of 768 amino acids (Fig. IA) was identified. The predicted protein sequence had 69% amino acid homology with ralGDS, which is a GDP/GTP exchange protein for ral p24, a member of small G- protein superfamily (Fig. IB) (Albright et al., supra). This protein was designated RGL. In the RGL cDNA, the 5' noncoding region was long and had a high percentage of G*C base pairs, which is typical in the 5' noncoding region. The neighboring sequence of the first ATG was consistent with the translation initiation start proposed by Kozak (Kozak, Nucleic Acids Res.. 15:8125-8148 (1987)).

The sequence of 290 amino acids (amino acids 210 to 499) of RGL was similar to a comparable region of CDC25, which is a GDP/GTP exchange protein for ras p21 (Lowy et al., Annu. Rev. Biochem. , 62:851-891 (1993); Martegani et al., EMBO J.. 11:2151-2157 (1992)) (Fig. 1C) . It is known that this region of CDC25 is important for GDS activity (Albright et al., supra; Lowy and Willumsen, supra; Martegani, supra) . RGL and ralGDS had an additional extensive region C terminal to the

CDC25-like domain (Albright, supra) . RID was located in this region of RGL. There was a region exhibiting 66% amino acid homology with RID in the C terminus of ralGDS. This strong homology in the overall sequence suggests that ralGDS and RGL constitute a family. Furthermore, this structural analysis suggests that ralGDS and RGL may interact with the effector loop of ras p21. Since ralGDS has been well characterized (Albright, supra) , it was convenient for testing as to whether it could be an effector protein of ras p21. Interaction of rαlGDB with rαs p21 in the yeast two- hybrid system.

Whether ralGDS interacts with ras p21 in the yeast two-hybrid system was examined (Table 2) . As assessed by filter assays of jS-galactosidase, ralGDS interacted with ras p21. Consistent with the data shown in Table 1, ralGDS interacted with ras p21^cι86s but not with ras p21^T35A. Furthermore, ralGDS interacted with rapl p21 as well as with ras p21 but not with ralB p24 or with racl p21^G12V.

Interaction of raGDS with rαs p21 in intact cells. To examine whether ralGDS interacts with ras p21 in intact cells, ralGDS was coexpressed with v-ras p21 in insect cells. The expression level of transfected ralGDS in Sf9 cells expressing ralGDS alone was similar to that in the cells coexpressing ralGDS with v-ras p21, as assessed by immunoblotting (Fig. 2A, lanes 1 to 3) . The lower band which is seen under ralGDS might be a degradation product of ralGDS. When the lysates coexpressing ralGDS with v-ras p21 were immunoprecipitated with the anti-ras p21 antibody, both ralGDS and ras p21 were detected in the ras p21 immune complex (Fig. 2B, lane 1) . When the lysates expressing ralGDS alone or v-ras p21 alone were immunoprecipitated with the anti-ras p21 antibody, ralGDS was not detected (Fig. 2B, lane 2; Fig. 2D, lane 1) . Neither ralGDS nor ras p21 was immunoprecipitated with nonimmune immunoglobulin in lysates expressing both proteins (Fig. 2B, lane 3) .

TABLE 2. Interaction of ralGDS with ras p21 in the yeast two-hybrid system³

GAL4 DNA-binding GAL4 transactivation .•-Galactosidase domain fusion domain fusion activity

Vector ralGDS _ ras p21 Vector - ras p21 ralGDS + ras p21^cι86s ralGDS + ras p21^T35A ralGDS - rapl p21 ralGDS + ralB p24 ralGDS - racl p21^G12V ralGDS -

^aYl53 was cotransformed with ralGDS and ras p2l mutants or other small G proteins and assayed for .-galactosidase activity. A blue signal, representing 3-galactosidase activity, is indicated by a +, and a white signal, indicating a lack of ^-galactosidase activity, is shown as a -. In each case, ^-galactosidase expression was not detected when cells were transformed with the DNA-binding domain (amino acids 1 to 147) or the transactivation domain (amino acids 768 to 881) fusion alone.

To characterize the interaction of ralGDS and ras p21 further, the ability of ralGDS to interact with a ras p21 mutant, ras p21^sl7N, was examined, ras p21^sl7N is well known as a dominant negative mutant that has higher affinity for GDP than GTP and strongly interacts with upstream molecules but not with downstream molecules (Barbacid, Annu. Rev. Biochem.. 56:779-827 (1987); Farnsworth et al., Mol. Cell Biol..

11:4822-4829 (1991); Lowy et al.. supra) . The expression level of ras p21^sl7N was similar to that of V-ras p21 (Fig. 2A, lanes 3 and 4) . When the lysates coexpressing ralGDS with ras p21^sl7N were immunoprecipitated with the anti- ras p21 antibody, ralGDS was not coprecipitated with ras p₂₁s^i7N _un(j_{er the sa}me conditions in which ralGDS was coprecipitated with v-ras p21 (Fig. 2C, lanes 1 and 2) . These results indicate that ralGDS makes a complex with v-ras p21 but not with ras p21^sl7N in intact cells.

Y13-238 was used as the anti-ras p21 antibody to immunoprecipitate ras p21 for these experiments. Another antibody, Y13-259, was tested for its ability to immunoprecipitate a ras p21-ralGDS complex. Y13-259 is known to be a neutralizing antibody (Mulcahy et al., Nature (London), 313:241-243 (1995)). In contrast to Y13-238, Y13- 259 could not immunoprecipitate the ras p21-ralGDS complex from the lysate coexpressing ralGDS with v-ras p21 under the same conditions (Fig. 2D, lanes 3 and 4) . Y13-259 and Y13-238 immunoprecipitated similar amounts of ras p21 from the lysates expressing v-ras p21 alone (Fig. 2D, lanes 1 and 2) . Interaction of raiGDS with rαs p21 in vitro.

The interaction of ralGDS and ras p21 from the cytosolic fraction of insect cells was direct, ralGDS and ras p21 were purified from the cytosolic fraction of insect cells. The purity of both proteins was more than 95% by Coomassie brilliant blue staining (Fig. 3A) . The GTPγS- or GDP-bound form of ras p21 was incubated with ralGDS, and this mixture was immunoprecipitated with the anti-ras p21 antibody. ralGDS was coprecipitated with the GTPγS-bound form of ras in a dose- dependent manner but not with the GDP-bound form (Fig. 3B) . Effect of rαlβDB on the GAP activity of NF1 for rαs p2l.

It has been reported that ralGDS does not affect the dissociation of GDP and GTP from ras p21 (Albright, supra) . The effect of ralGDS on the GTPase activity of ras p21 was examined. ralGDS did not alter the intrinsic GTPase activity of ras p21 (Fig. 4A) . In contrast, ralGDS inhibited the GAP activity of GST-NF1 for ras p21 at a 50% inhibitory dose (IC₅₀) of about 300 nM (Fig. 4). GST-N-Raf also inhibited the GAP activity of GST-NF1 at an IC₅₀ of about 50 nM under the same conditions (Fig. 4B) . The IC₅₀ value of GST-N-Raf to inhibit the GAP activity of GST-NF1 was similar to previous observations (Warne et al., Nature (London), 364:352-355 (1993)). ralGDS did not interact with GST-NF1 (data not shown) .

Effect of ra/GDS on the interaction of Raf with ras p21. Since Raf is an effector protein of ras p21, ralGDS was examined as to whether it inhibits the interaction of Raf with ras p21. GST-N-Raf interacted with ras p21 as described previously (Kikuchi et al., supra; Vojtek et al., Cell. 74:205-214 (1993); Warne, supra; Zhang et al., Nature (London), 364:308-313 (1993). ralGDS inhibited this interaction in a dose-dependent manner (Fig. 5) . The IC₅₀ value of ralGDS to inhibit the interaction of Raf-1 with ras p21 was about 250 nM.

Summary of experimental examples Using a yeast two-hybrid system, a novel protein which interacts with ras p21 was identified. This protein shares 69% amino acid homology with ral guanine nucleotide dissociation stimulator (ralGDS) , a GDP/GTP exchange protein for ral p24. Hence, the designation of this novel protein as RGL, for ralGDS-like. It was found that an effector loop mutant of ras p21 was defective in interacting with the ras p21-interacting domain of RGL, indicating that this domain binds to ras p21 through the effector loop of ras p21. Since ralGDS contained a region highly homologous with the ras p21- interacting domain of RGL, ralGDS was examined for possible interaction with ras p21. ralGDS failed to interact with an effector loop mutant of ras p21. In insect cells, ralGDS made a complex with v-ras p21 but not with a dominant negative mutant of ras p21, ralGDS interacted with the GTP-bound form of ras p21 but not with the GDP-bound form in vitro. ralGDS inhibited both the GTPase-activating activity of the neurofibro atosis gene product (NF1) for ras p21 and the interaction of Raf with ras p21 in vitro. These results demonstrate that ralGDS specifically interacts with the active form of ras p21 and that ralGDS can compete with NF1 and Raf for binding to the effector loop of ras p21. Therefore, ralGDS family members likely are effector proteins of ras p21 or will inhibit interactions between ras p21 and its effectors.

All the information contained in the references and patent documents cited above is incorporated herein by reference.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Williams, Lewis T. Demo, Susan

(ii) TITLE OF INVENTION: A Novel ras p21-Interacting Protein and

Uses Thereof

(iii) NUMBER OF SEQUENCES: 5

(iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Townsend and Townsend Khourie and Crew

(B) STREET: 379 Lytton Avenue

(C) CITY: Palo Alto

(D) STATE: California

(E) COUNTRY: USA (F) ZIP: 94301

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: To be assigned

(B) FILING DATE: 20-MAR-1995

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Dow, Karen B.

(B) REGISTRATION NUMBER: 29,684

(C) REFERENCE/DOCKET NUMBER: 02307K-56800

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 415-326-2400

(B) TELEFAX: 415-326-2422

(2) INFORMATION FOR SEQ ID NO:l:

<i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2671 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 160..2463

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

AATTCGGCAC GAGGCGGTCG CGCGCGGCGG CGGCGGCGGC AGTCGGGCAG CAAGGCGCGT 60

GGGAAGCGCG GGGACCCGGA GCCGGGCCAG AGAGACGCCC CGACCGCCTC GGAGCAGGGC 120

GCACCATGCA GCGTCCGTGT GCCGGAAAGA AAACTGAGA ATG AAA TTA CTT TGG 174

Met Lys Leu Leu Trp 1 5

CAA GCT AAA ATG AGC TCG ATT CAG GAC TGG GGT GAA GAG GTA GAG GAA 222 Gin Ala Lys Met Ser Ser lie Gin Asp Trp Gly Glu Glu Val Glu Glu

10 15 20

GGA GCT GTT TAC CAT GTC ACC CTC AAA AGA GTC CAG ATT CAA CAG GCG 270 Gly Ala Val Tyr His Val Thr Leu Lys Arg Val Gin He Gin Gin Ala 25 30 35

GCC AAT AAA GGA GCG AGA TGG CTA GGG GTT GAA GGG GAC CAG CTG CCT 318 Ala Asn Lys Gly Ala Arg Trp Leu Gly Val Glu Gly Asp Gin Leu Pro 40 45 50

CCA GGA CAC ACA GTC AGT CAG TAC GAG ACC TGC AAG ATC AGG ACC ATC 366 Pro Gly His Thr Val Ser Gin Tyr Glu Thr Cys Lys He Arg Thr He 55 60 65

AAA GCT GGT ACG CTG GAG AAG CTT GTG GAG AAC CTG CTG ACG GCT TTT 414 Lys Ala Gly Thr Leu Glu Lys Leu Val Glu Asn Leu Leu Thr Ala Phe 70 75 80 85

GGG GAC AAT GAC TTT ACC TAC ATC AGC ATC TTT TTG TCG ACA TAC AGA 462 Gly Asp Asn Asp Phe Thr Tyr He Ser He Phe Leu Ser Thr Tyr Arg

90 95 100 GGC TTT GCC TCG ACT AAG GAA GTG CTG GAG CTG CTG CTG GAC AGG TAT 510 Gly Phe Ala Ser Thr Lys Glu Val Leu Glu Leu Leu Leu Asp Arg Tyr 105 110 115

GGA AAC CTG ACA GGC CCA AAC TGT GAA GAC GAT GGA AGC CAA AGT TCA 558 Gly Asn Leu Thr Gly Pro Asn Cys Glu Asp Asp Gly Ser Gin Ser Ser 120 125 130

CCC GAG TCC AAG GCC GTG ATC CGG AAT GCC ATT GCT TCC ATC CTG AGG 606 Pro Glu Ser Lys Ala Val He Arg Asn Ala He Ala Ser He Leu Arg 135 140 145

GCC TGG CTT GAC CAG TGT GCG GAA GAC TTC CGG GAG CCC CCT CAC TTC 654 Ala Trp Leu Asp Gin Cys Ala Glu Asp Phe Arg Glu Pro Pro His Phe 150 155 160 165

CCT TGC CTT CAG AAG CTG CTG GAG TAC CTC AAA CAG ATG ATG CCT GGC 702 Pro Cys Leu Gin Lys Leu Leu Glu Tyr Leu Lys Gin Met Met Pro Gly 170 175 180

TCT GAC CCA GAG AGG AGA GCA CAG AAC CTT CTT GAA CAG TTT CAA AAG 750 Ser Asp Pro Glu Arg Arg Ala Gin Asn Leu Leu Glu Gin Phe Gin Lys 185 190 195

CAG GAC GTG GAT TCC GAC AAT GGA CTT CTC AAC ACC AGC TCC TTC AGC 798 Gin Asp Val Asp Ser Asp Asn Gly Leu Leu Asn Thr Ser Ser Phe Ser 200 205 210

CTG GAA GAG GAA GAG GAA CTG GAG AGC GGA GGG TCA GCA GAA TTC ACG 846 Leu Glu Glu Glu Glu Glu Leu Glu Ser Gly Gly Ser Ala Glu Phe Thr 215 220 225

AAC TTC TCA GAA GAT CTC GTG GCA GAA CAG CTG ACC TAC ATG GAC GCA 894 Asn Phe Ser Glu Asp Leu Val Ala Glu Gin Leu Thr Tyr Met Asp Ala 230 235 240 245

CAA CTA TTC AAG AAG GTA GTG CCT CAC CAT TGC CTG GGC TGT ATT TGG 942 Gin Leu Phe Lys Lys Val Val Pro His His Cys Leu Gly Cys He Trp 250 255 260

TCT CAG CGG GAT AAA AAG GAA AAC AAG CAT TTG GCT CCT ACG ATC CGT 990 Ser Gin Arg Asp Lys Lys Glu Asn Lys His Leu Ala Pro Thr He Arg 265 270 275

GCC ACC ATC TCT CAG TTT AAT ACG CTC ACC AAG TGT GTT GTC AGC ACC 1038 Ala Thr He Ser Gin Phe Asn Thr Leu Thr Lys Cys Val Val Ser Thr 280 285 290

GTC CTG GGG AGC AAG GAA CTC AAA ACT CAG CAG CGA GCC AGA GTC ATC 1086 Val Leu Gly Ser Lys Glu Leu Lys Thr Gin Gin Arg Ala Arg Val He 295 300 305

GAG AAG TGG ATC AAC ATT GCT CAC GAA TGT AGA ATC CTG AAG AAT TTT 1134 Glu Lys Trp He Asn He Ala His Glu Cys Arg He Leu Lys Asn Phe 310 315 320 325

TCC TCC TTG AGG GCC ATC GTT TCC GCA CTG CAG TCT AAT TCC ATC TAT 1182 Ser Ser Leu Arg Ala He Val Ser Ala Leu Gin Ser Asn Ser He Tyr

330 335 340

CGG TTG AAA AAG GCT TGG GCT GCT GTC CCG AAG GAC AGA ATG CTG ATG 1230 Arg Leu Lys Lys Ala Trp Ala Ala Val Pro Lys Asp Arg Met Leu Met 345 350 355

TTT GAA GAA CTT TCA GAT ATC TTC TCT GAT CAC AAT AAC CAT CTA ACC 1278 Phe Glu Glu Leu Ser Asp He Phe Ser Asp His Asn Asn His Leu Thr 360 365 370

AGT CGG GAG CTA CTA ATG AAG GAA GGA ACT TCA AAA TTT GCA AAC CTG 1326 Ser Arg Glu Leu Leu Met Lys Glu Gly Thr Ser Lys Phe Ala Asn Leu 375 380 385

GAC AGC AGC GTG AAA GAA AAC CAG AAG CGG ACC CAG AGG CGC CTG CAA 1374 Asp Ser Ser Val Lys Glu Asn Gin Lys Arg Thr Gin Arg Arg Leu Gin 390 395 400 405

CTG CAG AAG GAT ATG GGT GTG ATG CAG GGT ACC GTG CCT TAC CTG GGC 1422 Leu Gin Lys Asp Met Gly Val Met Gin Gly Thr Val Pro Tyr Leu Gly

410 415 420

ACC TTC CTG ACT GAC CTG ACC ATG CTG GAC ACT GCC CTG CAG GAC TAC 1470 Thr Phe Leu Thr Asp Leu Thr Met Leu Asp Thr Ala Leu Gin Asp Tyr 425 430 435

ATT GAG GGT GGA CTG ATC AAC TTC GAG AAA AGA AGA AGG GAA TTT GAA 1518 He Glu Gly Gly Leu He Asn Phe Glu Lys Arg Arg Arg Glu Phe Glu 440 445 450

GTC ATT GCC CAG ATA AAG CTC CTA CAG TCT GCT TGC AAC AGC TAC TGC 1566 Val He Ala Gin He Lys Leu Leu Gin Ser Ala Cys Asn Ser Tyr Cys 455 460 465 ATG GGC CCA GAC CAG AAG TTT ATC CAG TGG TTC CAG AGG CAG CAG CTT 1614 Met Gly Pro Asp Gin Lys Phe He Gin Trp Phe Gin Arg Gin Gin Leu 470 475 480 485

CTA TCA GAG GAG GAA AGC TAC GCC CTC TCG TGT GAG ATT GAA GCC GCT 1662 Leu Ser Glu Glu Glu Ser Tyr Ala Leu Ser Cys Glu He Glu Ala Ala

490 495 500

GCC GAC GCC AAC ACC ACT TCC CCT AAG CCT CGG AAA AGC ATG GTG AAG 1710 Ala Asp Ala Asn Thr Thr Ser Pro Lys Pro Arg Lys Ser Met Val Lys 505 510 515

AGG CTG AGC CTG CTA TTT CTG GGG TCT GAC ATC ATC CCC GGG AGC ACT 1758 Arg Leu Ser Leu Leu Phe Leu Gly Ser Asp He He Pro Gly Ser Thr 520 525 530

CCC ACC AAA GAG CAG CCC AAG TCC GCA GCC AGT GGG AGC TCT GGG GAG 1806 Pro Thr Lys Glu Gin Pro Lys Ser Ala Ala Ser Gly Ser Ser Gly Glu 535 540 545

AGT ATG GAC TCA GTC AGT GTG TCG TCC TGT GAA TCA AAC CAC TCC GAG 1854 Ser Met Asp Ser Val Ser Val Ser Ser Cys Glu Ser Asn His Ser Glu 550 555 560 565

GCT GAG GAG GGC CCC GTC ACA CCC ATG GAC ACA CCA GAT GAG CCC CAA 1902 Ala Glu Glu Gly Pro Val Thr Pro Met Asp Thr Pro Asp Glu Pro Gin

570 575 580

AAG AAG CTC TCT GAA TCC TCC TCT TCC TGT TCC TCC ATC CAT TCC ATG 1950 Lys Lys Leu Ser Glu Ser Ser Ser Ser Cys Ser Ser He His Ser Met 585 590 595

GAC ACG AAT TCC TCA GGG ATG TCG TCC CTA ATC AAC CCC CTG TCC TCC 1998 Asp Thr Asn Ser Ser Gly Met Ser Ser Leu He Asn Pro Leu Ser Ser 600 605 610

CCT CCA ACG TGC AAC AAC AAT CCT AAA ATC CAC AAG CGC TCC GTC TCC 2046 Pro Pro Thr Cys Asn Asn Asn Pro Lys He His Lys Arg Ser Val Ser 615 620 625

GTG ACA TCC ATT ACC TCC ACA GTA CTG CCT CCT GTT TAC AAT CAG CAG 2094 Val Thr Ser He Thr Ser Thr Val Leu Pro Pro Val Tyr Asn Gin Gin 630 635 640 645

AAC GAA GAC ACC TGC ATC ATC CGC ATC AGT GTA GAA GAC AAC AAT GGC 2142 Asn Glu Asp Thr Cys He He Arg He Ser Val Glu Asp Asn Asn Gly 650 655 660

CAC ATG TAC AAG AGC ATC ATG CTG ACA AGC CAG GAT AAG ACC CCC GCT 2190 His Met Tyr Lys Ser He Met Leu Thr Ser Gin Asp Lys Thr Pro Ala 665 670 675

GTG ATC CAG AGA GCG ATG TCG AAG CAC AAC CTG GAG TCG GAC CCC GCC 2238 Val He Gin Arg Ala Met Ser Lys His Asn Leu Glu Ser Asp Pro Ala 680 685 690

GAG GAG TAT GAG CTG GTG CAG GTC ATC TCG GAG GAC AAA GAA CTA GTG 2286 Glu Glu Tyr Glu Leu Val Gin Val He Ser Glu Asp Lys Glu Leu Val 695 700 705

ATC CCG GAC TCT GCA AAC GTC TTT TAC GCC ATG AAT AGC CAA GTG AAC 2334 He Pro Asp Ser Ala Asn Val Phe Tyr Ala Met Asn Ser Gin Val Asn 710 715 720 725

TTT GAT TTC ATT TTA CGC AAA AAG AAC TCG GTG GAG GAG CAG GTG AAG 2382 Phe Asp Phe He Leu Arg Lys Lys Asn Ser Val Glu Glu Gin Val Lys 730 735 740

TTG CGC AGT CGG ACC AGC CTG ACT TTG CCC AGG ACA GCT AAG CGG GGC 2430 Leu Arg Ser Arg Thr Ser Leu Thr Leu Pro Arg Thr Ala Lys Arg Gly 745 750 755

TGC TGG AGT AAC AGG CAC AGC AAG ATC ACC CTC T GAAAGGGACA 2474

Cys Trp Ser Asn Arg His Ser Lys He Thr Leu 760 765

GTACACTCCT ACTGCCCAAG GCAGAGTGAG GCTGAGCAAA AGCCATGGCG ACGCCAACCA 2534

CCACCCAGTG TTGAGCATCA TTGGTGAAAG CGACAGATAT TTATAGAATT CAGCTGTGCA 2594

GAGAGCACTG TGCAGGGGAG AGTGGAAGTG AATTTGACAT TAAAAGGATA AAAGGTTCAA 2654

AAAAAAAAAA AAAAAAA 2671

(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 768 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Lys Leu Leu Trp Gin Ala Lys Met Ser Ser He Gin Asp Trp Gly 1 5 10 15

Glu Glu Val Glu Glu Gly Ala Val Tyr His Val Thr Leu Lys Arg Val 20 25 30

Gin He Gin Gin Ala Ala Asn Lys Gly Ala Arg Trp Leu Gly Val Glu 35 40 45

Gly Asp Gin Leu Pro Pro Gly His Thr Val Ser Gin Tyr Glu Thr Cys 50 55 60

Lys He Arg Thr He Lys Ala Gly Thr Leu Glu Lys Leu Val Glu Asn 65 70 75 80

Leu Leu Thr Ala Phe Gly Asp Asn Asp Phe Thr Tyr He Ser He Phe 85 90 95

Leu Ser Thr Tyr Arg Gly Phe Ala Ser Thr Lys Glu Val Leu Glu Leu 100 105 110

Leu Leu Asp Arg Tyr Gly Asn Leu Thr Gly Pro Asn Cys Glu Asp Asp 115 120 125

Gly Ser Gin Ser Ser Pro Glu Ser Lys Ala Val He Arg Asn Ala He 130 135 140

Ala Ser He Leu Arg Ala Trp Leu Asp Gin Cys Ala Glu Asp Phe Arg 145 150 155 160

Glu Pro Pro His Phe Pro Cys Leu Gin Lys Leu Leu Glu Tyr Leu Lys 165 170 175

Gin Met Met Pro Gly Ser Asp Pro Glu Arg Arg Ala Gin Asn Leu Leu 180 185 190

Glu Gin Phe Gin Lys Gin Asp Val Asp Ser Asp Asn Gly Leu Leu Asn 195 200 205

Thr Ser Ser Phe Ser Leu Glu Glu Glu Glu Glu Leu Glu Ser Gly Gly 210 215 220

Ser Ala Glu Phe Thr Asn Phe Ser Glu Asp Leu Val Ala Glu Gin Leu 225 230 235 240

Thr Tyr Met Asp Ala Gin Leu Phe Lys Lys Val Val Pro His His Cys 245 250 255

Leu Gly Cys He Trp Ser Gin Arg Asp Lys Lys Glu Asn Lys His Leu 260 265 270

Ala Pro Thr He Arg Ala Thr He Ser Gin Phe Asn Thr Leu Thr Lys 275 280 285

Cys Val Val Ser Thr Val Leu Gly Ser Lys Glu Leu Lys Thr Gin Gin 290 295 300

Arg Ala Arg Val He Glu Lys Trp He Asn He Ala His Glu Cys Arg 305 310 315 320

He Leu Lys Asn Phe Ser Ser Leu Arg Ala He Val Ser Ala Leu Gin 325 330 335

Ser Asn Ser He Tyr Arg Leu Lys Lys Ala Trp Ala Ala Val Pro Lys

340 345 350

Asp Arg Met Leu Met Phe Glu Glu Leu Ser Asp He Phe Ser Asp His 355 360 365

Asn Asn His Leu Thr Ser Arg Glu Leu Leu Met Lys Glu Gly Thr Ser 370 375 380

Lys Phe Ala Asn Leu Asp Ser Ser Val Lys Glu Asn Gin Lys Arg Thr 385 390 395 400

Gin Arg Arg Leu Gin Leu Gin Lys Asp Met Gly Val Met Gin Gly Thr 405 410 415

Val Pro Tyr Leu Gly Thr Phe Leu Thr Asp Leu Thr Met Leu Asp Thr 420 425 430

Ala Leu Gin Asp Tyr He Glu Gly Gly Leu He Asn Phe Glu Lys Arg 435 440 445

Arg Arg Glu Phe Glu Val He Ala Gin He Lys Leu Leu Gin Ser Ala 450 455 460

Cys Asn Ser Tyr Cys Met Gly Pro Asp Gin Lys Phe He Gin Trp Phe 465 470 475 480

Gin Arg Gin Gin Leu Leu Ser Glu Glu Glu Ser Tyr Ala Leu Ser Cys 485 490 495

Glu He Glu Ala Ala Ala Asp Ala Asn Thr Thr Ser Pro Lys Pro Arg 500 505 510

Lys Ser Met Val Lys Arg Leu Ser Leu Leu Phe Leu Gly Ser Asp He 515 520 525

He Pro Gly Ser Thr Pro Thr Lys Glu Gin Pro Lys Ser Ala Ala Ser 530 535 540

Gly Ser Ser Gly Glu Ser Met Asp Ser Val Ser Val Ser Ser Cys Glu 545 550 555 560

Ser Asn His Ser Glu Ala Glu Glu Gly Pro Val Thr Pro Met Asp Thr 565 570 575

Pro Asp Glu Pro Gin Lys Lys Leu Ser Glu Ser Ser Ser Ser Cys Ser 580 585 590

Ser He His Ser Met Asp Thr Asn Ser Ser Gly Met Ser Ser Leu He 595 600 605

Asn Pro Leu Ser Ser Pro Pro Thr Cys Asn Asn Asn Pro Lys He His 610 615 620

Lys Arg Ser Val Ser Val Thr Ser He Thr Ser Thr Val Leu Pro Pro 625 630 635 640

Val Tyr Asn Gin Gin Asn Glu Asp Thr Cys He He Arg He Ser Val 645 650 655

Glu Asp Asn Asn Gly His Met Tyr Lys Ser He Met Leu Thr Ser Gin 660 665 670

Asp Lys Thr Pro Ala Val He Gin Arg Ala Met Ser Lys His Asn Leu 675 680 685 Glu Ser Asp Pro Ala Glu Glu Tyr Glu Leu Val Gin Val He Ser Glu 690 695 700

Asp Lys Glu Leu Val He Pro Asp Ser Ala Asn Val Phe Tyr Ala Met 705 710 715 720

Asn Ser Gin Val Asn Phe Asp Phe He Leu Arg Lys Lys Asn Ser Val

725 730 735

Glu Glu Gin Val Lys Leu Arg Ser Arg Thr Ser Leu Thr Leu Pro Arg 740 745 750

Thr Ala Lys Arg Gly Cys Trp Ser Asn Arg His Ser Lys He Thr Leu 755 760 765

(2) INFORMATION FOR SEQ ID NO:3:

_*

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 492 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:

TCGTCCCTAA TCAACCCCCT GTCCTCCCCT CCAACGTGCA ACAACAATCC TAAAATCCAC 60

AAGCGCTCCG TCTCCGTGAC ATCCATTACC TCCACAGTAC TGCCTCCTGT TTACAATCAG 120

CAGAACGAAG ACACCTGCAT CATCCGCATC AGTGTAGAAG ACAACAATGG CCACATGTAC 180

AAGAGCATCA TGCTGACAAG CCAGGATAAG ACCCCCGCTG TGATCCAGAG AGCGATGTCG 240

AAGCACAACC TGGAGTCGGA CCCCGCCGAG GAGTATGAGC TGGTGCAGGT CATCTCGGAG 300

GACAAAGAAC TAGTGATCCC GGACTCTGCA AACGTCTTTT ACGCCATGAA TAGCCAAGTG 360

AACTTTGATT TCATTTTACG CAAAAAGAAC TCGGTGGAGG AGCAGGTGAA GTTGCGCAGT 420

CGGACCAGCC TGACTTTGCC CAGGACAGCT AAGCGGGGCT GCTGGAGTAA CAGGCACAGC 480 AAGATCACCC TC 492

(2) INFORMATION FOR SEQ ID NO:4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 164 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 :

Ser Ser Leu He Asn Pro Leu Ser Ser Pro Pro Thr Cys Asn Asn Asn

1 5 10 15

Pro Lys He His Lys Arg Ser Val Ser Val Thr Ser He Thr Ser Thr 20 25 30

Val Leu Pro Pro Val Tyr Asn Gin Gin Asn Glu Asp Thr Cys He He 35 40 45

Arg He Ser Val Glu Asp Asn Asn Gly His Met Tyr Lys Ser He Met 50 55 60

Leu Thr Ser Gin Asp Lys Thr Pro Ala Val He Gin Arg Ala Met Ser 65 70 75 80

Lys His Asn Leu Glu Ser Asp Pro Ala Glu Glu Tyr Glu Leu Val Gin

85 90 95

Val He Ser Glu Asp Lys Glu Leu Val He Pro Asp Ser Ala Asn Val

100 105 110

Phe Tyr Ala Met Asn Ser Gin Val Asn Phe Asp Phe He Leu Arg Lys 115 120 125

Lys Asn Ser Val Glu Glu Gin Val Lys Leu Arg Ser Arg Thr Ser Leu 130 135 140

Thr Leu Pro Arg Thr Ala Lys Arg Gly Cys Trp Ser Asn Arg His Ser

145 150 155 160 Lys He Thr Leu

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 852 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Met Met Val Asp Cys Gin Ser Ser Thr Gin Glu He Gly Glu Glu Leu 1 5 10 15

He Asn Gly Val He Tyr Ser He Ser Leu Arg Lys Val Gin Leu His 20 25 30

Gin Gly Ala Thr Lys Gly Gin Arg Trp Leu Gly Cys Glu Asn Glu Ser 35 40 45

Ala Leu Asn Leu Tyr Glu Thr Cys Lys Val Arg Thr Val Lys Ala Gly 50 55 60

Thr Leu Glu Lys Leu Val Glu His Leu Val Pro Ala Phe Gin Gly Ser^' 65 70 75 80

Asp Leu Ser Tyr Val Thr Val Phe Leu Cys Thr Tyr Arg Ala Phe Thr

85 90 95

Thr Thr Gin Gin Val Leu Asp Leu Leu Phe Lys Arg Tyr Gly Arg Cys

100 105 110

Asp Ala Leu Thr Ala Ser Ser Arg Tyr Gly Cys He Leu Pro Tyr Ser 115 120 125

Ser Glu Asp Gly Gly Pro Gin Asp Gin Leu Lys Asn Ala He Ser Ser 130 135 140 He Leu Gly Thr Trp Leu Asp Gin Tyr Ser Glu Asp Phe Cys Gin Pro 145 150 155 160

Pro Asp Phe Pro Cys Leu Lys Gin Leu Val Ala Tyr Val Gin Leu Asn 165 170 175

Met Pro Gly Ser Asp Leu Glu Arg Arg Ala His Leu Leu Leu Ala Gin

180 185 190

Leu Glu Asp Leu Glu Pro Ser Glu Ala Glu Ser Glu Ala Leu Ser Pro 195 200 205

Ala Pro Val Leu Ser Leu Lys Pro Ala Ser Gin Leu Glu Pro Ala Leu 210 215 220

Leu Leu Thr Pro Ser Gin Val Val Thr Ser Thr Pro Val Arg Glu Pro 225 230 235 240

Ala Ala Ala Pro Val Pro Val Leu Ala Ser Ser Pro Val Val Ala Pro 245 250 255

Ala Pro Glu Leu Glu Pro Val Pro Glu Pro Pro Gin Glu Pro Glu Pro

260 265 270

Ser Leu Ala Leu Ala Pro Glu Leu Glu Pro Ala Val Ser Gin Ser Leu 275 280 285

Glu Leu Glu Ser Ala Pro Val Pro Thr Pro Ala Leu Glu Pro Ser Trp 290 295 300

Ser Leu Pro Glu Ala Thr Glu Asn Gly Leu Thr Glu Lys Pro His Leu 305 310 315 320

Leu Leu Phe Pro Pro Asp Leu Val Ala Glu Gin Phe Thr Leu Met Asp 325 330 335

Ala Glu Leu Phe Lys Lys Val Val Pro Tyr His Cys Leu Gly Ser He

340 345 350

Trp Ser Gin Arg Ala Lys Lys Gly Lys Glu His Leu Ala Pro Thr He 355 360 365

Arg Ala Thr Val Ala Gin Phe Asn Asn Val Ala Asn Cys Val He Thr 370 375 380

Thr Cys Leu Gly Asp Gin Ser Met Lys Ala Pro Asp Arg Ala Arg Val 385 390 395 400

Val Glu His Trp He Glu Val Ala Arg Glu Cys Arg Ala Leu Lys Asn 405 410 415

Phe Ser Ser Leu Tyr Ala He Leu Ser Ala Leu Gin Ser Asn Ala He 420 425 430

His Arg Leu Lys Lys Thr Trp Glu Glu Val Ser Arg Asp Ser Phe Arg 435 440 445

Val Phe Gin Lys Leu Ser Glu He Phe Ser Asp Glu Asn Asn Tyr Ser 450 455 460

Leu Ser Arg Glu Leu Leu He Lys Glu Gly Thr Ser Lys Phe Ala Thr

465 470 475 480

He Glu Met Asn Pro Arg Arg Ala Gin Arg Arg Gin Lys Glu Thr Gly 485 490 495

Val He Gin Gly Thr Val Pro Tyr Leu Gly Thr Phe Leu Thr Asp Leu 500 505 510

Val Met Leu Asp Thr Ala Met Lys Asp Tyr Leu Tyr Gly Arg Leu He 515 520 525

Asn Phe Glu Lys Arg Arg Lys Glu Phe Glu Val He Ala Gin He Lys 530 535 540

Leu Leu Gin Ser Ala Cys Asn Asn Tyr Ser He Ala Pro Glu Glu His

545 550 555 560

Phe Gly Thr Trp Phe Arg Ala Met Glu Arg Leu Ser Glu Ala Glu Ser 565 570 575

Tyr Thr Leu Ser Cys Glu Leu Glu Pro Pro Ser Glu Ser Ala Ser Asn 580 585 590

Thr Leu Arg Ser Lys Lys Ser Thr Ala He Val Lys Arg Trp Ser Asp 595 600 605

Arg Gin Ala Pro Ser Thr Glu Leu Ser Thr Ser Ser Ser Ala His Ser 610 615 620

Lys Ser Cys Asp Gin Leu Arg Cys Ser Pro Tyr Leu Gly Ser Gly Asp

625 630 635 640 He Thr Asp Ala Leu Ser Val His Ser Ala Gly Ser Ser Ser Ser Asp 645 650 655

Val Glu Glu He Asn Met Ser Phe Val Pro Glu Ser Pro Asp Gly Gin 660 665 670

Glu Lys Lys Phe Trp Glu Ser Ala Ser Gin Ser Ser Pro Glu Thr Ser

675 680 685

Gly He Thr Ser Ala Ser Ser Ser Thr Ser Ser Ser Ser Ala Ser Thr 690 695 700

Thr Pro Val Ser Thr Thr Arg Thr His Lys Arg Ser Val Ser Gly Val 705 710 715 720

Cys Ser Tyr Ser Ser Ser Leu Pro Leu Tyr Asn Gin Gin Val Gly Asp 725 730 735

Cys Cys He He Arg Val Ser Leu Asp Val Asp Asn Gly Asn Met Tyr 740 745 750

Lys Ser He Leu Val Thr Ser Gin Asp Lys Ala Pro Thr Val He Arg

755 760 765

Lys Ala Met Asp Lys His Asn Leu Asp Glu Asp Glu Pro Glu Asp Tyr 770 775 780

Glu Leu Val Gin He He Ser Glu Asp His Lys Leu Lys He Pro Glu 785 790 795 800

Asn Ala Asn Val Phe Tyr Ala Met Asn Ser Thr Ala Asn Tyr Asp Phe 805 810 815

He Leu Lys Lys Arg Thr Phe Thr Lys Gly Ala Lys Val Lys His Gly 820 825 830

Ala Ser Ser Thr Leu Pro Arg Met Lys Gin Lys Gly Leu Arg He Ala

835 840 845

Lys Gly He Phe

850

Claims

WHAT IS CLAIMED IS:

1. An isolated polynucleotide comprising at least 80% sequence identity with the nucleotide sequence of SEQ ID NO:l, an allelic or species variation thereof, or a fragment thereof.

2. The polynucleotide of claim 1, comprising the nucleotide sequence of SEQ ID NO:l.

3. The polynucleotide of claim 1, comprising the nucleotide sequence of SEQ ID NO:3.

. An isolated polypeptide comprising at least 80% sequence identity with the sequence of SEQ ID NO:2, an allelic or species variation thereof, or a fragment thereof.

5. The polypeptide of claim 4, comprising the sequence of SEQ ID NO:2.

6. The polypeptide of claim 4, comprising the sequence of SEQ ID NO:4.

7. The polypeptide of claim 4, said polypeptide capable of binding the effector loop of ras p21.

8. The polypeptide of claim 4, which is a fusion protein.

9. The polypeptide of claim 8, wherein the fusion protein is RID-GAL4.

10. The polypeptide of claim 8, wherein the fusion protein comprises a tag, or a product of a second gene or fragment thereof.

11. The polypeptide of claim 10, wherein the tag is GST, an epitope tag or an enzyme.

12. The polypeptide of claim 10, wherein the second gene is lacZ.

13. A recombinant DNA molecule comprising the nucleotide sequence of SEQ ID NO:l or a fragment thereof.

14. The recombinant DNA molecule of claim 13, wherein the nucleotide sequence encodes a RID-GAL4 transactivation domain fusion protein.

15. A recombination DNA molecule, pGAD/ralGDS, encoding a ralGDS-GAL4 transactivation domain fusion protein.

16. A cell containing the recombinant DNA molecule of claim 13.

17. An antibody that specifically binds to a polypeptide of claim 4.

18. The antibody of claim 17, wherein the polypeptide comprises the sequence of SEQ ID NO:2.

19. The antibody of claim 17, wherein the polypeptide comprises the sequence of SEQ ID NO:4.

20. The antibody of claim 17, which is a monoclonal antibody.

21. A hybridoma capable of producing the antibody of claim 20.

22. A kit comprising an antibody of claim 17.

23. A method of isolating a RGL gene or fragment thereof, comprising screening a DNA library using a RGL probe to identify a hybridizing clone and isolating said RGL gene or gene fragment from said hybridizing clone.

24. The method of claim 23, wherein said RGL probe comprises the nucleotide sequence of SEQ ID NO:l or a fragment thereof.

25. The method of claim 23, wherein said RGL gene is from a human.

26. A method of identifying a gene encoding a ras p21-binding protein, comprising screening a DNA library with a RID probe to identify a hybridizing clone containing a RID sequence, the presence of a RID sequence being indicative of a gene encoding a ras p21-binding protein.

27. The method of claim 26, wherein said RID probe comprises the sequence of SEQ ID NO:3 or a fragment thereof.

28. The method of claim 26, wherein said ras p21- binding protein is a regulator or an effector protein of ras p21.

29. A method of identifying a ras effector loop- binding protein, comprising the steps of: screening a gene library with an RID probe for a gene that is substantially homologous to the RGL gene; isolating said substantially homologous gene; producing a polypeptide encoded by said substantially homologous gene; determining if said polypeptide binds an effector loop of a ras protein, binding indicating that said polypeptide is a ras effector loop-binding protein.

30. A method of modulating or blocking ras p21 activity comprising providing a RID polypeptide in a cell expressing ras p21 protein wherein said RID polypeptide binds to said ras p21 protein to block ras p21 activity.

31. The method of claim 30, wherein said RID polypeptide is provided by introducing an expression vector encoding a RID polypeptide into said ras p21 expressing cell.

32. The method of claim 31, wherein the RID polypeptide is derived from RGL or ralGDS.

33. A method of blocking a ras p21 protein binding to a ras effector protein in a cell, comprising expressing a RID polypeptide from a RGL or a ralGDS protein in said cell.

34. The method of claim 33, wherein the ras effector protein is selected from the group consisting of Raf, GAP, NF1 and PI (3)K.

35. A method of isolating a RGL interacting protein, comprising contacting a cell lysate suspected of containing a RGL interacting protein with a RGL polypeptide and isolating any protein bound to said RGL polypeptide as a RGL interacting protein.

36. The method of claim 35, wherein said RGL interacting protein is ras p21 and said RGL polypeptide is a RID polypeptide.

37. A pharmaceutical composition useful in the treatment of a cell proliferative condition, comprising a RID polypeptide, and a pharmaceutically acceptable carrier.

38. A pharmaceutical composition useful in the treatment of a cell proliferative condition, comprising an expression vector capable of expressing a RID polypeptide in an affected cell and a pharmaceutically acceptable carrier.

39. A method of alleviating a patient suffering from a cell proliferative condition, comprising administering to said patient, a therapeutically effective amount of the pharmaceutical composition of claim 37.

40. The method of claim 39, wherein the cell proliferative condition is cancer or restenosis caused by ras dysfunction.