Novel Grb2 Associating Polypeptides And Nucleic Acids Encoding Therefor

Abstract

The present invention generally relates to novel GRB2 associating proteins and nucleic acids which encode these protein. In particular, these novel proteins possess inositol polyphosphate 5-phosphatase and phosphatidylinositol 5-phosphatase activities, important in growth factor mediated signal transduction. As such, the proteins, nucleic acids encoding the proteins, cells capable of expressing these nucleic acids and antibodies specific for these proteins will find a variety of uses in a variety of screening, therapeutic and other applications.

Description

[0002] The present invention generally relates to novel GRB2 associating polypeptides and nucleic acids which encode these polypeptides. In particular, these novel polypeptides possess inositol polyphosphate 5-phosphatase activity, important in growth factor mediated signal transduction. As such, the polypeptides, nucleic acids encoding the polypeptides, cells capable of expressing these nucleic acids and antibodies specific for the polypeptides will find a variety of uses in a wide range of screening, therapeutic and other applications.

BACKGROUND OF THE INVENTION

[0003] Receptor signaling pathways are the subject of widespread research efforts. A better understanding of these signaling pathways will lead to the design of new and more effective drugs in the treatment of many diseases. Of particular interest are the growth factor and related receptor signaling pathways and their role in cell growth and differentiation. Binding of a particular growth factor to its receptor on the cell plasma membrane can stimulate a wide variety of biochemical responses, including changes in ion fluxes, activation of various kinases, alteration of cell shape, transcription of various genes and modulation of enzymatic activities in cellular metabolism.

[0004] Growth factors play a role in embryonic development, cancer, atherosclerosis and the responses of tissues to injury. Growth factors are involved in several normal developmental processes as well as in pathological conditions. Many growth factor receptors are tyrosine kinases whose signalling is dependent upon tyrosine phosphorylation of both the receptor and other molecules. Specific phosphorylated tyrosine residues on these receptors recruit soluble intracellular signaling molecules to the complex upon growth factor stimulation, thus initiating the growth factor signaling cascade. The signal can then proceed through a series of steps to the nucleus and other subcellular locations where the final effects of activation by the extracellular ligand are produced. Recruitment of molecules is often carried out by adapter molecules containing only protein-protein interaction domains with no associated enzymatic activity. By examining the molecules that interact with these adapters, important parts of the signaling mechanism can be discovered, monitored and controlled. One such adapter protein is GRB2, a 24 kDa cytosolic adapter protein containing two SH3 domains flanking an SH2 domain, which is known to be involved in linking many important molecules in signal transduction.

[0005] Because disregulation of the cellular processes involved in cell growth can have disastrous effects, it is important to understand and gain control over these processes. This requires identifying the participants in the signaling events that lead to mitogenesis and elucidating their mechanism of function. The identification of these participants is important for a wide range of diagnostic, therapeutic and screening applications. In particular, by knowing the structure of a particular participant in a growth factor activation cascade, one can design compounds which affect that cascade, to either activate an otherwise inactive pathway, or inactivate an overly active pathway. Similarly, having identified a particular participant in a growth factor cascade, one can also identify situations where that cascade is defective, resulting in a particular pathological state. The identification of participants in particular growth factor activation cascades is thus of critical importance for screening compounds that affect these cascades and treating a variety of disorders resulting from anomalies in these cascades, both as therapeutic agents and as model systems for identification of compounds which affect the pathway and thus may be useful as therapeutic agents. The present invention meets these and many other needs.

SUMMARY OF THE INVENTION

[0006] The present invention generally provides substantially pure polypeptides, comprising an amino acid sequence that is substantially homologous to the amino acid sequence shown in FIG. 10 (SEQ ID NO:2), or biologically active fragments thereof.

[0007] The present invention also provides isolated nucleic acid segments, which encode a polypeptide having an amino acid sequence substantially homologous to the amino acid sequence shown in FIG. 10 (SEQ ID NO:2), or biologically active fragments thereof.

[0008] Also provided are isolated antibodies that are specifically immunoreactive with a polypeptide having an amino acid sequence substantially homologous to the amino acid sequence shown in FIG. 10 (SEQ ID NO:2) or its biologically active fragments.

[0009] In a further aspect, the present invention provides methods of using these polypeptides. In particular, the invention provides a method of determining whether a test compound is an agonist or antagonist of a GRB2/GA5Ptase interaction. The method comprises contacting GRB2 with GA5Ptase (SEQ ID NO:2) under conditions conducive to forming a GRB2/GA5Ptase complex, in the presence and absence of the test compound. The amount of GRB2/GA5Ptase complex formed in the presence and absence of the test compound is then determined. An increase or decrease in the amount of GRB2/GA5Ptase complex formed in the presence of the test compound is indicative that the test compound is an agonist or antagonist of GRB2/GA5Ptase interaction, respectively.

[0010] In a related aspect, the present invention provides a method for determining whether a test compound is an agonist or antagonist of an inositol polyphosphate 5-phosphatase activity. The method comprises incubating a mixture of inositol polyphosphate substrate and GA5Ptase, in the presence and absence of the test compound. The mixture is then assayed to determine the amount of GA5Ptase product formed in the presence and absence of the test compound. The amount of product of GA5Ptase activity in the presence of the test compound is compared to the amount of product of GA5Ptase activity in the absence of the test compound. An increase or decrease in the amount of product of GA5Ptase activity in the presence of the test compound is indicative that the test compound is an agonist or antagonist of an inositol polyphosphate 5-phosphatase activity, respectively.

[0011] The present invention also provides a method of identifying the presence of GRB2 in a sample. The method comprises incubating the sample with the polypeptide of the invention, and detecting binding between the polypeptide and a portion of the sample. This binding is indicative of the presence of GRB2 in the sample.

[0012] Also provided is a method of purifying GRB2 from a mixture of different proteins containing GRB2. The method comprises immobilizing the polypeptide of the invention, on a solid support. The mixture of proteins is then contacted with the solid support under conditions in which the polypeptide binds GRB2. The solid support is washed to remove unbound proteins, and GRB2 is eluted from the solid support.

[0013] The present invention also provides kits for practicing these methods.

[0014] In a further aspect, the present invention provides a method of treating a patient suffering from a proliferative disorder. The method comprises administering to the patient a therapeutically effective amount of the polypeptide of the invention.

[0015] The present invention also provides substantially pure polypeptides that are immunologically cross-reactive with antibodies to the GA5ptase polypeptides and fragments, described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 shows a schematic representation of the probe used to screen a .lambda.gt11 expression library.

[0017] FIG. 2 shows a ribbon diagram and dendrogram illustrating relative similarities between inositol polyphosphate 5-phosphatases. Numbers beside the nodes of the dendrogram indicate the percent identity and (similarity). The black bar above the ribbon representation of GA5Ptase indicates the region cloned by interaction with GRB2, whereas the "P" indicates the location of PXXP motifs.

[0018] FIG. 3 shows a schematic representation of GA5Ptase, GRB2 and GRB3.3 molecules used in Cos7 Immunoprecipitations.

[0019] FIG. 4 shows results of co-immunoprecipitation of GA5Ptase with wild type GRB2 ("GRB2 wt"), point mutations of GRB2 ("GRB2 P49L", "GRB2 E89K", "GRB2 S90N", "GRB2 G203R") and GRB3.3.

[0020] FIG. 5A shows c-Fos Serum Responsive Element (SRE) activation when co-expressed with various combinations of GRB2, c-Ras and GA5Ptase. The error bars indicate standard error of the mean of triplicate transfections.

[0021] FIG. 5B shows SRE activation when co-expressed as indicated with GA5Ptase, c-Ras and the various GRB2 point mutations used in the Cos7 co-immunoprecipitation experiments shown in FIG. 4.

[0022] FIG. 5C shows SRE activation when co-expressed as indicated with GA5Ptase, v-Ras and GrbRB2 point mutations.

[0023] FIG. 5D shows a comparison of SRE activation when co-expressed with GA5Ptase versus platelet inositol polyphosphate 5-phosphatase type-II ("5Ptase II").

[0024] FIG. 6 shows the effect of varying concentration of Ins(1,3,4,5)P.sub.4 on the rate of its hydrolysis by GA5Ptase.

[0025] FIGS. 7A and 7B show the immunoprecipitation of both GA5Ptase protein and Ins(1,3,4,5)P.sub.4 hydrolyzing activity of GA5Ptase. HA-tagged GA5Ptase was immunoprecipitated with .alpha.HA antiserum. Following contact with protein A sepharose, the supernatant (.cndot.) and protein A sepharose pellet (.degree.) were Western blotted against .alpha.HA antiserum (FIG. 7B) and assayed for ability to hydrolyze Ins(1, 3,4,5)P.sub.4 (FIG. 7A).

[0026] FIGS. 8A-C show HPLC analysis of reaction products from incubation of GA5Ptase with Ins(1,3,4,5)P.sub.4.

[0027] FIG. 8A shows conversion of .sup.3H-Ins(1,3,4,5)P.sub.4 to .sup.3H-Ins(1,3,4)P.sub.3 (peak) in the presence of GA5Ptase.

[0028] FIG. 8B shows the conversion of .sup.3H-Ins(1,3,4,5)P.sub.4 to .sup.3H-Ins(3,4)P.sub.2 (peak), in the presence of GA5Ptase and inositol polyphosphate 1-phosphatase.

[0029] FIG. 8C shows the conversion of .sup.3H-Ins(1,3,4,5)P.sub.4 to .sup.3H-Ins(1,3)P.sub.2 (peak) in the presence of GA5Ptase and inositol polyphosphate 4-phosphatase.

[0030] FIG. 9 shows the hydrolysis of PtdIns(3,4,5)P.sub.3 by recombinant inositol polyphosphate 5-phosphatases. Specifically shown is a graph showing hydrolysis with GA5Ptase (open squares) and human inositol polyphosphate 5-phosphatase II ("5-Ptase II") (closed squares). Also shown is a TLC autoradiogram indicating conversion of PtdIns(3,4,5)P.sub.3 to PtdInsP.sub.2 by GA5Ptase and 5-Ptase II (inset).

[0031] FIG. 10 shows the nucleotide sequence (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO:2) of the GRB2 associating protein GA5Ptase.

[0032] FIG. 11 shows a comparison between the amino acid sequence of GA5Ptase (SEQ ID NO:2) and that of a number of other inositol polyphosphate 5-phosphatases. Level of shading indicates similarity in residue structure. Black boxes indicate a consensus sequence. The sequences shown are C. elegans inositol polyphosphate 5-phosphatase ("celegptase") (SEQ ID NO:3), S. cereviseae inositol polyphosphate 5-phosphatase ("ysc5ptase") (SEQ ID NO:4), GA5Ptase (SEQ ID NO:2), human 51c ("51c") (SEQ ID NO:5), human inositol polyphosphate 5-phosphatase 75 kDa ("5ptaseii") (SEQ ID NO:6), human ocr1 protein responsible for human oculocerebrorenal syndrome ("ocr1") (SEQ ID NO:7), Arabidopsis inositol polyphosphate 5-phosphatase ("arab5ptase") (SEQ ID NO:8) and canine inositol polyphosphate 5-phosphatase 43 kDa ("h5ptase43")(SEQ ID NO:9). The identified consensus sequence is also provided ("consensus") (SEQ ID NO:10).

DESCRIPTION OF THE PREFERRED EMBODIMENT

[0033] I. General Description

[0034] The present invention generally provides novel GRB2 associating polypeptides. These polypeptides are generally involved in signal transduction pathways following growth factor activation. In particular, the polypeptides of the present invention contribute to the mediation of inositol polyphosphate based signal transduction pathways, following growth factor activation.

[0035] Also provided by the present invention are nucleic acids encoding these novel polypeptides, expression vectors containing these nucleic acids, cells capable of expressing these expression vectors, antibodies which specifically recognize and bind these polypeptides and methods of using these polypeptides and nucleic acids in screening and therapeutic applications.

[0036] The polypeptides of the present invention have been identified as possessing unique specificity for inositol polyphosphates. In particular, the polypeptides of the present invention have inositol polyphosphate 5-phosphatase activity, and more particularly, the ability to remove the 5-phosphate from D-myo-Inositol 1,3,4,5-tetrakisphosphate ("Ins(1,3,4,5)P.sub.4") and Phosphatidylinositol 3,4,5-trisphosphate ("PtdIns(3,4,5)P.sub.3"), but not D-myo-Inositol 1,4,5-trisphosphate ("Ins(1,4,5)P.sub.3") or Phosphatidylinositol (4,5)-bisphosphate ("PtdIns(4,5)P.sub.2"). Accordingly, the polypeptides of the present invention are generally referred to herein by the abbreviation GA5Ptase, for GRB2 Associating inositol polyphosphate 5-phosphatase.

[0037] Inositol polyphosphates have been broadly implicated in cell signalling pathways. For example, stimulation of cell surface receptors has been found to initiate hydrolysis of membrane-bound inositol lipid, which produces at least two second messengers: diacylglycerol (DAG) and inositol(1,4,5)trisphosphate. These messengers are generated by a membrane transduction process which comprises three main components: a receptor, a coupling G protein and phosphoinositidase C. DAG acts by stimulating protein kinase C, whereas Ins(1,4,5)P.sub.3 releases calcium from internal stores (see, Berridge and Irvine, Nature (1989) 341:197-205).

[0038] PtdIns(3,4,5)P.sub.3 in particular, is the product of phosphatidyl inositol 3-kinase ("PI3 kinase"), an important agonist activated signaling protein, stimulated in growth factor mediated signal transduction. PI3-kinase is known to be involved in the regulation of cell growth and oncogenic transformation (Cantley et al., Cell, 64:1657 (1993)). Upon growth factor receptor stimulation, the wild-type PI3-kinase is activated and can phosphorylate phosphatidylinositol ("PtdIns") at the 3' position of the inositol ring. These phosphatidylinositol 3-phosphates are candidate second messenger molecules. The PI3-kinase enzyme is found associated with receptor protein tyrosine kinases such as PDGF-R-.beta., CSF-1 receptor, Insulin receptor and IGF-1 receptor as well as non-receptor tyrosine kinase oncogenes, e.g., src, gag-abl and fyn. Studies on mutants of platelet-derived growth factor (PDGF) receptor have shown that PI3-kinase is a key mediator of PDGF-mediated mitogenic signaling (Fantl et al., Cell, 69:413 (1992); Valius et al., ibid., 73:321 (1993)). PDGF-R mutants that are unable to bind PI3-kinase are also unable to induce a mitogenic response after growth factor stimulation and unable to activate p21c-Ras (Ras). These data indicate that PI3-kinase acts upstream of Ras in PDGF-stimulated signaling. Studies also indicate that the PI3-kinase product, PtdIns(3,4,5)P.sub.3 is not the final product produced during the initial phases of signaling, indicating further processing of this signaling molecule. Stephens, et al., Nature 351:33-39 (1991), Hawkins, et al., Nature 358:157-159 (1992).

[0039] The action of the polypeptides of the present invention upon the specific product of PI3-kinase implicates these polypeptides as important downstream mediators of growth factor activation signaling cascades. Furthermore, in addition to inositol polyphosphate 5-phosphatase activity, the polypeptides of the invention also associate with GRB2, in cell culture. GRB2 is an intracellular signalling molecule that is recruited to the cell membrane/receptor complex upon growth factor stimulation. GRB2 is specifically recruited to the PDGF, EGF and other tyrosine growth factor receptors. It is also in the signaling pathway that activates Ras upon growth factor stimulation. GRB2 is a small protein (24 kDa) that functions as an adapter molecule using its two SH3 domains and single SH2 domain to provide a bridge between other important signaling molecules. Clark, et al., Nature 356:340-344 (1992), Stern, et al., Mol. Biol. Cell 4:1175-1188 (1993). The ability of the polypeptides of the invention to specifically associate with GRB2 further indicates the importance of these polypeptides as downstream mediators of growth factor activation signal transduction, generally.

[0040] The polypeptides of the present invention have also been shown to activate signaling through the Fos serum response element (SRE) in fibroblast cells when these polypeptides are co-expressed with GRB2 and c-Ras. This activation is four to six fold over the activation seen with GRB2, Ras or GRB2/Ras alone. The Fos SRE is a gene that is known to be turned on early in growth factor activation, and has been identified as an upstream event for many response elements for cell growth. See, e.g., Janknecht et al., Carcinogenesis 16(3)443-450 (1995), Piechaczyk et al., Crit. Rev. Oncol./Hematol. 17(2):93-131 (1994), Maruta et al., Bioessays 16(7):489-496 (1994). The Fos gene is also responsive to a large number of growth factors. In particular, the Fos SRE is believed to be the direct target induced by growth factor stimulation through the Ras oncogene.

[0041] The combination of the activities and specificities of the polypeptides of the present invention implicates these polypeptides as key elements in the activation of Ras and as downstream molecules generally, in agonist activated signal transduction cascades.

[0042] II. Proteins and Polypeptides of the Invention

[0043] In one aspect, the present invention provides substantially pure, or isolated polypeptides that are generally characterized by one or more of the following activities: inositol polyphosphate 5-phosphatase activity; the ability to associate with GRB2; and/or the ability to enhance activation of the Fos SRE when co-expressed with c-Ras or c-Ras and GRB2. In particular, the polypeptides of the present invention will generally possess inositol polyphosphate 5-phosphatase activity, and be capable of removing the 5-phosphate from D-myo-Inositol 1,3,4,5-tetrakisphosphate ("Ins(1,3,4,5)P.sub.4") and Phosphatidylinositol 3,4,5-trisphosphate ("PtdIns(3,4,5)P.sub.3"), but not D-myo-Inositol 1,4,5-trisphosphate ("Ins(1,4,5)P.sub.3") or Phosphatidylinositol (4,5)-bisphosphate ("PtdIns(4,5)P.sub.2").

[0044] The terms "substantially pure" or "isolated", when referring to proteins and polypeptides, denotes those polypeptides that are separated from proteins or other contaminants with which they are naturally associated. A protein or polypeptide is considered substantially pure when that protein makes up greater than about 50% of the total protein content of the composition containing that protein, and typically, greater than about 60% of the total protein content. More typically, a substantially pure protein will make up from about 75 to about 90% of the total protein. Preferably, the protein will make up greater than about 90%, and more preferably, greater than about 95% of the total protein in the composition.

[0045] Particularly preferred polypeptides will have an amino acid sequence that is substantially homologous to the amino acid sequence shown in FIG. 10 (SEQ ID NO:2), or biologically active fragments thereof. Still more preferred polypeptides include the GA5Ptase protein (SEQ ID NO:2) or biologically active fragments thereof.

[0046] In describing the polypeptides of the present invention, conventional amino acid abbreviations will generally be used as follows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I; Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; and Glycine is Gly or G. In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0047] The term "biologically active fragment" as used herein, refers to portions of the proteins or polypeptides which portions possess a particular biological activity. For example, such biological activity may include the ability to bind a particular protein or substrate, block or otherwise inhibit an interaction between two proteins or between an enzyme and its substrate, or may include a particular catalytic activity. With regard to the polypeptides of the present invention, particularly preferred polypeptides or biologically active fragments include, e.g., polypeptides that possess one or more of the biological activities described above, such as the ability to associate or bind GRB2 or affect the binding of GRB2 to its ligand, e.g., GA5Ptase. Also included are those fragments that bind the GA5Ptase substrates described above, are capable of affecting the binding of GA5Ptase to those substrates or that are capable of affecting the hydrolysis of those substrates. Fragments possessing this catalytic activity are also termed "catalytically active fragments." Fragments that are specifically recognized and bound by antibodies raised against the GA5Ptase polypeptides are also included in the definition of biologically active fragments. Such fragments are also referred to herein as "immunologically active fragments." Particularly preferred polypeptides or biologically active fragments are capable of enhancing the activation of Fos SRE when co-expressed with Ras or Ras and GRB2.

[0048] Biologically active fragments of the polypeptides of the invention will generally be useful where it is desired to analyze a single particular biological activity of the polypeptide. For example, where the fragment is used in a model to screen for agonists or antagonists of GA5Ptase/GRB2 interaction (discussed in greater detail, below), it may be desirable to utilize only the GRB2 binding portion of the polypeptides of the invention. Similarly, therapeutic applications will generally target a single biological activity of the GA5Ptase signaling operation, e.g. GRB2 binding, substrate binding or substrate catalysis, and as such, peptides having fewer than all of these activities will be desired, as discussed in greater detail, below. Alternatively, such fragments may be useful where use of a full length protein is unsuitable for the particular application, e.g. therapeutic treatments where administration of full length proteins is difficult.

[0049] Generally, biologically active fragments of the above described proteins will be from about 5 to about 1000 amino acids in length. Typically, these peptides will be from about 10 to about 500 amino acids in length, more typically about 20 to about 250 amino acids in length, and preferably from about 50 to about 200 amino acids in length. Generally, the length of the fragment may depend, in part, upon the application for which the particular peptide is to be used. For example, for raising antibodies, the peptides may be of a shorter length, e.g., from about 5 to about 50 amino acids in length, whereas for binding applications, the peptides will generally have a greater length, e.g., from about 50 to about 1000 amino acids in length, preferably, from about 100 to about 500 amino acids in length, and more preferably, from about 100 to about 200 amino acids in length.

[0050] The terms "substantially homologous" when referring to polypeptides, refer comparatively to two amino acid sequences which, when optimally aligned, are at least about 75% homologous, preferably at least about 85% homologous more preferably at least about 90% homologous, and still more preferably at least about 95% homologous. Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (USA) 85:2444, or by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.).

[0051] The polypeptides of the present invention may also be characterized by their ability to bind antibodies raised against proteins having the amino acid sequence shown in FIG. 10 (SEQ ID NO:2). These antibodies recognize polypeptides that are homologous to the GA5Ptase polypeptide (SEQ ID NO:2). A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein or domain. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Antibodies to the polypeptides of the present invention are discussed in greater detail, below.

[0052] The polypeptides of the present invention may generally be prepared using recombinant or synthetic methods well known in the art. Recombinant techniques are generally described in Sambrook, et al., Molecular Cloning: A Laboratory Manual, (2nd ed.) Vols. 1-3, Cold Spring Harbor Laboratory, (1989). Techniques for the synthesis of polypeptides are generally described in Merrifield, J. Amer. Chem. Soc. 85:2149-2456 (1963), Atherton, et al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press (1989), and Merrifield, Science 232:341-347 (1986). In preferred aspects, the polypeptides of the present invention may be expressed by a suitable host cell that has been transfected with a nucleic acid of the invention, as described in greater detail below.

[0053] Biologically active fragments of the above described polypeptides may generally be identified and prepared using methods well known in the art. For example, selective proteolytic digestion, recombinant deletional methods or de novo peptide synthesis methods may be employed to identify portions of the above described peptides that possess the desired biological activity, e.g., GRB2 binding, substrate binding, catalytic activity and the like. See, e.g. Sambrook, et al.

[0054] Isolation and purification of the polypeptides of the present invention can be carried out by methods that are generally well known in the art. For example, the polypeptides may be purified using readily available chromatographic methods, e.g., ion exchange, hydrophobic interaction, HPLC or affinity chromatography, to achieve the desired purity. Affinity chromatography may be particularly attractive in allowing the investigator to take advantage of the specific biological activity of the desired peptide, e.g., ligand binding, presence of antigenic determinants or the like. For example, the polypeptides of the present invention may be purified by taking advantage of their ability to associate with GRB2. Such affinity purification methods are well known in the art. In particular, GRB2 may be coupled to a suitable solid support and contacted with a mixture of proteins containing the polypeptides of the invention under conditions conducive the association of these polypeptides with GRB2. Once bound to the immobilized GRB2, the solid support is washed to remove unbound material and/or nonspecifically bound proteins. The polypeptides of the invention may then be eluted from the solid support in substantially pure form by, e.g. a change in salt, pH or buffer concentration. Suitable solid supports for affinity purifications are well known in the art and are generally commercially available from, e.g. Pharmacia, Inc., or Sigma Chemical Co. Examples of such solid supports include agarose, cellulose, dextran, silica, polystyrene or similar solid supports.

[0055] In addition to those polypeptides and fragments described above, the present invention also provides fusion proteins which contain these polypeptides or fragments. The term "fusion protein" as used herein, generally refers to a composite protein, i.e., a single contiguous amino acid sequence, made up of two distinct, heterologous polypeptides which are not normally fused together in a single amino acid sequence. Thus, a fusion protein may include a single amino acid sequence that contains two similar or identical polypeptide sequences, provided that these sequences are not normally found together in a single amino acid sequence. Fusion proteins may generally be prepared using either recombinant nucleic acid methods, i.e. as a result of transcription and translation of a gene fusion, which fusion comprises a segment encoding a polypeptide of the invention and a segment encoding a heterologous protein, or by chemical synthesis methods well known in the art.

[0056] These fusion proteins may be prepared to exhibit a combination of properties or activities of the derivative proteins. Typical fusion proteins may include a polypeptide of the invention fused to a reporter polypeptide, e.g., a substrate, cofactor, inhibitor, affinity ligand, antibody binding epitope tag, or an enzyme which is capable of being assayed. Because of their ability to associate with the GRB2 protein, the polypeptides of the invention, when included as a portion of the fusion protein, may act as an affinity ligand to direct the activity of the fused protein directly to the GRB2 protein. In the case of a fusion protein including a reporter group, this allows the presence and or location of the GRB2 protein to be determined. More importantly, such fusions can also be readily used as a marker for determining the level of fusion protein/GRB2 interaction. Examples of some useful fusion partners which can also serve as reporter groups include affinity ligands and antibody binding epitopes, such as the influenza virus hemagglutinin (IHA) epitope tag, or glutathione-S-transferase. Other typical fusion partners include bacterial .beta.-galactosidase, trpE, protein A, .beta.-lactamase, .alpha.-amylase, alcohol dehydrogenase and yeast .alpha.-mating factor. See, e.g., Godowski et al., Science 241:812-816 (1988).

[0057] Also included within the present invention are amino acid variants of the above described polypeptides. These variants may include insertions, deletions and substitutions with other amino acids. For example, in some aspects, amino acids may be substituted with different amino acids having similar structural characteristics, e.g. net charge, hydrophobicity, or the like. For example, phenylalanine may be substituted with tyrosine, as a similarly hydrophobic residue. Glycosylation modifications, either changed, increased amounts or decreased amounts, as well as other sequence modifications are also envisioned.

[0058] Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) may also be used to generate more stable peptides. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch (1992) Ann. Rev. Biochem. 61: 387; for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide. Similarly, modification of the amino or carboxy terminals may also be used to confer stabilizing properties upon the polypeptides of the invention, e.g., amidation of the carboxy-terminus or acylation of the amino-terminus. Substitution of amino acids involved in catalytic activity can be used to generate dominant negative inhibitors of signaling pathways.

[0059] Furthermore, although primarily described in terms of "proteins" or "polypeptides" one of skill in the art, upon reading the instant specification, will appreciate that these terms also include structural analogs and derivatives of the above-described polypeptides, e.g., polypeptides having conservative amino acid insertions, deletions or substitutions, peptidomimetics and the like. For example, in addition to-the above described polypeptides which consist only of naturally-occurring amino acids, peptidomimetics of the polypeptides of the present invention are also provided. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compounds are termed "peptide mimetics" or "peptidomimetics" (Fauchere, J. (1986) Adv. Drug Res. 15:29; Veber and Freidinger (1985) TINS p.392; and Evans et al. (1987) J. Med. Chem 30:1229, and are usually developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biological or pharmacological activity), such as naturally-occurring receptor-binding polypeptide, but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of: --CH.sub.2NH--, --CH.sub.2S--, --CH.sub.2--CH.sub.2--, --CH.dbd.CH-- (cis and trans), --COCH.sub.2--, --CH(OH)CH.sub.2--, and --CH.sub.2SO--, by methods known in the art and further described in the following references: Spatola, A. F. in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, "Peptide Backbone Modifications" (general review); Morley, J. S., Trends Pharm Sci (1980) pp. 463-468 (general review); Hudson, D. et al., Int J Pept Prot Res (1979) 14:177-185 (--CH.sub.2NH--, CH.sub.2CH.sub.2--); Spatola, A. F. et al., Life Sci (1986) 38:1243-1249 (--CH.sub.2--S); Hann, M. M., J Chem Soc Perkin Trans I (1982) 307-314 (--CH--CH--, cis and trans); Almquist, R. G. et al., J Med Chem (1980) 23:1392-1398 (--COCH.sub.2--); Jennings-White, C. et al., Tetrahedron Lett (1982) 23:2533 (--COCH.sub.2--); Szelke, M. et al., European Appln. EP 45665 (1982) CA: 97:39405 (1982) (--CH(OH)CH.sub.2--); Holladay, M. W. et al., Tetrahedron Lett (1983) 24:4401-4404 (--C(OH)CH.sub.2--); and Hruby, V. J., Life Sci (1982) 31:189-199 (--CH.sub.2--S--).

[0060] Peptide mimetics may have significant advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.

[0061] For many applications, it may be desirable to provide the polypeptides of the invention as labeled entities, i.e., covalently attached or linked to a detectable group, to facilitate identification, detection and quantification of the polypeptide in a given circumstance. These detectable groups may comprise a detectable protein group, e.g. an assayable enzyme or antibody epitope as described above in the discussion of fusion proteins. Alternatively, the detectable group may be selected from a variety of other detectable groups or labels, such as radiolabels (e.g., .sup.125I, .sup.32P or .sup.35S) or a chemiluminescent or fluorescent group. Similarly, the detectable group may be a substrate, cofactor, inhibitor or affinity ligand. Labeling of peptidomimetics usually involves covalent attachment of one or more labels, directly or through a spacer (e.g., an amide group), to non-interfering position(s) on the peptidomimetic that are predicted by quantitative structure-activity data and/or molecular modeling. Such non-interfering positions generally are positions that do not form direct contacts with the molecules to which the peptidomimetic binds (e.g., GRB2) to produce the therapeutic effect. Derivitization (e.g., labeling) of peptidomimetics should not substantially interfere with the desired biological or pharmacological activity of the peptidomimetic. Generally, peptidomimetics of peptides of the invention bind to their ligands (e.g., GRB2) with high affinity and/or possess detectable biological activity (i.e., are agonistic or antagonistic to one or more inositol polyphosphate 5-phosphatase mediated phenotypic changes).

[0062] III. Pharmaceutical Compositions

[0063] For a variety of applications, it may be desirable to provide the polypeptides or polypeptide fragments of the invention as part of a pharmaceutical composition, e.g., in combination with a pharmaceutically acceptable carrier. In such pharmaceutical compositions, the polypeptide of the present invention is also referred to as "the active ingredient." Pharmaceutical formulations suitable for use in the present invention are generally described in Remington's Pharmaceutical Sciences, Mack Publishing Co., 17th ed. (1985).

[0064] The pharmaceutical compositions of the present invention are intended for parenteral, topical, oral, or local administration. Where the pharmaceutical compositions are administered parenterally, the invention provides pharmaceutical compositions that comprise a solution of the agents described above, e.g., proteins or polypeptides of the invention, dissolved or suspended in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, saline, glycine and the like. These compositions may be sterilized by conventional, well known methods, e.g., sterile filtration. The resulting aqueous solutions may be packaged for use as is, or lyophilized for combination with a sterile solution prior to administration. The compositions may also 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.

[0065] 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 may be formed by incorporating any of the normally employed excipients, such as the previously listed carriers, and generally, 10-95% of active ingredient, and more preferably 25-75% active ingredient. In addition, for oral administration of peptide based compounds, the pharmaceutical compositions may include the active ingredient as part of a matrix to prevent proteolytic degradation of the active ingredient by digestive process, e.g., by providing the pharmaceutical composition within a liposomal composition, according to methods well known in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., 17th Ed. (1985).

[0066] For aerosol administration, the polypeptides are generally supplied in finely divided form along with a surfactant or propellant. Preferably, the surfactant will be soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids, with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.

[0067] The amount of the above compositions to be administered to the patient will vary depending upon what is to be administered to the patient, the state of the patient, and the manner of administration. Typically, the polypeptides are administered in an amount sufficient to affect the growth factor activation cascade, and thereby cure or at least partially arrest the symptoms of the disease which is sought to be treated, and its associated complications. An amount adequate to accomplish this is termed "a therapeutically effective amount" as described below. Amounts effective for this use will depend many factors, including the severity of the disorder and the weight and general state of the patient, but will generally be in the range of from about 1 mg to about 5 g of active agent per day, preferably from About 50 mg per day to about 500 mg per day, and more preferably, from about 50 mg to about 100 mg per day, for a 70 kg patient.

[0068] IV. Nucleic Acids and Expression Vectors

[0069] In addition to the above described polypeptides, the present invention also provides isolated nucleic acids encoding these polypeptides, as well as expression vectors which include these polynucleotides. Generally, the isolated nucleic acids of the present invention encode a polypeptide which is capable of associating with GRB2, and/or possesses inositol polyphosphate 5-phosphatase activity. In preferred aspects, the nucleic acids of the invention encode a polypeptide having an amino acid sequence that is substantially homologous to the amino acid sequence shown in FIG. 10. More preferred are those isolated nucleic acid sequences that are substantially homologous to the nucleotide sequence shown in FIG. 10, or fragments thereof, and most preferred are those nucleic acid sequences having the nucleotide sequence shown in FIG. 10.

[0070] "Nucleic acids" of the present invention include RNA, cDNA, genomic DNA, synthetic forms and mixed polymers, both sense and antisense strands. Furthermore, different alleles of each isoform are also included. The present invention also provides recombinant nucleic acids which are not otherwise naturally occurring. The nucleic acids described herein also include self replicating plasmids and infectious polymers of DNA or RNA. Unless specified otherwise, conventional notation for nucleic acids is used herein. For example, as written, the left hand end of a single stranded polynucleotide sequence is the 5'-end, whereas the right-hand end is the 3'-end. The left hand direction of double-stranded polynucleotide sequences is referred to as the 5' direction. The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences"; sequence regions on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences".

[0071] The nucleic acids of the present invention may be present in whole cells, cell lysates or in partially pure or substantially pure or isolated form. When referring to nucleic acids, the terms "substantially pure" or "isolated" generally refer to the nucleic acid separated from contaminants with which it is generally associated, e.g., lipids, proteins and other nucleic acids. The substantially pure or isolated nucleic acids of the present invention will be greater than about 50% pure. Typically, these nucleic acids will be more than about 60% pure, more typically, from about 75% to about 90% pure, and preferably, from about 95% to about 98% pure.

[0072] The DNA compositions will generally include a coding region which encodes a polypeptide possessing inositol polyphosphate 5-phosphatase activity and capable of associating with GRB2. Preferred nucleic acids will typically encode polypeptides having an amino acid sequence which is substantially homologous to the amino acid sequence shown in FIG. 10 (SEQ ID NO:2), or biologically active fragments thereof. More preferred nucleic acids will comprise a segment having more than about 20 contiguous nucleotides from the nucleotide sequence shown in FIG. 10 (SEQ ID NO:1), with still more preferred nucleic acids having a nucleotide sequence that is substantially homologous to the nucleotide sequence shown in FIG. 10 (SEQ ID No:1). Most preferred nucleic acids are those which include the nucleotide sequence shown in FIG. 10 (SEQ ID NO:1).

[0073] The phrase "nucleic acid sequence encoding" refers to a nucleic acid which directs the expression of a specific protein or peptide. The nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein. The nucleic acid sequences include both the full length nucleic acid sequences as well as non-full length sequences derived from the full length protein. It being further understood that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell.

[0074] Substantial homology in the nucleic acid context means that the segments, or their complementary strands, when compared, are the same when properly aligned, with the appropriate nucleotide insertions or deletions, in at least about 60% of the nucleotides, typically, at least about 70%, more typically, at least about 80%, usually, at least about 90%, and more usually, 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 of at least about 20 contiguous nucleotides derived from the nucleotide sequence shown in FIG. 10. However, larger segments will usually be preferred, e.g., at least about 30 contiguous nucleotides, more usually about 40 contiguous nucleotides, and preferably more than about 50 contiguous nucleotides. Selective hybridization exists when hybridization occurs which is more selective than total lack of specificity. See, Kanehisa, Nucleic Acid Res. 12:203-213 (1984).

[0075] There are various methods of isolating the nucleic acids which encode the polypeptides of the present invention. Typically, the DNA is isolated from a genomic or cDNA library using labeled oligonucleotide probes specific for sequences in the desired DNA. Restriction endonuclease digestion of genomic DNA or cDNA containing the appropriate genes can be used to isolate the DNA encoding the polypeptides of the invention. From the nucleotide sequence given in FIG. 10, a panel of restriction endonucleases can be constructed to give cleavage of the DNA in desired regions, i.e., to obtain segments which encode biologically active fragments of the polypeptides of the invention. Following restriction endonuclease digestion, DNA encoding the polypeptides of the invention is identified by its ability to hybridize with a nucleic acid probe in, for example a Southern blot format. These regions are then isolated using standard methods. See, e.g., Sambrook, et al., supra.

[0076] The polymerase chain reaction, or "PCR" can also be used to prepare nucleic acids which encode the polypeptides of the present invention. PCR technology is used to amplify nucleic acid sequences of the desired nucleic acid, e.g., the DNA which encodes the polypeptides of the invention, directly from mRNA, cDNA, or genomic or cDNA libraries. Alternatively, solid phase oligonucleotide synthesis methods may also be employed to produce the nucleic acids described herein. Such methods include the phosphoramidite method described by, e.g., Beaucage and Carruthers, Tetrahedron Lett. 22:1859-1862 (1981), or the triester method according to Matteucci, et al., J. Am. Chem. Soc., 103:3185 (1981), both incorporated herein by reference. A double stranded fragment may then be obtained, if desired, by annealing the chemically synthesized single strands together under appropriate conditions or by synthesizing the complementary strand using DNA polymerase with an appropriate primer sequence.

[0077] Appropriate primers and probes for amplifying the nucleic acids described herein, may be generated from analysis of the nucleic acid sequences described herein, e.g. at FIG. 10. Briefly, oligonucleotide primers complementary to the two 3' borders of the DNA region to be amplified are synthesized. The PCR is then carried out using the two primers. See, e.g., PCR Protocols: A Guide to Methods and Applications (Innis, M., Gelfand, D., Sninsky, J. and White, T., eds.) Academic Press (1990). Primers can be selected to amplify various sized segments from the nucleic acid sequence.

[0078] The present invention also includes fragments of the above described nucleic acids. Such fragments will generally comprise a segment of from about 15 to about 150 nucleotides. These fragments can be useful as oligonucleotide probes in the methods of the present invention, or alternatively to encode the polypeptides or biologically active fragments of the present invention, described herein. Also provided are substantially similar nucleic acid sequences, allelic variations and natural or induced sequences of the above described nucleic acids. Also included are chemically modified and substituted nucleic acids, e.g. those which incorporate modified nucleotide bases or which incorporate a labelling group.

[0079] In one aspect, cDNA encoding the polypeptides of the present invention, or fragments thereof, may be readily employed as nucleic acid probes useful for obtaining genes which encode the polypeptides of the present invention. "Nucleic acid probes" may be DNA or RNA fragments. DNA fragments can be prepared, for example, by digesting plasmid DNA, or by use of PCR, or synthesized by either the phosphoramidite method described above. Where a specific sequence for a nucleic acid probe is given, it is understood that the complementary strand is also identified and included. The complementary strand will work equally well in situations where the target is a double-stranded nucleic acid.

[0080] Typical nucleic acid probes may be readily derived from the nucleotide sequence shown in FIG. 10 (SEQ ID NO:1), or alternatively, may be prepared from the amino acid sequence of the GA5Ptase protein, as shown in FIG. 10 (SEQ ID NO:2). In particular, probes may be prepared based upon segments of the amino acid sequence which possess relatively low levels of degeneracy, i.e., few or one possible nucleic acid sequences which encode therefor. Suitable synthetic DNA fragments may then be prepared. Such cDNA probes may be used in the design of oligonucleotide probes and primers for screening and cloning genes which encode the polypeptides of the invention or related polypeptides, e.g., using well known PCR techniques. These nucleic acids, or fragments may comprise part or all of the cDNA sequence that encodes the polypeptides of the present invention. Effective cDNA probes may comprise as few as 15 consecutive nucleotides in the cDNA sequence, but will often comprise longer segments. Further, these probes may further comprise an additional nucleotide sequence, such as a transcriptional primer sequence for cloning, or a detectable group for easy identification and location of complementary sequences.

[0081] cDNA or genomic libraries of various types may be screened for new alleles or related sequences using the above probes. The choice of cDNA libraries normally corresponds to tissue sources which are abundant in mRNA for the desired polypeptides. Phage libraries are normally preferred, but plasmid libraries may also be used. Clones of a library are spread onto plates, transferred to a substrate for screening, denatured, and probed for the presence of the desired sequences.

[0082] In addition to comprising a segment which encodes one or more of the above described polypeptides or biologically active fragments, the nucleic acids of the present invention may also comprise a segment encoding a heterologous protein, such that the gene is expressed to produce the two proteins as a fusion protein, as substantially described above.

[0083] Typically, the nucleic acids of the present invention will be used in expression vectors for the preparation of the polypeptides of the present invention, namely those polypeptides which possess inositol polyphosphate 5-phosphatase activity and that are capable of associating with GRB2. The phrase "expression vector" generally refers to nucleotide sequences that are capable of affecting expression of a structural gene in hosts compatible with such sequences. These expression vectors typically include at least suitable promoter sequences and optionally, transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used as described herein. DNA encoding the polypeptides of the present invention will typically be incorporated into DNA constructs capable of introduction into and expression in an in vitro cell culture. Often, the nucleic acids of the present invention may be used to produce a suitable recombinant host cell. Specifically, DNA constructs will be suitable for replication in a prokaryotic host, such as bacteria, e.g., E. coli, or may be introduced into a cultured mammalian, plant, insect, yeast, fungi or other eukaryotic cell line. DNA constructs prepared for introduction into a particular host, e.g., bacteria or yeast, will typically include a replication system recognized by the host, the intended DNA segment encoding the desired polypeptide, and transcriptional and translational initiation and termination regulatory sequences operably linked to the polypeptide encoding segment. A DNA segment is operably linked when it is placed into a functional relationship with another DNA segment. For example, a promoter or enhancer is operably linked to a coding sequence if it stimulates the transcription of the sequence. DNA for a signal sequence is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide. Generally, DNA sequences that are operably linked are contiguous, and in the case of a signal sequence both contiguous and in reading phase. However, enhancers need not be contiguous with the coding sequences whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof. The selection of an appropriate promoter sequence will generally depend upon the host cell selected for the expression of the DNA segment. Examples of suitable promoter sequences include prokaryotic, and eukaryotic promoters well known in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed.), vols. 1-3 Cold Spring Harbor Laboratory (1989). The transcriptional regulatory sequences will typically include a heterologous enhancer or promoter which is recognized by the host. 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 and available. See Sambrook et al., (1989).

[0084] Conveniently available expression vectors which include the replication system and transcriptional and translational regulatory sequences together with the insertion site for the polypeptide encoding segment may be employed. Examples of workable combinations of cell lines and expression vectors are described in Sambrook et al., and in Metzger et al., Nature 334:31-36 (1988). For example, where an insect host cell is selected as-the host cell of choice to express the polypeptide, the cDNA encoding the polypeptides of the invention may be cloned into a baculovirus expression vector (e.g. pV-IKS). The recombinant baculovirus may then be used to transfect a suitable insect host cell, e.g., Sf9 cells, which may then express the polypeptide. See, e.g., D. K. Morrison et al., Cell 58:649-657 (1989), M. D. Summers and G. E. Smith, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Station, College Station, Tex. (1987).

[0085] V. Cell Lines

[0086] The vectors containing the DNA segments of interest, e.g., those encoding polypeptides of the invention as described above, can be transferred into the host cell by well known methods which may vary depending upon the type of host cell used. For example, calcium chloride transfection is commonly used for prokaryotic cells, whereas calcium phosphate treatment may be used for other hosts. See, Sambrook et al. The term "transformed cell" as used herein, includes the progeny of originally transformed cells, which progeny express the nucleic acids of the invention.

[0087] Techniques for manipulation of nucleic acids which encode the polypeptides of the present invention, i.e., subcloning the nucleic acids into expression vectors, labeling probes, DNA hybridization and the like, are generally described in Sambrook, et al., supra.

[0088] In recombinant methods, generally the nucleic acid encoding a peptide of the present invention is first cloned or isolated in a form suitable for ligation into an expression vector. After ligation, the vectors containing the nucleic acid fragments or inserts are introduced into a suitable host cell, for the expression of the polypeptide of the invention. The polypeptides may then be purified or isolated from the host cells. Methods for the synthetic preparation of oligonucleotides are generally described in Gait, Oligonucleotide Synthesis: A Practical Approach, IRL Press (1990).

[0089] VI. Antibodies

[0090] The nucleic acids and polypeptides of the present invention or fragments thereof, are also useful in producing antibodies, either polyclonal or monoclonal, which are specifically immunoreactive with the polypeptides of the present invention.

[0091] The phrase "specifically immunoreactive," when referring to the interaction between an antibody of the invention and a particular protein, refers to an antibody that specifically recognizes and binds with relatively high affinity to the particular protein, such that this binding is determinative of the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0092] For production of polyclonal antibodies, an appropriate target immune system is selected, typically a mouse or rabbit, but also including goats, sheep, cows, guinea pigs, monkeys and rats. The substantially purified antigen or plasmid is presented to the immune system in a fashion determined by methods appropriate for the animal. These and other parameters are well known to immunologists. Typically, injections are given in the footpads, intramuscularly, intradermally or intraperitoneally. The immunoglobulins produced by the host can be precipitated, isolated and purified by routine methods, including affinity purification.

[0093] For monoclonal antibodies, appropriate animals will be selected and the desired immunization protocol followed. After the appropriate period of time, the spleens of these animals are excised and individual spleen cells are 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 the production of an appropriate antibody specific for the desired region of the antigen. Techniques for producing antibodies are well known in the art. See, e.g., Goding et al., Monoclonal Antibodies: Principles and Practice (2d ed.) Acad. Press, N.Y., and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988). Other suitable techniques involve the in vitro exposure of lymphocytes to the antigenic polypeptides or alternatively, to selection of libraries of antibodies in phage or similar vectors. Huse et al., Generation of Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda, Science 246:1275-1281 (1989). Monoclonal antibodies with affinities of 10.sup.8 liters/mole, preferably 10.sup.9 to 10.sup.10 or stronger, will be produced by these methods.

[0094] The antibodies generated can be used for a number of purposes, e.g., as probes in immunoassays, for inhibiting GRB2/GA5Ptase interaction, or interaction with other ligands, thereby inhibiting or reducing the growth factor signaling cascade, in diagnostics or therapeutics, or in research to further elucidate the mechanism of growth factor activation pathways, and particularly, the growth factor activation of Ras. Where the antibodies are used to block the interaction between two signaling molecules, e.g. GRB2 and GA5Ptase, the antibody will generally be referred to as a "blocking antibody."

[0095] The antibodies of the present invention can be used with or without modification. Frequently, the antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. Such labels include those that are well known in the art, such as the labels described previously for the polypeptides of the invention. Additionally, the antibodies of the invention may be chimeric, human-like or humanized, in order to reduce their potential antigenicity, without reducing their affinity for their target. Chimeric, human-like and humanized antibodies have generally been described in the art. Generally, such chimeric, human-like or humanized antibodies comprise hypervariable regions, e.g., complementarity determining regions (CDRs) from a mammalian animal, i.e., a mouse, and a human framework region. See, e.g., Queen, et al., Proc. Nat'l Acad. Sci. USA 86:10029 (1989), Verhoeyan, et al., Science 239:1534-1536 (1988). By incorporating as little foreign sequence as possible in the hybrid antibody, the antigenicity is reduced. Preparation of these hybrid antibodies may be carried out by methods well known in the art.

[0096] Preferred antibodies are those monoclonal or polyclonal antibodies which specifically recognize and bind the polypeptides of the invention. Accordingly, these preferred antibodies will specifically recognize and bind the polypeptides which have an amino acid sequence that is substantially homologous to the amino acid sequence shown in FIG. 10 (SEQ ID NO:2). Still more preferred are antibodies which are capable of forming an antibody-ligand complex with the polypeptides of the invention, whereby the ability of the polypeptide to associate with GRB2, in vitro, is reduced, e.g. blocking antibodies.

[0097] VII. Methods of Use

[0098] The polypeptides, antibodies and nucleic acids of the present invention may be used in a variety of important applications. Such applications include but are not limited to screening applications for identifying compounds that affect the growth factor signal transduction pathways, also termed "signaling cascades," and therapeutic applications for the treatment of proliferative cell disorders.

[0099] A. Screening Applications

[0100] In a particular aspect, the present invention provides methods of screening test compounds to determine whether the test compounds are capable of affecting growth factor activation signal transduction pathways. More particularly, the methods described herein are used to screen compounds for there ability to affect the interaction of the polypeptides of the invention, and their respective substrates and ligands, as these interactions are involved in growth factor activation signal transduction pathways, and particularly, the growth factor activation of Ras.

[0101] In one aspect, the present invention provides methods of screening whether a test compound is an agonist or antagonist of GRB2-mediated signal transduction. More specifically, the polypeptides of the present invention can be used as a model system of GRB2/GA5Ptase interaction, to screen for compounds which affect this interaction. An agonist, antagonist or test compound may be a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal cells or tissues. Typically, test compounds may include structural analogs or peptidomimetics which are derived from the polypeptides described herein, and particularly the biologically active fragments. Test compounds are evaluated for potential activity as agonists or antagonists of functions which result in signal transduction, by inclusion in screening assays described herein. An "agonist" will enhance the particular observed activity, e.g. GRB2 association or 5-phosphate cleavage, while an "antagonist" will diminish the particular observed activity. The terms "agonist" and "antagonist", as used herein, do not imply a particular mechanism of function. Particularly targeted test compounds include polypeptide fragments of the polypeptides of the present invention and structural analogs or peptidomimetics of these peptides.

[0102] In a first aspect, the screening methods of the present invention typically involve the incubation of a polypeptide of the present invention, or a GRB2 associating fragment thereof, in the presence of GRB2 as well as a particular test compound. The mixture is then assayed to determine the levels of GRB2/polypeptide interaction by determining the amount of GRB2/polypeptide complex formed in the presence and absence of the test compound. Where the presence of the test compound results in an increase or decrease in the amount of complex formed, it will be indicative that the test compound is an agonist or antagonist of GRB2-mediated signal transduction, respectively.

[0103] For determination of the amount of GRB2/polypeptide complex formed, one may employ any number of a variety of well known assay methods. For example, immunoprecipitation of one polypeptide or protein that participates in the complex, followed by assaying the immunoprecipitate for the other participant, will generally indicate the amount of complex formed. For example, following co-incubation of the polypeptide of the invention with GRB2, in the presence of the test compound, the polypeptide may be immunoprecipitated using an antibody that recognizes and specifically binds an epitope in the polypeptide's sequence. This epitope may be a sequence that is endogenous to the polypeptide or may be exogenously introduced as a labeling group, e.g., an antibody binding epitope tag or assayable enzyme. Following immunoprecipitation, the precipitate may be assayed for the presence of the other participant in the complex, e.g., GRB2, which may also be labelled, albeit in a distinguishing manner, e.g., a radiolabel, separately assayable enzyme, distinct antibody binding epitope tag or the like.

[0104] In an alternative method, one of the participants in complex formation, e.g., a polypeptide of the invention, may be coupled with an appropriate reporter group, while another participant, e.g., GRB2, is immobilized upon a solid support. Useful reporter groups, or labels have been previously described herein, including, e.g. radiolabels, such as, .sup.125I, .sup.32P or .sup.35S, fluorescent or chemiluminescent groups, substrates, cofactors, inhibitors, affinity ligands, antibody binding epitope tags, or enzymes which are capable of being assayed, e.g. horseradish peroxidase, luciferase, or other readily assayable enzymes. These enzyme groups may be attached to the polypeptide of the present invention by chemical means or expressed as a fusion protein, as already described.

[0105] Screening is then carried out by contacting the labeled participant with the immobilized participant in the presence and absence of the test compound. The amount of reporter group that binds to the solid support-bound participant is indicative of the amount of complex formed. The level of polypeptide bound in the presence of the test compounds may then be compared to the levels bound in control experiments, e.g., in the absence of the test compounds.

[0106] A variety of solid supports may be used in these screening methods. For example, blot formats may be employed where the protein or polypeptide is spotted on an appropriate substrate, e.g., nitrocellulose, PVDF and the like. Alternatively, resin or bead formats may also be used as the solid supports, including beads of agarose, cellulose, silica, polystyrene, divinylbenzene and the like.

[0107] Where the test compound results in an increase in the level of polypeptide which associates with GRB2, it is indicative that the test compound is an agonist of GRB2/GA5Ptase interaction, and more particularly, the GRB2-mediated signal transduction pathway. Similarly, where the presence of the test compound results in a decrease in the level of polypeptide GRB2 complex formed, it is indicative that the test compound is an antagonist of that interaction and signal transduction pathway.

[0108] In another aspect, the polypeptides of the present invention can be used as a model system for screening test compounds to identify agonists or antagonists of inositol polyphosphate 5-phosphatase activity generally, and in particular the inositol polyphosphate 5-phosphatase activity of GA5Ptase. Because the processing of phosphatidylinositols and particularly, the cleavage of the 5-phosphate from PtdIns(3,4,5)P.sub.3 has been linked to the activation of Ras, it will be desirable to provide a model system for screening compounds which block or otherwise inhibit this conversion, and thereby block Ras activation.

[0109] The methods for determining whether a test compound is an agonist or antagonist of the inositol polyphosphate 5-phosphatase activity of the polypeptides of the invention, are generally similar to the above described methods. In particular, these methods comprise incubating a polypeptide having the desired inositol polyphosphate 5-phosphatase activity, e.g., GA5Ptase or catalytically active fragments thereof, with its substrate in the presence and absence of the test compound. This incubation may be carried out in vitro or in vivo, e.g., using a transgenic animal model which has been engineered to express the polypeptides of the invention. For the polypeptides of the present invention, the appropriate substrate may generally be selected from, e.g. D-myo-Inositol 1,3,4,5-tetrakisphosphate ("Ins(1,3,4,5)P.sub.4") and Phosphatidylinositol 3,4,5-trisphosphate ("PtdIns(3,4,5)P.sub.3"). Following a prescribed reaction time the reaction mixture is assayed for the production of the products of inositol polyphosphate 5-phosphatase activity on these substrates. Assaying for production of the various reaction products, e.g. Ins(1,3,4)P.sub.3 from Ins(1,3,4,5)P.sub.4, or PtdIns(3,4)P.sub.2 from PtdIns(3,4,5)P.sub.3, may be carried out by a variety of methods known in the art. For example, HPLC analysis can be readily used to quantitatively identify the above described reaction products, using, e.g. tritiated substrates, and the like (see Example 2, below). Similarly, on a more qualitative level, thin layer chromatography (TLC) can also be used to identify reaction products. The levels of the above described reaction products produced in the presence and absence of the test compound are then compared. Where the presence of the test compound results in an increase or decrease in the level of the reaction product produced by the polypeptide, it is indicative that the test compound is an agonist or antagonist of inositol polyphosphate 5-phosphatase activity, respectively, and more particularly, the inositol polyphosphate 5-phosphatase activity described herein.

[0110] In a related embodiment, the present invention also provides kits for carrying out the above described screening methods. The kits of the present invention generally include a polypeptide of the present invention, e.g. the GA5Ptase polypeptide or a biologically active fragment thereof, as well as a ligand of the polypeptide where the binding activity is to be screened, e.g., GRB2, or a substrate of that polypeptide where the inositol polyphosphate 5-phosphatase activity is to be screened, e.g., Ins(1,3,4,5)P.sub.4, or PtdIns(3,4,5)P.sub.3. One or more of these components may generally be provided in premeasured aliquots. The aliquots can be contained in any suitable container such as a vial or a tube. The polypeptide component can be provided in solution or in lyophilized form, and may be immobilized. The polypeptide preparation may also contain 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 also be included to stabilize the polypeptide. The solution form of GA5Ptase may contain up to 50% glycerol if the enzyme is to be stored frozen, e.g., at -20.degree. C. to -70.degree. C. If the GA5Ptase is provided in lyophilized form, the kit can include a reconstitution buffer to reconstitute the polypeptide, as well as a reaction buffer. Alternatively, the polypeptide can be added to the reaction buffer and the solution freeze dried. This form can be readily reconstituted in distilled water with the necessary salt components already present for the particular reaction to be screened, so that no additional reaction buffer need be supplied. Thus, depending on the form and composition of the polypeptide preparation, different buffers may be included in the kit and they may be provided in more than one aliquot. Although described in substantial detail herein, these buffers are generally optional. The appropriate substrate or ligand, depending upon the particular screening method used, may be provided in a similar fashion to that of the polypeptide component. The kits will also typically include additional reagents for carrying out the particular method, e.g. stains for detection, antibodies, solid supports, and the like, as well as detailed operating specifications for their use. For example, where binding interactions are being screened, the ligand component may generally be supplied within the kit, already coupled to an appropriate support.

[0111] Once identified, particular agonists or antagonists may then be used to enhance or block the activity of the polypeptides of the present invention. This may be particularly useful in therapeutic applications (see discussion, below).

[0112] B. Therapeutic Applications

[0113] In addition to the above described uses, the polypeptides and nucleic acids of the present invention may also be used in therapeutic applications for the treatment of human or non-human mammalian patients. The term "treatment" refers to the full spectrum of treatment for a given disorder from which the patient is suffering, including alleviation of some, most or all symptoms resulting from that disorder, to an outright cure for the particular disorder to prevention of the onset of the disorder.

[0114] As described previously herein, the polypeptides of the present invention have been implicated as providing a critical step in the growth factor activation cascade, and particularly the activation of Ras. Activation of Ras has been associated with a variety of proliferative disorders including atherosclerosis, inflammatory joint diseases, psoriasis, restenosis following angioplasty, and cancer.

[0115] Accordingly, treatment of the above described disorders can generally be carried out by blocking or inhibiting activation of Ras. This may generally be accomplished by blocking or inhibiting one or more of the activities of the GA5Ptase polypeptide which are involved in the signal transduction pathway which activates Ras, e.g., the polypeptide's ability to bind GRB2, or the polypeptide's ability to bind to or catalyze the dephosphorylation of its substrate.

[0116] Generally, inhibition of the particular activity may be carried out by providing a polypeptide of the invention which will compete with the endogenous GA5Ptase protein. For example, by administering to a patient an effective amount of a GRB2 associating fragment of the polypeptides, as described herein, one can out compete the endogenous GRB2 associating activity of the endogenous GA5Ptase protein, and thereby reduce the level of Ras activation. Similarly, by administering to the patient an effective amount of a substrate binding, although non-catalytic, fragment of the GA5Ptase peptide, as described herein, one can effectively out compete the naturally occurring GA5Ptase protein, and thus block cleavage of the substrate, and the ensuing activation cascade reactions.

[0117] The quantities of reagents necessary for effective therapy, also referred to herein as an "effective amount," or "therapeutically effective amount," will depend upon many different factors, including means of administration, target site, physiological state of the patient and other medicants administered. Thus, treatment doses will need to be titrated to optimize safety and efficacy. Typically, dosages used in vitro 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. Generally, therapeutically effective amounts of the GA5Ptase containing polypeptides of the present invention will be from about 0.0001 to about 10 mg/kg, and more usually, from about 0.001 to about 0.1 mg/kg of the host's body weight. Various considerations are described, e.g., in Gilman et al., (Eds.), Goodman and Gilman's: The Pharmacological Basis of Therapeutics, (8th ed. 1990), Pergamon Press, and Remington's Pharmaceutical Sciences (7th ed. 1985) Mack Publishing Co., Easton, Penn. Methods of administration, also discussed in the above references, include, e.g., oral, intravenous, intraperitoneal or intramuscular administration, and local administration, including topical, transdermal diffusion and aerosol administration, for therapeutic, and/or prophylactic treatment. The active agent, i.e., the polypeptide component, will generally be administered in a composition additionally comprising a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include water, saline, buffers and other compounds described in, e.g., the Merck Index, Merck and Co., Rahway, N.J.

[0118] Constituents of pharmaceutical compositions, in addition to the active agents, include those generally known in the art for the various administration methods used. For example, oral forms generally include powders, tablets, pills, capsules, lozenges and liquids. Similarly, intravenous, intraperitoneal or intramuscular formulations will generally be dissolved or suspended in a pharmaceutically acceptable carrier, e.g., water, buffered water, saline and the like. Additionally, these compositions may include additional constituents which may be required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like. For solid compositions, conventional nontoxic solid carriers may be used which include, e.g., pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate and the like.

[0119] Administration may also be carried out by way of a controlled release composition or device, whereby a slow release of the active ingredient allows continuous administration over a longer period of time.

[0120] Additionally, inositol polyphosphates play important roles in cell signaling pathways, the present invention can provide an exogenous regulatory mechanism in the treatment of disorders where these regulatory mechanisms are disfunctional. In particular, the treatment of a particular disorder may comprise gene therapy techniques involving the mutation, dysregulation or augmentation of levels of GA5ptase. For example, gene therapy techniques may involve the introduction into afflicted cells, of genes which encode a protein or polypeptide which possesses the activity of GA5ptase. This exogenously introduced protein may then augment existing levels of this activity in cells that may be otherwise deficient.

[0121] Strategies for gene therapy are reviewed in Friedmann, Science 244:1275 (9189). Genetic constructs encoding the PTB domain or functional derivative of that domain, can be used in these gene therapy techniques. Delivery of the genetic construct of interest, i.e., the nucleic acid encoding a GA5ptase protein or fragment, may be accomplished in vivo by administering the therapy vector to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial administration). Alternatively, the vector may be used to deliver nucleic acids to cells ex vivo, such as cells explanted from an individual patient or universal donor hematopoietic stem cells, neurons, etc, e.g., by transfection of the cells with nucleic acids of interest cloned into retroviruses. Following transfection, the cells are reimplanted into the patient, usually after selection for cells which have incorporated the nucleic acid. The infusion into the patient of transfected cells can replace cells which are dysfunctional for the particular regulatory scheme which results in the disorder being treated.

[0122] C. Affinity Probes

[0123] Because the polypeptides of the present invention associate with GRB2 proteins, and specifically, via the SH3 domains, these proteins or their biologically active fragments may be particularly useful as affinity probes or ligands. In particular, the proteins can be used to identify or capture GRB2 proteins from a mixture of different proteins.

[0124] Typically, use of the polypeptides of the present invention in identifying GRB2 proteins in a mixture of proteins may be carried out using a Western blotting format. In particular, the mixture of proteins may be immobilized on a solid support, as described above. Immobilization may include simple spotting, electroblotting of SDS-PAGE gels and the like. The blot is then blocked using a nonspecific protein, i.e., BSA. Labeled polypeptides of the present invention may then be used to interrogate the blot, binding to the immobilized GRB2.

[0125] The polypeptides of the present invention may also be used as affinity ligands to purify Grb2 proteins from a mixture of proteins. In particular, the polypeptide of the invention is coupled to a solid support. The support bound polypeptide is then contacted with the mixture of proteins containing the GRB2 protein under conditions that are conducive to GA5Ptase/GRB2 binding. The support is then washed to remove unbound and nonspecifically bound proteins. Substantially pure GRB2 may then be readily eluted from the support by, e.g. a change in salt, pH or buffer concentrations.

[0126] The present invention is further illustrated by the following examples. These examples are merely to illustrate aspects of the present invention and are not intended as limitations of this invention.

VIII. EXAMPLES

Example-1

Cloning and Sequence Analysis of GA5Ptase

[0127] A bacterially expressed GST-GRB2 fusion protein containing a 5 amino acid RRASV heart muscle kinase site was purified and radioactively labeled with [.sup.32P]ATP. The purified protein was used to screen a human placental .lambda.gt11 oligo dT primed cDNA library (Clonetech) using the guanidine-HCl denaturation/renaturation screening technique initially described by Blanar and Rutter, Science 256:1014-1018 (1992). A schematic of this protein is shown in FIG. 1. Because all GRB2, interacting clones obtained in the first round of screening encoded catalytic regions of protein tyrosine kinases, duplicate filters were probed with antiphosphotyrosine antibody to screen against SH2 domain interactions. One clone was identified that specifically interacted with GRB2 and was not tyrosine phosphorylated. Sequencing of this clone indicated that it was a partial cDNA clone with no similarity to any other protein or cDNA. Multiple PXXP motifs were present, indicating the likely contact region with the SH3 domains of GRB2. Northern blot analysis indicated a 4.3 kb long transcript with broad tissue distribution, the highest being in placenta. Purified GST-GA5Ptase fragment was produced by cloning the original .lambda.gt11 cDNA fragment (nucleotides 2687-4146) into pGex1. Smith, et al., Gene 67:31-40 (1988).

[0128] Binding specificity of this protein fragment for GRB2 was evaluated by Far-Western blots of the protein with roughly equal amounts of radioactively labeled GRB2, Vav SH3-SH2--SH3 domains (amino acids 648-844, Katzav, et al., Embo J. 8:2283-2290 (1989)), Nck SH3--SH3--SH3 domains (amino acids 1-249, Hu, et al., Mol. Cell. Biol. 15:1169-1174 (1995)) and p85 SH3 domain (amino acids 1-81, Klippel, et al., Mol. Cell. Biol. 12:1451-1459 (1992)). Only GRB2 bound specifically to the GST fused fragment of the newly cloned protein and no proteins bound GST itself. A full length cDNA clone was obtained by screening a .lambda.gt10 human placental cDNA library. This 4146 bp clone contained an open reading frame encoding a 976 amino acid protein. The predicted 110 kDa protein showed that it has significant homology to a family of proteins known as inositol polyphosphate 5-phosphatases. FIG. 2 shows a ribbon diagram and dendrogram indicating relative homology of GA5Ptase to a number of inositol polyphosphate 5-phosphatases. FIG. 11 also shows a direct sequence comparison of GA5Ptase to these other phosphatase sequences. Included in the comparison are the C. elegans inositol polyphosphate 5-phosphatase ("celegptase") (SEQ ID NO:3), S. cereviseae inositol polyphosphate 5-phosphatase ("ysc5ptase") (SEQ ID NO:4), GA5Ptase (SEQ ID NO:2), human 51c ("51c") (SEQ ID NO:5), the 75 kDa human platelet inositol polyphosphate 5-phosphatase type-II ("5ptaseii") (SEQ ID NO:6), the human ocr1 protein responsible for human oculocerebrorenal syndrome ("ocr1") (SEQ ID NO:7), Arabidopsis inositol polyphosphate 5-phosphatase ("arab5ptase") (SEQ ID NO:8) and canine inositol polyphosphate 5-phosphatase 43 kDa ("h5ptase43") (SEQ ID NO:9). The identified consensus sequence is also provided ("consensus") (SEQ ID NO:10).

Example-2

Characterization of Enzymatic activity of GA5Ptase

[0129] The next step was to characterize the nature of the activity of the GA5ptase protein. FIG. 6 illustrates the effect of varying concentration of Ins(1,3,4,5)P.sub.4 on the rate of its hydrolysis by GA5Ptase. FIG. 7 illustrates the coprecipitation of GA5ptase and IP.sub.4 hydrolyzing activity.

[0130] To ensure that GA5Ptase was in fact an inositol polyphosphate 5-phosphatase, .sup.3H-Ins(1,3,4,5)P.sub.4 (200 pmoles) was incubated with GA5Ptase (1 .mu.g) for 1 hour at 37.degree. C. An aliquot of the reaction mix was quenched with 500 .mu.l cold water, mixed with 300 cpm .sup.32P-Ins(1,4,5)P.sub.3 as an internal standard, and analyzed by Absorbosphere.TM. Sax HPLC using a NaPO.sub.4 gradient. FIG. 8A shows the resulting chromatogram showing conversion of the Ins(1,3,4,5)P.sub.4 to Ins(1,3,4)P.sub.3 by GA5Ptase.

[0131] An aliquot of the reaction mix was then incubated with a purified recombinant inositol polyphosphate 1phosphatase (York, et al., Proc. Nat'l Acad. Sci. USA 87:9548-9552 (1990)), quenched with 500 .mu.l cold water, mixed with 300 cpm .sup.32P-Ins(1,4)P.sub.2 as an internal standard, and analyzed using Partisil.TM. 10 Sax HPLC using an NH.sub.4COOH gradient. FIG. 8B shows that the product of GA5Ptase was converted by the inositol polyphosphate 1 phosphatase to Ins(3,4)P.sub.2.

[0132] A further aliquot of the original reaction mix was incubated with a purified recombinant inositol polyphosphate 4-phosphatase, quenched with 500 .mu.l cold water, mixed with .sup.32P-Ins(3,4)P.sub.2 as an internal standard and again analyzed using Partisil 10 Sax HPLC using an NH.sub.4COOH gradient. FIG. 8C shows that the product of GA5Ptase action on Ins(1,3,4,5)P.sub.4 was converted by inositol polyphosphate 4-phosphatase to Ins(1,3)P.sub.2. These assays confirm that GA5Ptase has inositol polyphosphate 5-phosphatase activity.

[0133] GA5Ptase (20 ng) and 5ptase II (31 ng) were separately incubated with 1400 cpm .sup.32P-PtdIns(3,4,5)P.sub.3 in phosphatidylserine vesicles for 1, 3 or 10 minutes at 37.degree. C. The reaction was stopped by addition of 30 .mu.l chloroform/methanol (1:1). The chloroform layer was spotted on an oxalate-dipped silica gel TLC plate and developed using a solvent mixture of chloroform/acetone/methanol/acetic acid/water from the TLC plate and quantified by Cerenkov radiation. Production of PtdInsP.sub.2 from PtdIns(3,4,5)P.sub.3 is shown in FIG. 9, plotted as a function of time. Each point shown is the average of quadruplicate assays. The inset shows an autoradiogram of a TLC plate indicating conversion of PtdIns(3,4,5)P.sub.3 to PtdInsP.sub.2 by both GA5Ptase and 5ptase II.

Example-3

Co-Immunoprecipitation of GA5Ptase

[0134] Lysates from Balb 3T3 cells were immunoprecipitated with GRB2 antibody (Transduction Laboratories) and blotted with preimmune (P) and immune (I) GA5Ptase polyclonal antibodies. Co-immunoprecipitation of endogenous GA5Ptase with endogenous GRB2 was detected in unstimulated Balb 3T3 cells using antibodies raised against two different regions of GA5Ptase (amino acids 47-231 and 891-983). To define the interaction of GA5Ptase with GRB2, both molecules were co-expressed in Cos cells and co-immunoprecipitated (FIG. 4).

[0135] Cos7 cells were transiently transfected with GA5Ptase and either wild type or single point mutations of GRB2 or GRB3.3. Schematic illustrations of the sequence structure of each of these proteins is shown in FIG. 3. Point mutations were the human counterparts of natural C. elegans Sem-5 point mutations. After 2 days of growth, cell lysates were immunoprecipitated with either myc antibody 9E10 (FIG. 4, odd numbered lanes) or HA antibody 12CA5 (even numbered lanes) and blotted with the same HA and myc antibodies. Wild type GRB2 or molecules having 2 intact SH3 domains (E89K, S9ON, GRB3.3) did bind to full length GA5Ptase (closed circles) but not shorter GA5Ptase proteins. Mutations that disrupt binding of either SH3 domain (P49L, G203R) markedly reduced (shaded triangle) or eliminated (open triangle) full length GA5Ptase binding. This illustrates that GRB2 associates with GA5Ptase through both of its SH3 domains.

Example-4

Activation of Serum Response Element Upon co-Expression With Ras and GRB2

[0136] NIH 3T3 cells were transiently transfected with constructs encoding GRB2, GA5Ptase, c-Ras, GRB2/c-Ras, GA5Ptase/c-Ras, GA5Ptase/GRB2 and GA5Ptase/c-Ras/GRB2, as listed in FIGS. 5A-D, and the luciferase indicator plasmid p2FTL. This plasmid contains two copies of the c-fos serum response element (SRE) (-357 to -276) and the herpes simplex virus (HSV) thymidine kinase (TK) gene promoter (-200 to +70) driving the firefly luciferase gene. After growth for two days in serum depleted media, the cells were harvested and endogenous luciferase activity was measured in relative light units. Each value is the average of triplicate transfections, error bars represent standard error of the mean. Point mutations in GRB2 are the same as those indicated above.

[0137] Substantial synergistic activation of the Fos promoter occurred when all three cDNAs were expressed. GRB2 mutants that reduce or eliminate the binding to GA5Ptase did not activate the Fos SRE as well as wild type (FIGS. 5B and 5C), indicating the importance of the interaction between GA5Ptase and GRB2. Platelet inositol polyphosphate 5-phosphatase type II ("5Ptase II"), a 5ptase family member also possessing Ins(1,3,4,5)P.sub.4 and PtdIns(3,4,5)P.sub.3 hydrolyzing activity can substitute for GA5Ptase in its activation of cFos transcription (FIG. 5D). These results indicate the importance of GA5Ptase activity in Fos SRE activation.

[0138] While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. All publications and patent documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent document were so individually denoted.

Sequence CWU 1

1

Claims

What is claimed is:

1. A substantially pure polypeptide, comprising an amino acid sequence that is substantially homologous to the amino acid sequence shown in FIG. 10 (SEQ ID NO:2), or biologically active fragments thereof.

2. The substantially pure polypeptide of claim 1, wherein said polypeptide comprises the amino acid sequence shown in FIG. 10 (SEQ ID NO:2) or biologically active fragments thereof.

3. The substantially pure polypeptide of claim 1, wherein said polypeptide further comprises a heterologous protein fused to said amino acid sequence that is substantially homologous to the amino acid sequence shown in FIG. 10 (SEQ ID NO:2), or biologically active fragments thereof.

4. A pharmaceutical composition, comprising an effective amount of the polypeptide of claim 1 in combination with a pharmaceutically acceptable carrier.

5. The substantially pure polypeptide of claim 1, wherein said polypeptide comprises an inositol polyphosphate 5-phosphatase activity, whereby said polypeptide is capable of hydrolyzing a 5-phosphate from Ins(1,3,4,5)P.sub.4 and PtdIns(3,4,5)P.sub.3, but not Ins(1,4,5)P.sub.3 or PtdIns(4,5)P.sub.2.

6. The substantially pure polypeptide of claim 1, wherein said polypeptide is capable of associating with GRB2.

7. The substantially pure polypeptide of claim 1, wherein said polypeptide is capable of binding a GRB2--SH3 domain.

8. An isolated nucleic acid segment, said nucleic acid segment encoding a polypeptide having an amino acid sequence substantially homologous to the amino acid sequence shown in FIG. 10 (SEQ ID NO:2), or biologically active fragments thereof.

9. The isolated nucleic acid segment of claim 8, wherein said nucleic acid segment comprises at least about 15 contiguous nucleotides from a nucleotide sequence that is substantially homologous to the nucleotide sequence shown in FIG. 10 (SEQ ID NO:1).

10. The isolated nucleic acid segment of claim 9, wherein said nucleic acid segment comprises at least about 15 contiguous nucleotides from the nucleic acid segment as shown in FIG. 10 (SEQ ID NO:1).

11. The isolated nucleic acid segment of claim 8, wherein said nucleic acid segment encodes a polypeptide having an inositol polyphosphate 5-phosphatase activity, whereby said polypeptide is capable of hydrolyzing a 5-phosphate from Ins(1,3,4,5)P.sub.4 and PtdIns(3,4,5)P.sub.3, but not Ins(1,4,5)P.sub.3 or PtdIns(4,5)P.sub.2.

12. The isolated nucleic acid segment of claim 8, wherein said nucleic acid further comprises a segment which encodes a heterologous protein, whereby said nucleic acid is expressed as a fusion protein.

13. An expression vector, said expression vector comprising a nucleic acid segment operably linked to a promoter sequence, wherein said nucleic acid segment is the nucleic acid of claim 8.

14. A recombinant host cell, wherein said host cell has been transfected with the expression vector of claim 13, whereby said host cell is capable of expressing said nucleic acid segment.

15. The recombinant host cell of claim 14, wherein said host cell is selected from the group consisting of bacterial, mammalian, plant, fungal and insect cells.

16. The recombinant host cell of claim 15, wherein said host cell is an Sf9 insect cell.

17. An isolated antibody, said antibody being specifically immunoreactive with a polypeptide having an amino acid sequence substantially homologous to the amino acid sequence shown in FIG. 10 (SEQ ID NO:2) or its biologically active fragments.

18. The isolated antibody of claim 17, wherein said antibody is capable of blocking or inhibiting one or more biological activities of the polypeptide having the amino acid sequence shown in FIG. 10 (SEQ ID NO:2).

19. The isolated antibody of claim 17, wherein said antibody is specifically immunoreactive with a polypeptide having the amino acid sequence shown in FIG. 10 (SEQ ID NO:2).

20. The isolated antibody of claim 17, wherein said antibody is a monoclonal antibody.

21. A pharmaceutical composition, comprising an effective amount of the isolated antibody of claim 17 in combination with a pharmaceutically acceptable carrier.

22. A method of preparing a substantially pure polypeptide having inositol polyphosphate 5-phosphatase activity, comprising: inserting the nucleic acid segment of claim 8, into an expression vector; transfecting a host cell capable of expressing said nucleic acid segment with said expression vector; culturing said host cell under conditions which result in expression of said nucleic acid segment; and recovering said protein having inositol polyphosphate 5-phosphatase activity.

23. A method of determining whether a test compound is an agonist or antagonist of a GRB2/GA5Ptase interaction, the method comprising: contacting GRB2 with GA5Ptase or a biologically active fragment thereof, under conditions conducive to forming a GRB2/GA5Ptase complex, in the presence and absence of the test compound; and determining the amount of GRB2/GA5Ptase complex formed in the presence and absence of said test compound, an increase or decrease in the amount of GRB2/GA5Ptase complex formed in the presence of said test compound being indicative that the test compound is an agonist or antagonist of GRB2/GA5Ptase interaction, respectively.

24. A method for determining whether a test compound is an agonist or antagonist of an inositol polyphosphate 5-phosphatase activity, comprising: incubating a mixture of inositol polyphosphate substrate and GA5Ptase, in the presence and absence of the test compound; assaying the mixture to determine an amount of a product of GA5Ptase activity on said inositol polyphosphate substrate in the presence and absence of the test compound; and comparing the amount of product of GA5Ptase activity in the presence of the test compound to the amount of product of GA5Ptase activity in the absence of the test compound, an increase or decrease in the amount of product of GA5Ptase activity in the presence of the test compound being indicative that the test compound is an agonist or antagonist of an inositol polyphosphate 5-phosphatase activity, respectively.

25. The method of claim 24, wherein said inositol polyphosphate substrate is selected from the group consisting of Ins(1,3,4,5)P.sub.4 and PtdIns(3,4,5)P.sub.3.

26. A method of identifying the presence of GRB2 in a sample, comprising incubating said sample with the polypeptide of claim 6, and detecting binding between said polypeptide and a portion of said sample, said binding being indicative of the presence of GRB2 in said sample.

27. A method of purifying GRB2 from a mixture of different proteins containing GRB2, comprising: immobilizing the polypeptide of claim 6, on a solid support; contacting said mixture of proteins with said solid support under conditions in which said polypeptide binds GRB2; washing said solid support to remove unbound proteins; and eluting GRB2 from said solid support in substantially pure form.

28. A kit for determining whether a test compound is an agonist or antagonist of GA5Ptase activity, comprising: a GA5Ptase polypeptide, or catalytically active fragment thereof; a GA5Ptase substrate; and instructions for assaying for the presence of said substrate and a product of GA5Ptase.

29. The kit of claim 26, wherein said GA5Ptase substrate is selected from the group consisting of Ins(1,3,4,5)P.sub.4 and PtdIns(3,4,5)P.sub.3.

30. The kit of claim 26, wherein said GA5Ptase and said substrate are lyophilized, and further comprising a buffer solution for reconstituting said GA5Ptase and said substrate.

31. A method of treating a patient suffering from a proliferative disorder, the method comprising administering to said patient a therapeutically effective amount of the polypeptide of claim 6.

32. A substantially pure polypeptide, said polypeptide being immunologically cross-reactive with an antibody to a GA5ptase protein, or biologically active fragment thereof.