AU727305B2 - A potential effector for the GRB7 family of signalling proteins - Google Patents

Description

WO 99/15647 PCT/AU98/00795 1 A POTENTIAL EFFECTOR FOR THE GRB7 FAMILY OF SIGNALLING

PROTEINS

Field of the Invention: The present invention relates to a novel polynucleotide molecule encoding a candidate effector protein for the Grb7 family of signalling proteins. Detection of the encoded protein in a tissue sample should provide a useful tumour marker and/or prognostic indicator. Furthermore.

antagonism of the interaction between Grb7 family members and the encoded protein should provide a novel treatment strategy for human diseases exhibiting aberrant receptor tyrosine kinase (RTK) signalling (e.g.

cancer).

Background of the Invention RTKs play a major role in the regulation of cellular growth, differentiation, motility and metabolism by converting an extracellular signal in the form of the binding of a specific hormone or growth factor to the activation of specific signalling pathways and hence modes of intracellular communication (Schlessinger and Ullrich, Neuron 9. 383-391. 1992).

Activation of RTKs results in both autophosphorylation of the receptor and the phosphorylation of downstream targets on tyrosine residues. It has become evident over the last decade that key elements in receptor-substrate and other protein-protein interactions in RTK signalling are src homology (SH)2 domains. SH2 domains are conserved modules of approximately 100 amino acids found in a wide variety of signalling molecules which bind to short tyrosine-phosphorylated peptide sequences. The specificity of interaction is determined both by the nature of the amino acids flanking the phosphotyrosine residue in the target peptide and residues in the SH2 domain which interact with these sites (Pawson, Nature 373, 573-580, 1995).

SH2-domain containing proteins can be divided into two classes: those which possess a catalytic function the cytoplasmic tyrosine kinase c-src and the tyrosine phosphatase SH-PTP2) and those which consist entirely of non-catalytic protein domains (eg Grb2). the adaptor sub-class. The function of the latter class is to link separate catalytic subunits to a tyrosine- WO 99/15647 PCT/AU98/00795 2 phosphorylated receptor or signalling intermediate, and other non-catalytic protein modules are often involved in these interactions. For example. SH3 and WW domains (conserved regions of approximately 50 and 40 amino acids, respectively) bind proline-rich peptide ligands. and pleckstrin homology domains (approximately 100 amino acids) interact with both specific phospholipid and protein targets (Pawson, 1995 supra).

The Grb7 family represents a family of SH2 domain-containing adaptors which currently contains three members: Grb7. 10 and 14 (Margolis et al, Proc. Natl. Acad. Sci. USA 89, 8894-8898. 1992: Stein et al. EMBO 13, 1331-1340, 1994; Ooi et al, Oncogene 10. 1621-1630. 1995; Daly et al, J. Biol.

Chem. 271, 12502-12510, 1996). These proteins share a common overall architecture, consisting of an N-terminal region containing a highly conserved proline-rich decapeptide motif, a central region harbouring a PH domain and a C-terminal SH2 domain. The central region of approximately 300 amino acids bears significant homology to the C. elegans protein miglO, which is required for long range neuronal migration in embryos, otherwise the Grb7 family and miglO are structurally distinct. However, they exhibit differences in both SH2 selectivity towards RTKs (Janes et al, J. Biol. Chem.

272, 8490-8497, 1997) and tissue distribution. The family has therefore evolved to link particular receptors to downstream effectors in a tissuespecific manner. Interestingly, the genes encoding this family appear to have co-segregated with ERBB family genes during evolution. Thus GRB7, 10 and 14 are linked to ERBB2, ERBB1 (epidermal growth factor receptor) and ERBB4, respectively (Stein et al 1994 supra; Ooi et al, 1995 supra: Baker et al, Genomics 36, 218-220, 1996). The juxtaposition of GRB7 and ERBB2 leads to common co-amplification in human breast cancers. and since the two gene products are functionally linked, likely up-regulation of an undefined erbB2 signalling pathway. Furthermore, GRB14 also exhibits differential expression in human breast cancers (Daly et al, 1996 supra). These two proteins may therefore modulate RTK signalling in this disease.

In order to identify proteins which bind to this family and therefore identify candidate effectors, we performed a genetic screen using the yeast two hybrid system and Grbl4 "bait". This application describes the cloning and characterization of a novel interacting protein, currently designated 2.2412.

WO 99/15647 PCT/AU98/00795 3 Disclosure of the Invention: Thus, in a first aspect, the present invention provides an isolated polynucleotide molecule encoding a candidate effector protein for the Grb7 family of signalling proteins, wherein the polynucleotide molecule comprises a nucleotide sequence having at least 75% sequence identity to that shown as SEQ ID NO: 1.

Preferably, the polynucleotide molecule comprises a nucleotide sequence having at least 85%, more preferably at least 95%. sequence identity to that shown as SEQ ID NO: 1. Most preferably, the polynucleotide molecule comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 2.

In a preferred embodiment of the invention of the first aspect, the polynucleotide molecule comprises a nucleotide sequence which substantially corresponds to that shown as SEQ ID NO: 1.

The polynucleotide molecule may be a dominant negative mutant which encodes a gene product causing an altered phenotype by, for example, reducing or eliminating the activity of endogenous effector proteins of the Grb7 family of signalling proteins.

The polynucleotide molecule may be incorporated into plasmids or expression vectors (including viral vectors), which may then be introduced into suitable host cells such as bacterial, yeast, insect and mammalian host cells. Such host cells may be used to express the protein encoded by the polynucleotide molecule.

Accordingly, in a second aspect, the present invention provides a host cell transformed with the polynucleotide molecule of the first aspect.

In a third aspect, the present invention provides a method of producing a protein, comprising culturing the host cell of the second aspect under conditions suitable for the expression of the polynucleotide molecule and optionally recovering the protein.

Preferably, the host cell is mammalian or of insect origin. Where the cell is mammalian, it is presently preferred that it be a Chinese hamster ovary (CHO) cell or human embryonic kidney (HEK) 293 cell. Where the host cell is of insect origin, it is presently preferred that it be an insect Sf9 cell.

WO 99/15647 PCT/AU98/00795 4 In a fourth aspect, the present invention provides a purified protein encoded by the polynucleotide molecule of the first aspect.

In a preferred embodiment of this aspect, the purified protein comprises an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 2.

In a fifth aspect. the present invention provides a fusion protein comprising an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 2.

Fusion proteins according to the fifth aspect may include an Nterminal fragment of a protein such as [3-galactosidase to assist in the expression and selection of host cells expressing candidate effector protein.

or may include a functional fragment of any other suitable protein to confer additional activity(ies).

In a sixth aspect, the present invention provides an antibody or fragment thereof which specifically binds to the protein of the fourth aspect.

The antibody may be monoclonal or polyclonal. however, it is presently preferred that the antibody is a monoclonal antibody. Suitable antibody fragments include Fab, and scFv.

In a seventh aspect. the present invention provides an oligonucleotide probe comprising a nucleotide sequence of at least 12 nucleotides, the oligonucleotide probe comprising a nucleotide sequence such that the oligonucleotide probe selectively hybridises to the polynucleotide molecule of the first aspect under high stringency conditions (Sambrook et al., Molecular Cloning: a Laboratory Manual, Second Edition. Cold Spring Harbor Laboratory Press).

In a preferred embodiment of this aspect, the oligonucleotide probe is labelled. In a further preferred embodiment of this aspect, the oligonucleotide probe comprises a nucleotide sequence of at least 18 nucleotides.

In an eighth aspect, the present invention provides a method of detecting in a sample the presence of an effector protein for the Grb7 family of proteins, the method comprising reacting the sample with an antibody or fragment thereof the sixth aspect, and detecting the binding of the antibody or fragment thereof.

WO 99/15647 PCT/AU98/00795 The method of the eighth aspect may be conducted using any immunoassays well known in the art ELISA). The sample may be, for example, a cell lysate or homogenate prepared from a tissue biopsy.

In a ninth aspect, the present invention provides a method of detecting in a sample the presence of mRNA encoding an effector protein for the Grb7 family of proteins, the method comprising reacting the sample with an oligonucleotide probe of the seventh aspect, and detecting the binding of the probe.

The method of the ninth aspect may be conducted using any hybridisation assays well known in the art Northern blot). The sample may be a poly(A) RNA preparation or homogenate prepared from a tissue biopsy.

Grb7 family proteins exhibit differential expression in certain human cancers (particularly breast and prostate cancer) and may therefore be involved in tumour progression. Detection of the protein encoded by the cDNA 2.2412 in a sample should provide a useful tumour marker and/or prognostic indicator for these cancers. Furthermore, the interaction of Grb7 family members with 2.2412 may provide a novel target for therapeutic intervention.

It is to be understood that methods of detecting suitable agonists and methods of therapy utilising detected agonists also form part of the present invention.

The term "substantially corresponds" as used herein in relation to the nucleotide sequence shown as SEQ ID NO: 1 is intended to encompass minor variations in the nucleotide sequence which due to degeneracy in the DNA code do not result in a change in the encoded protein. Further, this term is intended to encompass other minor variations in the sequence which may be required to enhance expression in a particular system but in which the variations do not result in a decrease in biological activity of the encoded protein.

The term "substantially corresponding" as used herein in relation to the amino acid sequences shown as SEQ ID NO: 2 is intended to encompass minor variations in the amino acid sequences which do not result in a decrease in biological activity of the protein. These variations may include conservative amino acid substitutions. The substitutions envisaged are:- G,A,V,I,L,M: D.E; N,Q; S,T; K,R,H: F, Y, W, H and WO 99/15647 PCT/AU98/00795 6 P, No.-alkalamino acids.

The terms "comprise", "comprises" and "comprising" as used throughout the specification are intended to refer to the inclusion of a stated step, component or feature of group of steps, components of features with or without the inclusion of a further step. component or feature or group of steps, components or features.

The invention will hereinafter be described with reference to the accompanying figure and the following. non-limiting example.

Brief description of the accompanving figure: Figure 1 provides the nucleotide and amino acid (single letter code) sequence of 2.2412. Numbers refer to distances in base pairs. Ankvrin-type repeat sequences are underlined. An additional repeat sequence is indicated by italics.

The stop codon is represented by an asterisk. The original cDNA clone 2.2412 isolated by the two hybrid screen spans nucleotides 694-2664 of this sequence.

Figure 2 provides a map of the 2.2412-binding region on Grbl4.

A. Structure of the deletion constructs used in the analysis. Gal4 DNA-BD fusion constructs encoding full length Grbl4 the N-terminal central region (C) and N-terminal central region (N C) were generated in the vector pAS2.1.

B. Results of P-galactosidase activity assays following transformation of the above plasmids into yeast strain Y190 together with the original 2.2412 cDNA clone in pACT-2.

Example: CLONING AND CHARACTERISATION OF 2.2412 Yeast two hybrid screen The yeast two hybrid system exploits protein-protein interactions to reconstitute a functional transcriptional activator which can then be detected using a gene reporter system (Fields and Sternglanz. TIG. 10. 286-292. 1994).

The technique takes advantage of the properties of the Gal4 protein of the yeast S. cerevisiae. The Gal4 DNA binding domain (DNA-BD) or activation domain (AD) alone are incapable of inducing transcription. However, an interaction between two proteins synthesized as DNA-BD- and AD-fusions.

respectively, brings the Gal4 domains into close proximity and results in WO 99/15647 PCT/AU98/00795 7 transcriptional activation of two reporter genes (HIS3 and LacZ) which can be monitored by growth on selective medium and biochemical assays.

A plasmid construct encoding a Gal4 DNA-BD-Grbl4 fusion was generated as follows. The plasmid GRB14/pRcCMVF containing full length GRB14 cDNA (Daly et al, 1996) was restricted with HindIII and Klenow treated to create blunt ends, and then digested with BclI to release three fragments of approximately 1.1, 4.2 and 1.7 kb. The 1.7 kb fragment was isolated and cloned into the Ndel (Klenow treated) and BamHI sites of the yeast expression vector pAS2.1 (Clontech) to generate GRB14/pAS2.1 containing an in-frame fusion of full length Grbl4 with the GAL4 DNA-BD.

This construct was introduced by electroporation into the yeast strain CG1945 (MATa, ura3-52, his3-200, ade2-101, lys2-801,trpl-901, leu2-3, 112, gal4-542, gal80-538, cyvh2, LYS2::GAL1 UAS-GAL1 TA7A-HIS3, URA3::GAL4 1 7mers(x3)-CYCITATA-lacZ) selecting for tryptophan prototrophy. The expression of the fusion protein was verified by Western blot analysis with antibodies directed against the Flag epitope and the Gal4 DNA-BD. The recipient strain was then grown to mid-log phase and a human liver cDNA library in the vector pACT2 (Clontech) introduced using the LiAc procedure (Schiestl and Gietz, Curr. Genet. 16, 339-346. 1989). Transformants were then selected for tryptophan, leucine and histidine prototrophy in the presence of 5nmM 3-aminotriazole.

From a screen of 1x10 6 clones, 39 colonies were initially selected on synthetic complete (SC)-leu-his-trp +3AT medium and were then tested for P-galactosidase activity. 12 clones scored positive in the latter assay and were subjected to cycloheximide (CHX) curing to remove the bait plasmid by streaking out on SC-leu media containing 10ug/ml CHX (pAS2-1 contains the CYH2 gene which restores CHX sensitivity to CG1945 cells). This enabled confirmation of the bait dependency of LacZ activation and subsequent isolation of the pACT2 plasmids encoding interacting proteins by standard methodology (Philippsen et al, Methods in Enzymologv 194, 170-177). Back transformations were then performed in which these pACT2 plasmids were introduced into CG1945 strains containing the bait plasmid (GRB14/pAS2-1) or constructs encoding non-related Gal4 DNA-BD fusions in order to confirm the specificity of the interactions.

The DNA sequences of the cDNA inserts were then obtained by cycle sequencing (f-mol kit. Promega) using pACT2-specific and/or clone-specific WO 99/15647 WO 99/ 5647PCT/AU98/00795 8 primers. Based on their nucleotide sequences the 12 interacting clones were classified into 6 independent groups (see Table I).

TABLE 1: Characterization of cDNA clones isolated by the yeast two hybrid screen.

Class No. of Identity Mean RLU Colour intensity clones (Liquid assay) (Filter assay) 1 6 Nedd4 2.86x10 6 2 2 Htk 1.86x10'++ 3 1 2.2412 5.18x10"....

4 1 Proleosorne 3.88x 10 2 1 Soniatostatin 1.45xl0 3 +1 receptor 6 1 L-arginine:glycine 8.61x10 2 amidinotransferase Tbn 1 2 Cl o nes exilii ill-i act ivalil o o 011 I lie HI1S" an on repor-ter goi s wore dilvidned into fU grou~ps byv sequenc,(e anairlysis or their cI)NA inser-ts. Results rA(-aatrihs ciiyassays peifl1ned using two mrethodiologies are shown. Thell liquid culturr-rinived 1inettior (Gaiacto-Liglrt.

'IRUPIX) is mnorn quantiitative: rnsults 11r0 givell ill 11Wani 11el1tive light un1its (IAJ) 1and are 1101-ura~lu for ille protein content of the samples. lfluo/whlt s croeerng oflm ie :1A clones was also periormned using a colony lift filter assay (Clontecli). The intensity of 11ue Colour ulervelopilenit over approxinnatelv 211 is scredl from1 (very weak) to (st ronigl).

Six clones were p~artial cDNAs corresponding to Nedd4, a multidomain protein: containing a calciumn-dependent phospholi pid binding (CaLB) domain, four WW domains and a C-terminal region homologous to the E6-AP carboxyl-terniinus (Kumar et al, Biocliem. Biopiwjs. Res. Comm un. 185. 115 1161, 1992; Sudol et a] 1. Biol. Chem. 270, 14733-14741. 1995; Huibregtse et a] Proc. Nat]. Acad. Sci. USA 92.2563-2567, 1995). The latter is likely to confer E3 ubiquitin-protein ligase activity on Nedd4. The pACT2 clones isolated encoded the CaLB domnain together with the first 22 amino acids of the first WW domain.

WO 99/15647 PCT/AU98/00795 9 Two clones encoded the intracellular region and part of the extracellular domain of Htk. which is a RTK of the Eph family (Bennett et al J. Biol. Chem. 269. 14211-14218. 1994). The recruitment of Grbl4 by Htk is of interest for two reasons. First. the expression profile of both Htk and the murine homologue mvk-1 are indicative of a potential role in mammary gland development and neoplasia (Andres et al Oncogene 9. 1461-1467. 1994: Berclaz et al Biochem. Biophys. Res. Comm. 226. 869-875. 1996). Second. Eph family members may be involved in the regulation of cell migration (Tessier- Lavigne, Cell 82, 345-348. 1995). which is intriguing given the homology of the Grb7 family to the C. elegans protein miglO (Stein et al. 1994 supra).

A novel cDNA of 1971 bp. designated 2.2412. was also isolated. This clone encoded a polypeptide of 657 amino acids in frame with the Gal4 DNA-BD. The cDNA did not contain a stop codon. and this. together with the Northern analysis described below, indicated that it was incomplete. This DNA fragment was therefore used as a probe to screen a human placental cDNA library STRETCH PLUS. Clontech. in Xgtl0). This resulted in the isolation of two clones, designated clone 8 and clone 12. Clone 8 was approximately 2 kh and overlapped the original 2.2412 clone by 900 bp at the 3' end. This clone provided the carboxv-terminal end of the 2.2412 protein sequence (Figure Clone 12 was approximately 3.5 kb and to date has provided an additional 692 bp of sequence information in the direction. The nucleotide and protein sequence for 2.2412 provided by these overlapping clones is shown in Figure 1. Since a 5' initiation codon has yet to be identified the coding sequence still appears to be incomplete.

Further characterization of 2.2412 Database searches using the 2.2412 cDNA sequence revealed significant homology with a large number of proteins containing ankyrin-like repeats. These sequences were first identified as homologous regions between certain cell cycle regulatory proteins and the Drosophila protein Notch (Breeden and Nasmyth. Nature 329. 651-654. 1987) but subsequently they have been identified in a wide variety of other proteins where they are thought to function in proteroteiotein interactions (Bork. Proteins 17, 363- 374, 1993). Subsequent analysis of the protein sequence identified 18 consecutive ankyrin repeats and an additional repetitive element (Figure 1).

The ankyrin repeat region is followed by a stretch of approximately 40 amino WO 99/15647 PCT/AU98/00795 acids rich in serine residues. The remaining C-terminal region has a relatively high content of charged amino acids.

Northern analysis of 2.2412 mRNA expression Northern blot analysis of multiple tissue northerns (Clontech) was performed using the original 2.2412 cDNA as a probe. This resulted in the detection of a single mRNA transcript of approximately 7 kb in all tissues examined with the exception of the kidney. Expression was particularly high in skeletal muscle and placenta. The size of this transcript compared to that of the 2.2412 clone indicates that the latter represents only a partial cDNA.

Genomic localization of the 2.2412 gene Fluorescence in situ hybridization of the original 2.2412 cDNA to normal metaphases (Baker et al, 1996 supra) and reference to the FRA1OA fragile site at 10q23.32 localized the gene to between chromosome 10q23.2 and proximal 10q23.32. Interestingly, deletions in the 10q22-25 region of chromosome 10 have been detected in a variety of human cancers including breast, prostate, renal, small cell lung and endometrial carcinomas, glioblastoma multiforme, melanoma and meningiomas, suggesting the presence of one or more tumour suppressive loci in this region (Li et al, Science 275, 1943-1947. 1997; Steck et al, Nature Genetics 15, 356-362. 1997, and references therein). Two candidate tumour suppressor genes have been identified in this region (MMAC1/PTEN and MXI1. Li et al 1997 supra: Steck et al 1997 supra; Albarosa et al, Hum. Genet. 95, 709-711. 1995).

Analysis of the interaction between 2.2412 and Grb7 family members cDNAs encoding the full length and N- and C-terminal regions of the original 2.2412 cDNA clone (nucleotides 694-2664, 694-1614 and 1615-2664 of the sequence shown in Figure 1, respectively) were cloned into the vector pGEX4T2 (Pharmacia). The full length construct was generated by subcloning from the pACT2 clone as a NdeI fragment, whereas the shorter constructs were synthesized by directional cloning of PCR products. The corresponding GST-fusion proteins were purified from IPTG-induced bacterial cultures using glutathione-agarose beads (Smith and Johnson, Gene 67, 31-40, 1988). These immobilized fusion proteins were then incubated with lysates from cells expressing Flag epitope-tagged Grbl4 (Daly et al, 1996 WO 99/15647 PCT/AU98/00795 11 supra) or human breast cancer cells expressing high levels of Grb7 (SK-BR-3: Stein et al, 1994) as described previously (Daly et al. 1996). Following washing, bound proteins were detected by Western blot analysis. The results indicated that 2.2412 bound specifically to both Grbl4 and Grb7 in vitro, and that the N-terminal fusion protein bound more strongly than that derived from the C-terminus. These data, obtained using a different methodology for detecting protein-protein interactions to the yeast two hybrid system. confirm that 2.2412 interacts with Grb14. Furthermore, 2.2412 also binds Grb7.

Consequently 2.2412 appears to represent a general effector for the Grb7 family.

Mapping of the 2.2412 binding region on Grbl4 In order to identify the region of Grbl4 that interacts with 2.2412, a series of Grbl4 deletion mutants were generated by cloning PCR fragments synthesized using the appropriate flanking primers into the vector pAS2.1.

These fragments spanned the following regions: N-terminus amino acids 1-110), the central region encompassing the miglO homology and the "between PH and SH2" (BPS) domain (amino acids 110-437) and the Nterminal and central regions amino acids 1-437). These plasmids were individually transformed into the yeast strain Y190 (MATa, ura3-52, his3-200, ade2-101, 1ys 2 8 01, trpl-901, leu2-3, 112, gal4A, gal80A, cylhr2, LYS2::GAL1UAS-HIS3TATA-HIS3, URA3::GAL1UAS-GAL1TATA-lacZ) and expression of the appropriately sized Gal4 DNA-BD fusion proteins confirmed by Western blotting. Following transformation of the resulting yeast strains with the original 2.2412 cDNA clone in pACT-2, the strength of the interaction was determined by either liquid- or filter-based 3galactosidase assays. The results are presented in Figure 2, and demonstrate that the N-terminal region of Grbl4 is not only required, but is also sufficient, for binding 2.2412. This supports the hypothesis that 2.2412 represents a general effector for the Grb7 family, since the N-terminal region of these proteins contains a highly conserved proline-rich motif which may mediate this interaction.

WO 99/15647 PCT/AU98/00795 12 It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

WO 99/15647 WO 99/ 5647PCT/AU98/00795 13 Sequence listings: SEQUENCE LISTING Applicant: Garvan Institute of Medical Research Title of Invention: A potential effector for the Grb7 family of signalling proteins.

Current Application Numb~er: Current Filing Date: Prior Application Number:P09388 Prior Application Filing Date: 1997-09-23 Number of ID SEQ Nos: 2 Software: Patentln Ver. SEQ ID NO: 1 Length: 3400 Type: DNA Organism: Homo sapiens Sequence:I attcctcttc catggtgcag attaaaggaa cgaaatacag actggtgaat atgatggctc actccattac catggacgtg t ctta tggtc atggacttgt tgttctcttc gctatagact cactcgttgc ctggaaatgg gcatctccat atcaatgaaa aatgatgttg ggtcagactt ctgagctatg ggaaatgaaa gacagacaat actgttcaga gcagctgggt catgctaaag gaagttgcag tttacacctt cagcatggtg aaagatggag gccaagaagg cgcgataccc gaagttgcag cttattcctt ataatgcatg accccaatgc agattgatgt atggaaggac ataagaaaga tactcacacc atttggcagc atgtccatgc attatgaagt ggcaattcac tcttaagtta tggctcccac tgcaagctgc tgaatttcaa atcccaaaag agactaaaga ttgaagtagt ctctacacag ggtgtgatcc atgtacagca tgctggaagc gtgtcaactg ataacagagt ataaaggagg aacttcttgt tacatgaagc cagaccctac atacagatat gttgtttagc aaggcagaca agtatttgtt tacataatgc ctcttttggt tcgagataat ttgcattgtg agcattggat tgaactctta attaaatgtc aggatataac taaagataaa aactgaactt tcctcttcat tggtgcagac accacagtta acgagaagct gcatcctcaa aaagcaaata attcttgact ggtgaaacat agctgcatat taacattata actcctccaa tgcaaaggct cagagacatt gtccgtggtg ccttgtacct taaacatgga agcagcaaaa aaaaaaaaac tcaagatctg cagagtgaag ttcaacacct acaacacgga agcatcttac catgctgaag tggaattata ctgttacagc ttagcagatc gaaagtgcca aactgccacg agagtaaaga ggtgatctgg ttggtcaagc gaggcagctt ccaacactgc aaagaaagat gatgttactc acacatgaaa tgtgaactgt cctctgcacg gaagcaaagg tgtggtcatc tcccttcagg gagggtatct ggagatgtcg gaagggcgtc gaatatctgc ttgcacaatg gcagtagtta ggaaaatatg agggatggaa cttaggggag aagttgtctt ttacatttag gctgatgtga gggcatgtag tagtcaatct ctcctctcca atggagctga catctgccaa ggagtggcaa caagtgatgg ttgtacagct taccattaca atggtggctg ctaagaacag tcaattgtaa tagcatatga gaatcaaaaa cagcattgca tgctaagaaa tggcatctga ttaatgctct tacaaacctg gctttactgc cat taggtaai aaactgtaaa agtctacacc tacagcatgg catgttctta atgtagctga aaatttgca a atactccttt atgcagcttt ctcctgataa cagctggtta atgcccaaga atgtagcagc ccttttgcga tgaagctgca gccaaccatc agcagtgctt tgaagaaaaa cagaaagtca gttactgcaa caatgcctgt tgtaaatgca ggttgaagta gaataaaagt atttaaaggc acatctctct ttgtgctgct aggagcaaac gaaagctcat ggataatctt ccgcctactc tttacagatg ttcagaggca aaaactgtgt acttcatttt agctgatgtg cggacattat tttatggaaa acttctgctc ggatcttgtt gctagatgct tgtaaattgc taataattta caaaggagga tctactaata 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 WO 99/15647 WO 99/ 5647PCT/AU98/00795 aaqtataatg gcccaaaagg cttaaaaatc cttctgacag aatggtgtga tcaagccttt tcagtagtta ggagtagatt atatttgaga aaggagattg cttatctccg acaa ttctta caaagtacag aatattctca agaaaagaag tctccttttg ggtatgtttg tatggaattg cacaggcagc atgaaaatgg ggcctagcat attacttacc aactaattcc tgctgaaaaa gtttaacatt catctc-tcaa gacgaacaca aggaaggaca cagccatgcc gaagcccagg ctgcagccag gttcaagtgg ttagcataac gagaacagat gaatcaatgc gacaacaagg tagatctgtc ttcgagagca agattcagaa tttctgaaga tgaatgcaat gagctggcat gaggaggtac tgctcttttg cacattctcc tagctgaata agattatgag actgaaccta aaatcatctt ctgacttgat tgccacggac gctttgtgct aacaccttta cccatctgct agccactgca cagtcttgac aacagagggt t caat tcg ta cactttggat ttatgga cat tcttaaccca tcctgatgat cagagatgga ggtttgtaac aaaccacaac tatccacaaa ttattttgct tgggtgtcca ccgggtaacc tccaggtcat tgttatttac gcctgaaggt aaatcatcaa gcccacaggc aaagctttaa aaatgggctt ttgttgctag gatttagttt ctgccctctt gatgctctct aacttatctg gcttccagtt aggaatcttg gtattagttg aggcacaaac tatttaactt aaagagtttc ggtcatgcag aagaaactat catgccaatg ggctttgatg gaaaactctt gttcacaaag ttgggaaagt cactcagtca agaggagaac atggtcgatg agcagcagtg ctgtggcaaa taatgtacag tcacacct tt cccatggagc cagcagatqa gttacaagcc cttcaggtrc ggagtttttc tggagaaaaa gacttgagca aqatgggqca taattaaagg tgaacacctc agtctgtgga gt ggaat c tt gggaaaqata aacgaatqct aaaggcatuc ccaaaagcaa acagatcttq ctttcctgca ctgqtaggcc aggcttatcc gataaataqt gcctctacgt aggataaa aa gcacgaagca tgacccgact tgtcagcgct tcaagtgctc atctagccca agaactgtct ggaggttcca cctaatggat caaggagctg agtcgagaga tggtagtgga ggaagagatg caacagatac cactcaccgg atttcatggg gtacataggt tcaatatgta ttacatttgc cttcagtgca cagtgtaaat tgagtattta tattttaaga tttactcctt tgtgaacgaa 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3400 SEQ ID NO: 2 Length: 1074 Type: PRT Organism: Homo sapiens- Sequence: 2 Ile Pro Leu His Asn 1 5 Ala Cys Ser Phe Gi y 10 His Ala Glu Val Val Asn Leu Leu Leu Tyr Thr Pro Arg His Gly Ala Asp Pro 25 Asn Ala Arg Asp Asn Trp Asn Asp Val Cys Leu His Glu Ala Ile Lys Gly Lys Ile Ile Val Leu Leu Gin His Gi y Ala Glu Pro Thr Arg Asn Thr Asp Gly Arg Thr Ala Leu Leu Ala Asp Pro Ser 75 Ala Lys Ala Val Leu Thr Gly Glu Tyr Lys Lys Asp Glu Leu Glu Ser Ala Arg Ser Gly Asn Glu Glu His Ala Ser 115 Met Met Ala Leu Leu 105 Thr Pro Leu Asn Val Asn Cys .1.10 Ala Ala Gly Asp Gly Arg Lys Thr Pro Leu His Leu 125 Tyr Asn 130 Arg Val Lys Ile Val1 135 Gin Leu Leu Leu His Gly Arg Asp Val His Ala Lys Asp Lys Gly Asp Leu Val Pro Leu His Asn Ala Cys WO 99/15647 WO 9915647PCT/AU98/00795 145 Ser Cys Al. a Al a Al a 225 His Lys Giu Gin Thr 305 As n Leu His Ile Val 385 Asp Lys Arg Val Tyr Val Ser Asp 210 Pro Ser His Thr Ile 290 Lys Asp Asp Leu Ile 370 Gin Ar g Lys Gin Val1 450 Gly As n Lys 195 Pro Thr Leu Leu Al a 275 Cys Giu Val1 As n Gin 355 Ser Gin Gin Leu Ser 435 Gi u His Al a 180 As n Thr Pro Leu Ser 260 Leu Giu Phe Val Leu 340 Th r Leu Leu Leu Cys 420 Thr Tyr Tyr 165 Met Arg Leu Gin Gin 245 Le u His Leu Leu Gi u 325 Gi y Cys Gin Leu Leu 405 Thr Pro Leu 150 Glu Asp Val1 Leu Leu 230 Al a Giu Cys Leu Thr 310 Val1 Gin Arg Gi y Gin 390 Giu Val1 Leu Leu Val Leu Glu As n 215 Lys Al a Met Al a Leu 295 Pro Val Th r Leu Phe 375 Gi u Al a Gin His Gin 455 Thr Trp Val 200 Cys Gi u Ar g Val1 Al a 280 Ar g Leu Va. 1 Ser Leu 360 Thr GI y Al a Ser Phe 440 His Giu Gin 185 Cys Lys Arg Giu As n 265 Al a Lys His Lys Leu 345 Leu Al a Ile Lys Val1 425 Al a Gi y Leu 170 Phe Ser As n Leu Al a 250 Phe Ser Gly Val1 His 330 His Ser Leu S er Al a 410 As n Al a Al a 155 Leu Thr Leu Lys Al a 235 Asp Lys Pro Al a Al a 315 Giu Arg Tyr Gin Leu 395 Gly Cys Gly Asp Val1 Pro Leu Ser 220 Tyr Val1 His Tyr As n 300 Ser Al a Al a Gi y Met 380 Gi y Asp Ar g Tyr Val1 460 Lys Leu Leu 205 Al a Giu Thr Pro Pro 285 Ile Glu Lys Al a Cys 365 Gi y As n Val Asp As n 445 His His His 190 Ser l.i.e Phe Arg Gin 270 Lys As n Lys Val1 Tyr 350 Asp As n Ser Giu Ile 430 Arg Ala Gi y 175 Gi u Tyr Asp Lys Ile 255 Th r Arg Giu Al a As n 335 Cys Pro Giu Gi u Th r 415 Giu Val Lys 160 Gi y Al a Gl y Leu Gi y 240 Lys His Lys Lys His 320 Al a Gi y As n As n Al a 400 Val Gly Ser Asp Lys Gly Gly Leu Val Pro Leu His Asn Ala Cys Ser Tyr Gly His Tyr WO 99/15647 WO 99/ 5647PCT/AU98/00795 Glu Asp Tyr Lys Thr 545 Al a As n Leu His His 625 Lys Leu Leu Pro Al a 705 As n Pro Ser Glu Ser Al a Trp Ile 515 Arg Ile Lys As n Al a 595 Al a Al a As n GI u His 675 Asp Pro Val Ser Ser 755 Al a Thr Giu Lys 500 Cys Asp Gin Gly Cys 580 Gly Asp Ala Al a Al a 660 Gly Leu Pro Arg Pro 740 Phe Ser Gin Leu Thr Leu As n Leu 550 Leu Asp As n As n Tyr 630 Leu Gin Asp Ser Ala 710 Pro Ser Glu Leu Val Val1 Pro Le u Tb r 535 Leu Al a Thr As n Al a 615 Gi y As n Lys Pro Al a 695 Leu Gi y Leu Leu Gi u 775 Ar g Lys Leu Leu 520 Pro Arg Ar g Gin Leu 600 Gin His Al a Gi y Tb r 680 Asp Pro Al a Se r Ser 760 Lys As n His His 505 Gin Leu Gly Val Gly 585 Glu Asp Val1 Thr Ar g* 665 Leu Asp Ser Th r Al a 745 Ser Lys Leu Gly 490 Gi u His Asp Asp Lys 570 Arg Val Lys Asp Asp 650 Thr Lys Val Cys Al a 730 Al a Val1 Gi u Gly Val1 Al a Al a Val1 540 Al a Leu Ser Glu Gly 620 Al a Trp Leu Gin Al a 700 Lys Al a Ser Ser Pro 780 Glu Val Al a Asp 525 Lys Leu Ser Thr Tyr 605 Leu Al a Al a Cys Glu 685 Leu Pro Leu Le u Ser 765 Gi y His Val1 495 Gi y Thr Gi y Asp Pro 575 Leu Leu Pro Leu Th r 655 Leu Gin Thr Val1 Ser 735 As n Gi y Asp Met WO 99/15647 WO 99/ 5647PCT/AU98/00795 785 Ile Phe His Lys Lys Leu Asn Pro 850 Asp Leu 865 Gi~n Ser Phe Asn Leu Trp His Asn 930 Asn Ala 945 Gly Met Asn Gin Lys Asp Val Thr 1010 His Set 1025 Giy Leu Gi u Giu Ile 835 Tyr Ser Th r Arg Giu 915 His Ile Phe Tyr Arg 995 Leu Pro Al a 790 Arg Giu Gin Ile Thr 805 Leu Lys Giu Ile Gly 820 Lys Giy Val Giu Arg 840 Leu Thr Leu Asn Thr 855 Pro Asp Asp Lys Giu 870 Val Arg Giu His Arg 885 Tyr Asn Ile Leu Lys 900 Arg Tyr Thr His Arg 920 Ala Asn Glu Arg Met 935 Ile His Lys Gly Phe 950 Gly Ala Gly Ile Tyr 965 Val Tyr Gly Ile Gly 980 Ser Cys Tyr Ile Cys 1000 Gly Lys Ser Phe Leu 1015 Pro Gly His His Ser 1030 Leu Ala Glu Tyr Val 1045 795 Leu Asp Val Leu Val.

810 Ile Asn Ala Tyr Gly 825 Leu Ile Set Gly Gin 845 Ser Gly Ser Gly Thr 860 Phe Gin Ser Val Giu 875 Asp Gly Gly His Ala 890 Ile Gin Lys Val Cys 905 Arg Lys Giu Val Set 925 Leu Phe His Gly Set 940 Asp Giu Atg His Ala 955 Phe Ala Glu Asn Set 970 Gly Gly Thr Gly Cys 985 His Arg Gin Leu Leu 1005 Gin Phe Set Aia Met 1020 Vai Thr Gly Arg Pro 1035 Ile Tyr Arg Giy Glu 1050 800 Giu Met Gly 815 His Arg His 830 Gin Gly Leu Ile Leu Ile Giu Glu Met 880 G:l-y Gly Ile 895 Asn Lys Lys 9.10 Giu Giu Asn Pro Phe Val Tyr Ile Gly 960 Set Lys Set 975 Pro Val His 990 Phe Cys Arg Lys Met Ala Set Val Asn 1040 Gin Ala Tyt 1055 Gly Met Val 1070 Pro Giu Tyt Leu Ile Tht Tyr Gin Ile Met Arg Pro Giu 1060 1065 Asp Gly

Claims (13)

1. An isolated polynucleotide molecule encoding a candidate effector protein for the Grb7 family of signalling proteins, wherein the polynucleotide molecule comprises a nucleotide sequence having at least 75% sequence identity to that shown as SEQ ID NO: 1.

2. A polynucleotide molecule according to claim 1. wherein the polynucleotide molecule comprises a nucleotide sequence having at least 85% sequence identity to that shown as SEQ ID NO: 1.

3. A polynucleotide molecule according to claim 1, wherein the polynucleotide molecule comprises a nucleotide sequence having at least sequence identity to that shown as SEQ ID NO: 1.

4. A polynucleotide molecule according to claim 1, wherein the polynucleotide molecule comprises a nucleotide sequence which substantially corresponds to that shown as SEQ ID NO: 1.

5. A host cell transformed with a polynucleotide molecule according to any one of the preceding claims.

6. A host cell according to claim 5, wherein the host cell is a mammalian, insect, yeast or bacterial host cell.

7. A method of producing a protein, comprising culturing the host cell of claim 5 or 6 under conditions suitable for the expression of the polynucleotide molecule and optionally recovering the protein.

8. A purified protein encoded by a polynucleotide molecule according to any one of claims 1 to 4.

9. A purified protein according to claim 8, wherein the protein comprises an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 2. WO 99/15647 PCT/AU98/00795 19 A fusion protein comprising an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 2.

11. An antibody or fragment thereof which specifically binds to a protein according to claim 8 or 9.

12. An oligonucleotide probe comprising a nucleotide sequence of at least 12 nucleotides, the oligonucleotide probe comprising a nucleotide sequence such that the oligonucleotide probe selectively hybridises to the polynucleotide molecule of any one of claims 1 to 4 under high stringency conditions.

13. An oligonucleotide probe according to claim 12. wherein the oligonucleotide probe comprises a nucleotide sequence of at least 18 nucleotides.

14. A method of detecting in a sample the presence of an effector protein for the Grb7 family of proteins, the method comprising reacting the sample with an antibody or fragment thereof according to claim 11. A method of detecting in a sample the presence of mRNA encoding an effector protein for the Grb7 family of proteins, the method comprising reacting the sample with an oligonucleotide probe of claim 12 or 13.