Analysis of Grb7 Recruitment by Heregulin-activated erbB Receptors Reveals a Novel Target Selectivity for erbB3

Abstract

Heregulin-mediated activation of particular erbB receptor combinations was used as a model system to investigate the interaction of erbB3 and erbB4 with the adaptor protein growth factor receptor-bound (Grb)7. In human breast cancer cell lines, co-immunoprecipitation of Grb7 with both receptors was detected upon heregulin stimulation. This association was direct and mediated by the Grb7 Src homology (SH)2 domain. Coexpression of erbB2 with erbB3 point mutants was used to map Grb7 binding sites. This demonstrated that tyrosine 1180 and 1243 represent the major and minor sites of Grb7 interaction, respectively. Although these recognition sequences possess an Asn residue at ؉2 relative to the phosphotyrosine and therefore represent potential Grb2 binding sites, phosphopeptide competition and "pull-down" experiments demonstrated that they interact preferentially with the Grb7 versus the Grb2 SH2 domain. Substitution analysis indicated that an Arg residue at ؉3 could act as a selectivity determinant, but the effect was context-dependent. Consequently, the Grb2 and Grb7 SH2 domains possess overlapping, but distinct, specificities. These studies therefore identify Grb7 as an in vivo target of erbB3 and erbB4 and provide an underlying mechanism for the ability of erbB3 to recruit Grb7 and not Grb2, a property unique among erbB receptors.Recently it has become evident that a complex series of interactions governs signaling by the erbB family of receptor tyrosine kinases. This family currently contains four members, the epidermal growth factor receptor (EGFR) 1 or erbB1, erbB2, erbB3, and erbB4, which differ both qualitatively and quantitatively in their signaling potential (1-3) and biological activities (4 -7). A variety of ligands exhibit distinct specificities for one or more of these receptors. For example, the EGFR possesses multiple ligands, including EGF, transforming growth factor-␣, amphiregulin, and betacellulin, but betacellulin also binds erbB4 (8), and both erbB3 and erbB4 provide receptors for the heregulin/neuregulin 1 and neuregulin 2 families of ligands (9 -16). Furthermore, ligand-induced erbB receptor heterodimerization, first detected between the EGFR and erbB2 (17,18), diversifies the signals that can be generated by particular ligands (6,19,20). Importantly, this occurs in a hierarchical fashion and also with directionality (20). In particular, erbB2-containing heterodimers are preferred, and this receptor favors interaction with erbB3. Moreover, heterodimerization is critical for the activity of the erbB3 receptor because it is kinase-impaired and hence reliant on transphosphorylation by other receptors for signal generation (2, 21-23).Dimerization of erbB receptors leads to kinase activation and phosphorylation of their cytoplasmic domains on specific tyrosine residues, thus creating binding sites for signaling molecules containing phosphotyrosine binding or Src homology (SH) 2 domains (24). The latter are conserved noncatalytic regions of approximately 100 amino acids which, along with other modu...

Evidence for an interaction between the insulin receptor and Grb7. A role for two of its binding domains, PIR and SH2

“…Insulin induces the IR-Grb7 interaction in cells expressing physiological levels of the proteins, suggesting that Grb7 is implicated in insulin signaling. Grb7 was already known to bind various tyrosine kinase receptors after ligand activation, like the EGF receptor, erbB2, erbB3, the PDGF receptor and Ret (Fiddes et al, 1998;Margolis et al, 1992;Pandey et al, 1996;Stein et al, 1994;Yokote et al, 1996), and our study suggests that Grb7 preferentially binds the insulin receptor. Grb7 is not a substrate of the insulin receptor tyrosine kinase activity.…”

“…Grb7 was already known to bind di erent tyrosine kinase receptors, like the EGF receptor (Fiddes et al, 1998;Margolis et al, 1992) and Ret (Pandey et al, 1996), but this is the ®rst evidence for an interaction with the insulin receptor (IR). To compare the Grb7/ IR interaction with the interaction of Grb7 with various tyrosine kinase receptors we quantitated them in the two-hybrid system.…”

Combination treatment with Grb7 peptide and Doxorubicin or Trastuzumab (Herceptin) results in cooperative cell growth inhibition in breast cancer cells

“…Since Grb7 associates with many oncogenic protein tyrosine kinases, such as EGFR (Margolis et al, 1992), ErbB2 (Stein et al, 1994), Erbb3 (Fiddes et al, 1998) and ErbB4 (Fiddes et al, 1998), combination therapy with protein tyrosine kinase targeting agents and the Grb7 peptide inhibitor may be a novel therapeutic intervention. Here we show that the Grb7 peptide in combination with Herceptin treatment enhances the inhibitory effect on SK-BR-3 proliferation.…”

“…Grb7 is an especially promising cancer target as the encoding gene maps closely to the ErbB2 gene on human chromosome 17q12 and is found co-amplified and overexpressed in a subset of breast, esophageal and gastric cancers (Stein et al, 1994;Kishi et al, 1997;Tanaka et al, 1997;Fiddes et al, 1998;Kauraniemi et al, 2001;Varis et al, 2002;Andrechek et al, 2003;Bieche et al, 2003). Analysis of chromosome 17q12 in a variety of different breast cancer cell lines Grb7 was found to have a high DNA copy number with a high level of ErbB2 co-expression (Kauraniemi et al, 2001).…”

Coamplification and coexpression of GRB7 and ERBB2 is found in high grade intraepithelial neoplasia and in invasive Barrett's carcinoma

“…5 GRB7 was initially identified as an EGF receptor binding protein. 13 Thereafter, many other binding partners have been reported for GRB7 including ERBB2/Shc, 5 PDGFR, 19 erbB2 receptor, 20 erbB3 receptor, 21 cKit, 22 FAK, 15 Tek/Tie2 23 or c-Kit/SCFR. 22 Most, if not all, of these interactions are through the interaction of the SH2 domain of Grb7 with a phosphotyrosine motif in its binding partners in a signal dependent manner.…”