Grb10 Interacts Differentially with the Insulin Receptor, Insulin-like Growth Factor I Receptor, and Epidermal Growth Factor Receptor via the Grb10 Src Homology 2 (SH2) Domain and a Second Novel Domain Located between the Pleckstrin Homology and SH2 Domains

Abstract

The Grb10 protein appears to be an adapter protein of unknown function that has been implicated in insulin receptor (IR) signaling. The interaction of this protein with the IR has been shown to be mediated in part by the Src homology 2 (SH2) domain of Grb10. Here we demonstrate the existence of a second novel domain within Grb10 that interacts with the IR and insulin-like growth factor receptor in a kinase-dependent manner. This domain was localized to a region of approximately 50 amino acids, and we term it the BPS domain to denote its location between the PH and SH2 domains. The BPS domain does not bear any obvious resemblance to other known protein interaction domains but is highly conserved among the Grb10-related proteins Grb7 and Grb14. We show that the BPS domain interaction is dependent upon receptor tyrosine kinase activity. Furthermore, interaction of the BPS domain requires the kinase domain of the IR, since mutation of the paired tyrosine residues (Y1150F/Y1151F) within the IR activation loop dramatically reduced the interaction. Last, our data suggest that the presence of two distinct protein interaction domains may help to determine the specificity by which Grb10 interacts with different receptors. Specifically, the IR, which appears to interact most strongly with Grb10, interacts well with both the SH2 and BPS domains. Conversely, the insulin-like growth factor receptor and EGFR, which interact less avidly with Grb10, interact well only with the BPS domain or the SH2 domain, respectively. In summary, our findings demonstrate the existence of a previously unidentified tyrosine kinase activity-dependent binding domain located between the Pleckstrin homology and SH2 domains of Grb10.The binding of hormones to their receptor tyrosine kinases triggers receptor tyrosine autophosphorylation (1). In the case of the insulin receptor (IR) 1 and insulin-like growth factor I receptor (IGFIR), phosphorylation of multiple tyrosine residues leads to activation of receptor kinase activity (2) and, in addition, creates docking sites for downstream adaptor proteins such as SHC, insulin receptor substrate-1 (IRS-1), and insulin receptor substrate-2 (IRS-2) (3-5). Phosphorylation of IRS-1 upon multiple tyrosines results in its association with Src homology 2 (SH2) domain-containing proteins including Grb2, Syp, NCK, and the p85 subunit of phosphatidylinositol 3-kinase, thus activating various signaling cascades (6 -10). Tyrosyl phosphorylation of SHC also leads to its interaction with Grb2 and mediates the activation of the guanine nucleotide exchange factor SOS and subsequent activation of the Ras signaling pathway (11-14).SHC, IRS-1, and IRS-2 proteins have been shown to interact with the IR or the related IGFIR through phosphotyrosine binding domains, which recognize the phosphorylated NPXY motif in the juxtamembrane domains of receptors (3,5,15). In addition, a second poorly understood receptor binding domain has also been identified within the central domain of IRS-2 (5, 16). Both SHC and IRS-1 have been implica...

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

“…Mutation of the tyrosyl residues in the juxtamembrane or the C-terminus regions did not alter the IR ± PIR interaction. These e ects are similar to those observed with the PIR of Grb14 and Grb10 (He et al, 1998;Kasus-Jacobi et al, 1998). This suggests that the PIR of the three proteins display similar binding speci®cities, and speci®cally interact with the phosphorylated kinase loop of the insulin receptors.…”

“…It has been reported previously for Grb10 and Grb14 that both SH2 and PIR (also called BPS) domains are able to mediate the interaction with the insulin receptor (He et al, 1998;Kasus-Jacobi et al, 1998). To assess if the same domains of Grb7 bind the Starved rats were injected with saline or insulin.…”

“…Di erent results were obtained concerning the respective role of the PIR and SH2 domains in the interaction with the insulin receptors (He et al, 1998;Kasus-Jacobi et al, 1998). To decipher the implication of the PIR and SH2 domains of Grb7 and Grb14 in the insulin receptor binding, we used the GST pulldown experiments to compare the amount of insulin receptors retained by the di erent domains.…”

“…The SH2 domain is responsible for the interaction of these Grbs proteins with all their identi®ed partners. However, it has recently been shown that the region located between the PH and the SH2 domains of Grb10 and Grb14 is a new binding domain, which has been called BPS or PIR and is implicated in the receptor tyrosine kinase binding (He et al, 1998;Kasus-Jacobi et al, 1998). The relative role of the BPS/PIR and of the SH2 domains depends on the Grb isoform and the tyrosine kinase receptor considered.…”

“…The relative role of the BPS/PIR and of the SH2 domains depends on the Grb isoform and the tyrosine kinase receptor considered. In the case of Grb10, the SH2 domain mediates the EGF receptor interaction, while the BPS/ PIR is responsible for the IGF-1 receptor binding, and both domains can associate with the insulin receptor (He et al, 1998). Interestingly, the interaction between Grb14 and the insulin receptor is mainly due to the PIR, whereas the SH2 domain is dispensable for it (Kasus-Jacobi et al, 1998).…”