Interaction between the Grb10 SH2 Domain and the Insulin Receptor Carboxyl Terminus

Abstract

Grb10 is a member of a recently identified family of adapter proteins that are thought to play a role in receptor tyrosine kinase-mediated signal transduction. We identified and isolated the Grb10 SH2 domain based on its interaction with the intracellular domain of the insulin receptor ␤-subunit using the yeast two-hybrid system. The interaction was specific for the insulin receptor and the insulin-like growth factor-1 receptor, and it required a catalytically active receptor kinase domain and an intact Grb10 SH2 domain. Glutathione S-transferase fusion proteins containing the Grb10 SH2 domain associated in an insulin-dependent manner with insulin receptors from cell lysates and with purified insulin receptors. Co-precipitation experiments revealed the association of cellular Grb10 with hormone-stimulated insulin receptors in cell extracts. The Grb10 SH2 domain did not bind to an insulin receptor lacking 43 amino acids at the carboxyl terminus, and it exhibited highest affinity for a phosphopeptide containing Tyr(P)-1322. Unlike p85 and Syp, which also bind to Tyr(P)-1322, Grb10 was not found to associate with insulin receptor substrate-1. These results suggest that Grb10 is a novel insulin receptor interactive protein and provide direct evidence for an insulin receptor substrate-1-independent function of the insulin receptor carboxyl terminus in protein binding.The insulin receptor plays important roles in metabolism and growth regulation of target tissues (1-3). Upon insulin stimulation the receptor becomes autophosphorylated on at least six or seven tyrosine residues located in the juxtamembrane, kinase, and carboxyl-terminal regions of its ␤-subunit (4 -7). Many signals are relayed through the insulin receptor substrate-1 (IRS-1), 1 which is phosphorylated by the receptor on various tyrosine residues (8). Phosphorylation of a tyrosine at position 960 in the juxtamembrane region of the insulin receptor is essential for signaling through IRS-1 (9, 10). Among the proteins that bind to IRS-1 are the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), Grb2, the tyrosine phosphatase Syp/SHPTP2, and Nck (2). IRS-1 is required for insulin-mediated mitogenesis (11, 12); however, studies using homozygous IRS-1 knockout mice predict important IRS-1-independent insulin signaling pathways (13,14). A promising candidate, the structurally closely related protein IRS-2, was recently identified, which has signaling functions similar to IRS-1 (15, 16). The Shc proteins represent additional receptor tyrosine kinase substrates that mediate p21 ras activation in the mitogenic pathway of insulin action (17). We employed the yeast two-hybrid system to identify signaling mediators for alternative pathways analogous to signals emerging from other receptor tyrosine kinases. Here we describe the identification of Grb10 as an IRS-1-independent interactive protein of the activated insulin receptor, and we define the sites of interaction in the receptor and Grb10. MATERIALS AND METHODSAntibodies-The antibody against the insulin re...

The Grb7 family proteins: structure, interactions with other signaling molecules and potential cellular functions

“…Grb10 has also been shown to associate with several proteins including the EGF receptor (Frantz et al, 1997;He et al, 1998;Ooi et al, 1995), IR (Hansen et al, 1996;Liu and Roth, 1995;Dong et al, 1997;Frantz et al, 1997;O'Neill et al, 1996), Ret (Pandey et al, 1995), IGFR (Dey et al, 1996;He et al, 1998;Morrione et al, 1996;O'Neill et al, 1996), Elk/EphB1 (Stein et al, 1996), PDGF receptor (Frantz et al, 1997), Tek/Tie (Mano et al, 1998), Raf/MEK1 (Nantel et al, 1998), Nedd4 (Morrione et al, 1999), BCR-ABL (Bai et al, 1998), and GH receptor (Moutoussamy et al, 1998). In contrast, Grb14 has been reported to have fewer binding partners including EGFR/PDGFR (Daly et al, 1996), IR (Kasus-Jacobi et al, 1998), Tek/ Tie2 (Jones et al, 1999), FGF receptor (Reilly et al, 2000).…”

Insulin receptor substrates 1 and 2 but not Shc can activate the insulin receptor independent of insulin and induce proliferation in CHO-IR cells

“…In addition to the recruitment of receptor substrates, phosphorylation of the NPEY motif in the juxtamembrane region of the IR is required for insulin-induced receptor endocytosis [5]. The role of the third tyrosine cluster in the COOH-terminal tail of the IR is not fully understood but it seems to be required for fine-tuning of the kinase activity [6], and eventually recruits signalling components with SH2 domains such as Grb10 [7].…”

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

“…The members of this family were originally cloned by interaction with EGF receptor, using the CORT (cloning of receptor target) system (Daly et al, 1996;Margolis, 1994;Ooi et al, 1995), and the use of the yeast two-hybrid technology has emphasized their implication in signal transduction (Daly, 1998). These adapters bind also other tyrosine kinase receptors, like erbB2, Ret, Elk, PDGF receptors, insulin receptors and IGF-1 receptors (Daly et al, 1996;Dey et al, 1996;Hansen et al, 1996;Kasus-Jacobi et al, 1998;Dong et al, 1997;Laviola et al, 1997;Liu and Roth, 1995;Morrione et al, 1997;O'Neill et al, 1996;Pandey et al, 1995Stein et al, 1994Wang et al, 1999;Yokote et al, 1996). In addition, Grb10 and Grb7 can interact with other families of proteins.…”