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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 beta-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.
Highlights
The insulin receptor plays important roles in metabolism and growth regulation of target tissues [1,2,3]
Many signals are relayed through the insulin receptor substrate-1 (IRS-1),1 which is phosphorylated by the receptor
We identified a truncated sequence of Grb10 lacking 30 amino acids of the Src homology-2 (SH2) domain at the carboxyl terminus when we randomly analyzed transformants from the two-hybrid screen
Summary
The insulin receptor plays important roles in metabolism and growth regulation of target tissues [1,2,3]. 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), which is phosphorylated by the receptor. Phosphorylation of a tyrosine at position 960 in the juxtamembrane region of the insulin receptor is essential for signaling through IRS-1 [9, 10]. The Shc proteins represent additional receptor tyrosine kinase substrates that mediate p21ras 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. We describe the identification of Grb as an IRS-1-independent interactive protein of the activated insulin receptor, and we define the sites of interaction in the receptor and Grb
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