SITE-DIRECTED MUTAGENESIS AND YEAST 2-HYBRID STUDIES OF THE INSULIN AND INSULIN-LIKE GROWTH-FACTOR-I RECEPTORS - THE SRC HOMOLOGY-2 DOMAIN-CONTAINING PROTEIN HGRB10 BINDS TO THE AUTOPHOSPHORYLATED TYROSINE RESIDUES IN THE KINASE DOMAIN OF THE INSULIN-RECEPTOR

To characterize the structural basis for the interaction between hGrb1 0 and the insulin receptor and the insulin-like growth factor-1 recept or, different mutant receptors containing a segment of deletion in eit her the juxtamembrane domain or in the C terminus of the receptors, or containing tyrosine-to-phenylalanine point mutations in these regions of the insulin receptor, were generated. Yeast two-hybrid and in vitr o binding studies of the interaction between the mutant receptors and hGrb10 revealed that tyrosine residues in these regions are not essent ial for the binding of hGrb10. To further identify the binding site fo r hGrb10, all conserved tyrosine residues in the kinase domain of the insulin receptor were replaced with either phenylalanine or alanine by site-directed mutagenesis. Mutations of all tyrosine residues in this region, except at positions 1162/1163, did not inhibit the binding of the receptor to hGrb10. The binding of the Src homology 2 domain of h Grb10 to the receptors was significantly enhanced in the presence of a n intact pleckstrin homology domain. Our findings suggest that, unlike other Src homology 2 domain-containing proteins, hGrb10 binds to the autophosphorylated tyrosine residues in the kinase domain of the insul in receptor, and the pleckstrin homology domain plays an important rol e in hGrb10/receptor interaction. Because the autophosphorylated tyros ine residues are critical for the autophosphorylation and kinase activ ity of the receptor, the binding of hGrb10 at these sites may suggest a role for the protein in the transduction or regulation of insulin re ceptor signaling.