title: Site-directed mutagenesis and yeast two-hybrid studies of the insulin and insulin-like growth factor-1 receptors: the Src homology-2 domain-containing protein hGrb10 binds to the autophosphorylated tyrosine residues in the kinase domain of the insulin receptor.
abstract: To characterize the structural basis for the interaction between hGrb10 and the insulin receptor and the insulin-like growth factor-1 receptor, different mutant receptors containing a segment of deletion in either 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 vitro binding studies of the interaction between the mutant receptors and hGrb10 revealed that tyrosine residues in these regions are not essential for the binding of hGrb10. To further identify the binding site for 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 hGrb10 to the receptors was significantly enhanced in the presence of an 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 insulin receptor, and the pleckstrin homology domain plays an important role in hGrb10/receptor interaction. Because the autophosphorylated tyrosine residues are critical for the autophosphorylation and kinase activity of the receptor, the binding of hGrb10 at these sites may suggest a role for the protein in the transduction or regulation of insulin receptor signaling.
authors.affiliation: Department of Pharmacology, The University of Texas Health Science Center at San Antonio 78284-7764, USA.
authors.forename: L Q
authors.initials: LQ
authors.lastname: Dong
authors.forename: S
authors.initials: S
authors.lastname: Farris
authors.forename: J
authors.initials: J
authors.lastname: Christal
authors.forename: F
authors.initials: F
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001798:Blood Proteins / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D066246:ErbB Receptors / Q000235:genetics*; D051383:GRB10 Adaptor Protein; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism*; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D015003:Yeasts; D018909:src Homology Domains / Q000235:genetics*
chemical_list: D001798:Blood Proteins; D010750:Phosphoproteins; D011506:Proteins; C039077:platelet protein P47; D051383:GRB10 Adaptor Protein; D019000:Phosphotyrosine; D007334:Insulin-Like Growth Factor I; D066246:ErbB Receptors; D011972:Receptor, Insulin