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

Lily Q. Dong, Sarah Farris, Jeff Christal, Feng Liu

Research output: Contribution to journalArticlepeer-review

38 Scopus citations

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 identity 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.

Original languageEnglish (US)
Pages (from-to)1757-1765
Number of pages9
JournalMolecular Endocrinology
Volume11
Issue number12
DOIs
StatePublished - 1997

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology

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