Inhibition of hGrb10 binding to the insulin receptor by functional domain-mediated oligomerization

hGrb10 is a newly identified Src homology 2 (SH2) and pleckstrin homology (PH) domain-containing protein that binds to autophosphorylated receptor tyrosine kinases, including the insulin and insulin-like growth factor receptors. To identify potential downstream proteins that interact with hGrb10, we screened a yeast two-hybrid cDNA library using the full-length hGrb10 gamma as bait. A fragment of hGrb10, which included the IPS (insert between the PH and SH2 domain) and the SH2 domains, was found to bind with high affinity to the full-length protein. The interaction between the IPSI SH2 domain and the full-length hGrb10 was further confirmed by in, vitro glutathione S-transferase fusion protein binding studies. Gel filtration assays showed that hGrb10 underwent tetramerization in mammalian cells. The interaction involved at least two functional domains, the IPS/SH2 region and the PH domain, both of which interacted with the NH2-terminal amino acid sequence of hGrb10 gamma (hGrb10 gamma Delta C, residues 4-414), Competition studies showed that hGrb10 gamma Delta C inhibited the binding of hGrb10 to the tyrosine-phosphorylated insulin receptor, suggesting that this region may play a regulatory role in hGrb10/insulin receptor interaction. We present a model for hGrb10 tetramerization and its potential role in receptor tyrosine kinase signal transduction.