PHOSPHOTYROSINE-DEPENDENT INTERACTION OF SHC AND INSULIN-RECEPTOR SUBSTRATE-1 WITH THE NPEY MOTIF OF THE INSULIN-RECEPTOR VIA A NOVEL NON-SH2 DOMAIN

The SHC proteins have been implicated in insulin receptor (IR) signali ng. In this study, we used the sensitive two-hybrid assay of protein-p rotein interaction to demonstrate that SHC interacts directly with the IR. The interaction is mediated by SEC amino acids 1 to 238 and is th erefore independent of the Src homology 2 domain. The interaction is d ependent upon IR autophosphorylation, since the interaction is elimina ted by mutation of the IR ATP-binding site. In addition, mutational an alysis of the Asn-Pro-Glu-Tyr (NPEY) motif within the juxtamembrane do main of the IR showed the importance of the Asn, Pro, and Tyr residues to both SHC and IR substrate 1 (IRS-1) binding. We conclude that SHC interacts directly with the IR and that phosphorylation of Tyr-960 wit hin the IR juxtamembrane domain is necessary for efficient interaction . This interaction is highly reminiscent of that of IRS-1 with the IR, and we show that the SHC IR-binding domain can substitute for that of IRS-1 in yeast and COS cells. We identify a homologous region within the IR-binding domains of SHC and IRS-1, which we term the SAIN (SHC a nd IRS-1 NPXY-binding) domain, which may explain the basis of these in teractions. The SAIN domain appears to represent a novel motif which i s able to interact with autophosphorylated receptors such as the IR.