TYROSINE PHOSPHORYLATION OF INSULIN-RECEPTOR SUBSTRATE-1 IN-VIVO DEPENDS UPON THE PRESENCE OF ITS PLECKSTRIN HOMOLOGY REGION

To characterize the structural basis for the interactions between the insulin receptor (IR) and its major substrate, insulin receptor substr ate-1 (IRS-1), a segment of the NH2-terminal region of IRS-1 (Pro(5)-P ro(65)) was deleted. This region contains the first four conserved box es of a pleckstrin homology (PH) domain, located at the NH2-terminal p art of IRS-1. COS-7 cells were then cotransfected with the genes codin g for IR and a wild-type (WT) or a mutated form of IRS-1. IRS-1(Delta PH) underwent significantly reduced insulin-dependent tyrosine phospho rylation compared with WT IRS-1. The reduced in. vivo tyrosine phospho rylation of IRS-1(Delta PH) was accompanied by reduced association bet ween IRS-1(Delta PH) and its downstream effector p85 regulatory subuni t of phosphatidylinositol-3 kinase. In contrast, both WT IRS-1 and IRS -1(Delta PH) underwent comparable in insulin-dependent tyrosine phosph orylation in vitro when incubated with partially purified insulin rece ptor ki nase. These findings suggest that the overall structure of IRS -1 is not altered by deletion of its PH domain and that the PR domain is not the main site for protein-protein interactions between the insu lin receptor and IRS-1, at least in vitro. In conclusion, the PH regio n might facilitate in vivo binding of IRS-1 to membrane phospholipids or other cellular constituents in close proximity to the IR, whereas t he actual interactions with the IR are presumably mediated through oth er domains of the IRS-1 molecule. This could account for the fact that partial deletion of the PH domain selectively impairs the in vivo int eractions between the insulin receptor and IRS-1, whereas their in vit ro interactions remain unaffected.