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 substrate-1 (IRS-1), a segment of the NH2-terminal region of IRS-1 (Pro(5)-Pro(65)) was deleted. This region contains the first four conserved boxes of a pleckstrin homology (PH) domain, located at the NH2-terminal part of IRS-1. COS-7 cells were then cotransfected with the genes coding for IR and a wild-type (WT) or a mutated form of IRS-1. IRS-1(Delta PH) underwent significantly reduced insulin-dependent tyrosine phosphorylation compared with WT IRS-1. The reduced in. vivo tyrosine phosphorylation of IRS-1(Delta PH) was accompanied by reduced association between IRS-1(Delta PH) and its downstream effector p85 regulatory subunit of phosphatidylinositol-3 kinase. In contrast, both WT IRS-1 and IRS-1(Delta PH) underwent comparable in insulin-dependent tyrosine phosphorylation in vitro when incubated with partially purified insulin receptor 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 insulin receptor and IRS-1, at least in vitro. In conclusion, the PH region might facilitate in vivo binding of IRS-1 to membrane phospholipids or other cellular constituents in close proximity to the IR, whereas the actual interactions with the IR are presumably mediated through other domains of the IRS-1 molecule. This could account for the fact that partial deletion of the PH domain selectively impairs the in vivo interactions between the insulin receptor and IRS-1, whereas their in vitro interactions remain unaffected.