Insulin receptor substrate-1 pleckstrin homology and phosphotyrosine-binding domains are both involved in plasma membrane targeting

The localization of insulin receptor substrate (IRS) molecules may be respo nsible for the differential biological activities of insulin and other pept ides such as platelet-derived growth factor. The subcellular localization o f IRS-1 is controversial, with some reports suggesting association with the cytoskeleton and other studies reporting membrane localization. In this st udy, we used immunofluorescence microscopy to define the localization of IR S-1. In the basal state, recombinant IRS-1 was localized predominantly in t he cytoplasm. In response to insulin, recombinant IRS-1 translocated to the plasma membrane. We have also studied the localization of green fluorescen t protein (GFP) fusion proteins. Unlike native IRS-1, a fusion protein cont aining GFP plus full-length IRS-1 appeared to localize in inclusion bodies. In contrast, when GFP was fused to the N terminus of IRS-1 (i.e. the pleck strin homology and phosphotyrosine-binding domains), this fusion protein wa s targeted to the plasma membrane. Mutations of phosphoinositide-binding si tes in both the pleckstrin homology and phosphotyrosine-binding domains sig nificantly reduced the ability of Myc-tagged IRS-1 to translocate to the pl asma membrane following insulin stimulation. However, these mutations did n ot cause a statistically significant impairment of tyrosine phosphorylation in response to insulin. This raises the possibility that IRS-1 tyrosine ph osphorylation may occur prior to plasma membrane translocation.