title: Insulin receptor substrate-1 pleckstrin homology and phosphotyrosine-binding domains are both involved in plasma membrane targeting.
abstract: The localization of insulin receptor substrate (IRS) molecules may be responsible for the differential biological activities of insulin and other peptides such as platelet-derived growth factor. The subcellular localization of IRS-1 is controversial, with some reports suggesting association with the cytoskeleton and other studies reporting membrane localization. In this study, we used immunofluorescence microscopy to define the localization of IRS-1. In the basal state, recombinant IRS-1 was localized predominantly in the cytoplasm. In response to insulin, recombinant IRS-1 translocated to the plasma membrane. We have also studied the localization of green fluorescent protein (GFP) fusion proteins. Unlike native IRS-1, a fusion protein containing 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 pleckstrin homology and phosphotyrosine-binding domains), this fusion protein was targeted to the plasma membrane. Mutations of phosphoinositide-binding sites in both the pleckstrin homology and phosphotyrosine-binding domains significantly reduced the ability of Myc-tagged IRS-1 to translocate to the plasma membrane following insulin stimulation. However, these mutations did not cause a statistically significant impairment of tyrosine phosphorylation in response to insulin. This raises the possibility that IRS-1 tyrosine phosphorylation may occur prior to plasma membrane translocation.
authors.affiliation: Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1758, USA.
authors.forename: A R
authors.initials: AR
authors.lastname: Jacobs
authors.forename: D
authors.initials: D
authors.lastname: LeRoith
authors.forename: S I
authors.initials: SI
authors.lastname: Taylor
mesh_terms: D000818:Animals; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism*; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins; D007328:Insulin / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D001798:Blood Proteins; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D008164:Luminescent Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C039077:platelet protein P47; D049452:Green Fluorescent Proteins; D019000:Phosphotyrosine