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 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.
Jacobs
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AR
Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1758, USA.
LeRoith
D
D
Taylor
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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
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