title: Intracellular localization of phosphatidylinositide 3-kinase and insulin receptor substrate-1 in adipocytes: potential involvement of a membrane skeleton.
abstract: Phosphatidylinositide (PI) 3-kinase binds to tyrosyl-phosphorylated insulin receptor substrate-1 (IRS-1) in insulin-treated adipocytes, and this step plays a central role in the regulated movement of the glucose transporter, GLUT4, from intracellular vesicles to the cell surface. PDGF, which also activates PI 3-kinase in adipocytes, has no significant effect on GLUT4 trafficking in these cells. We propose that this specificity may be mediated by differential localization of PI 3-kinase in response to insulin versus PDGF activation. Using subcellular fractionation in 3T3-L1 adipocytes, we show that insulin- and PDGF-stimulated PI 3-kinase activities are located in an intracellular high speed pellet (HSP) and in the plasma membrane (PM), respectively. The HSP is also enriched in IRS-1, insulin-stimulated tyrosyl-phosphorylated IRS-1 and intracellular GLUT4-containing vesicles. Using sucrose density gradient sedimentation, we have been able to segregate the HSP into two separate subfractions: one enriched in IRS-1, tyrosyl-phosphorylated IRS-1, PI 3-kinase as well as cytoskeletal elements, and another enriched in membranes, including intracellular GLUT4 vesicles. Treatment of the HSP with nonionic detergent, liberates all membrane constituents, whereas IRS-1 and PI 3-kinase remain insoluble. Conversely, at high ionic strength, membranes remain intact, whereas IRS-1 and PI 3-kinase become freely soluble. We further show that this IRS-1-PI 3-kinase complex exists in CHO cells overexpressing IRS-1 and, in these cells, the cytosolic pool of IRS-1 and PI 3-kinase is released subsequent to permeabilization with Streptolysin-O, whereas the particulate fraction of these proteins is retained. These data suggest that IRS-1, PI 3-kinase, as well as other signaling intermediates, may form preassembled complexes that may be associated with the actin cytoskeleton. This complex must be in close apposition to the cell surface, enabling access to the insulin receptor and presumably other signaling molecules that somehow confer the absolute specificity of insulin signaling in these cells.
authors.affiliation: The Center for Molecular and Cellular Biology, and the Department of Physiology and Pharmacology, University of Queensland, Brisbane, Queensland, 4072, Australia.
authors.forename: S F
authors.initials: SF
authors.lastname: Clark
authors.forename: S
authors.initials: S
authors.lastname: Martin
authors.forename: A J
authors.initials: AJ
authors.lastname: Carozzi
authors.forename: M M
authors.initials: MM
authors.lastname: Hill
authors.forename: D E
authors.initials: DE
authors.lastname: James
mesh_terms: D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D002498:Centrifugation; D006224:Cricetinae; D003902:Detergents / Q000494:pharmacology; D051380:GRB2 Adaptor Protein; D051275:Glucose Transporter Type 4; D005960:Glucosides / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D051379:Mice; D008854:Microscopy, Electron; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins; D017830:Octoxynol / Q000494:pharmacology; D009994:Osmolar Concentration; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000378:metabolism / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism*; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013347:Subcellular Fractions
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D003902:Detergents; D051380:GRB2 Adaptor Protein; D051275:Glucose Transporter Type 4; D005960:Glucosides; C495949:Grb2 protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D010750:Phosphoproteins; D010982:Platelet-Derived Growth Factor; D011506:Proteins; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; C495693:Slc2a4 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; C018619:octyl-beta-D-glucoside; D017830:Octoxynol; D019869:Phosphatidylinositol 3-Kinases; D011972:Receptor, Insulin