title: Insulin-dependent interactions of proteins with GLUT4 revealed through stable isotope labeling by amino acids in cell culture (SILAC).
abstract: The insulin-regulated glucose transporter (GLUT4) translocates to the plasma membrane in response to insulin in order to facilitate the postprandial uptake of glucose into fat and muscle cells. While early insulin receptor signaling steps leading to this translocation are well defined, the integration of signaling and regulation of GLUT4 traffic remains elusive. Several lines of evidence suggest an important role for the actin cytoskeleton and for protein-protein interactions in regulating GLUT4 localization by insulin. Here, we applied stable isotope labeling by amino acids in cell culture (SILAC) to identify proteins that interact with GLUT4 in an insulin-regulated manner. Myc-tagged GLUT4 (GLUT4myc) stably expressed in L6 myotubes was immunoprecipitated via the myc epitope from total membranes isolated from basal and insulin-stimulated cells grown in medium containing normal isotopic abundance leucine or deuterated leucine, respectively. Proteins coprecipitating with GLUT4myc were analyzed by liquid chromatography/ tandem mass spectrometry. Of 603 proteins quantified, 36 displayed an insulin-dependent change of their interaction with GLUT4myc of more than 1.5-fold in either direction. Several cytoskeleton-related proteins were elevated in immunoprecipates from insulin-treated cells, whereas components of the ubiquitin-proteasome degradation system were generally reduced. Proteins participating in vesicle traffic also displayed insulin-regulated association. Of cytoskeleton-related proteins, alpha-actinin-4 recovery in GLUT4 immunoprecipitates rose in response to insulin 2.1 +/- 0.5-fold by SILAC and 2.9 +/- 0.8-fold by immunoblotting. Insulin caused GLUT4 and alpha-actinin-4 co-localization as revealed by confocal immunofluorescence microscopy. We conclude that insulin elicits changes in interactions between diverse proteins and GLUT4, and that cytoskeletal proteins, notably alpha-actinin-4, associate with the transporter, potentially to facilitate its routing to the plasma membrane.
authors.affiliation: Center for Experimental BioInformatics (CEBI), Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
authors.forename: Leonard J
authors.initials: LJ
authors.lastname: Foster
authors.forename: Assaf
authors.initials: A
authors.lastname: Rudich
authors.forename: Ilana
authors.initials: I
authors.lastname: Talior
authors.forename: Nish
authors.initials: N
authors.lastname: Patel
authors.forename: Xudong
authors.initials: X
authors.lastname: Huang
authors.forename: L Michelle
authors.initials: LM
authors.lastname: Furtado
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Bilan
authors.forename: Matthias
authors.initials: M
authors.lastname: Mann
authors.forename: Amira
authors.initials: A
authors.lastname: Klip
mesh_terms: D000185:Actinin / Q000032:analysis / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000032:analysis / Q000378:metabolism; D003903:Deuterium / Q000737:chemistry / Q000378:metabolism; D051275:Glucose Transporter Type 4 / Q000032:analysis / Q000378:metabolism*; D047468:Immunoprecipitation; D007328:Insulin / Q000494:pharmacology*; D007553:Isotope Labeling; D007930:Leucine / Q000737:chemistry / Q000378:metabolism; D008840:Microfilament Proteins / Q000032:analysis / Q000378:metabolism; D032448:Myoblasts, Skeletal / Q000187:drug effects* / Q000378:metabolism; D025941:Protein Interaction Mapping; D021381:Protein Transport; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D051381:Rats
chemical_list: C491751:Actn4 protein, rat; D003598:Cytoskeletal Proteins; D051275:Glucose Transporter Type 4; D007328:Insulin; D008840:Microfilament Proteins; D011506:Proteins; C495694:Slc2a4 protein, rat; D000185:Actinin; D003903:Deuterium; D007930:Leucine