Immunological analysis of GLUT4-enriched vesicles. Identification of novel proteins regulated by insulin and diabetes.
In adipocytes and muscle, insulin stimulates the translocation of glucose transporter proteins from an intracellular vesicle pool to the plasma membrane. To study the molecular basis of this process, we used the anti-GLUT4 antibody 1F8 to isolate intracellular vesicles from rat adipocytes that are enriched in the muscle/fat glucose transporter isoform. These vesicles were then used as immunogens to generate monoclonal antibodies against their protein components. We isolated an antibody, 3F8, that recognizes three polypeptides, designated GTV3, migrating in the 36-40-kDa range as analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting. These proteins are enriched in GLUT4-containing vesicles, and the two smallest of the polypeptides recognized by 3F8 translocate to the cell surface in response to insulin. GTV3 proteins are also present in plasma membranes of fat cells and liver as well as in a wide number of tissues, red blood cells being the only exception. In adipocytes from streptozotocin-induced diabetic rats, GTV3 protein levels decrease dramatically and return to normal levels when animals are treated with insulin. The localization of GTV3 in glucose transporter-containing vesicles as well as their wide tissue distribution suggests that these proteins may be involved in vesicle mediated transport and regulated trafficking between membrane compartments.
Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118.
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D000273:Adipose Tissue / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D009124:Muscle Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley
D000911:Antibodies, Monoclonal; D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495693:Slc2a4 protein, mouse; C495694:Slc2a4 protein, rat