Endocytosis of the glucose transporter GLUT4 is mediated by the GTPase dynamin.
To study the role of the GTPase dynamin in GLUT4 intracellular recycling, we have overexpressed dynamin-1 wild type and a GTPase-negative mutant (K44A) in primary rat adipose cells. Transfection was accomplished by electroporation using an hemagglutinin (HA)-tagged GLUT4 as a reporter protein. In cells expressing HA-GLUT4 alone, insulin results in an approximately 7-fold increase in cell surface anti-HA antibody binding. Studies with wortmannin indicate that the kinetics of HA-GLUT4-trafficking parallel those of the native GLUT4 and in addition, that newly synthesized HA-GLUT4 goes to the plasma membrane before being sorted into the insulin-responsive compartments. Short term (4 h) coexpression of dynamin-K44A and HA-GLUT4 increases the amount of cell surface HA-GLUT4 in both the basal and insulin-stimulated states. Under conditions of maximal expression of dynamin-K44A (24 h), most or all of the intracellular HA-GLUT4 appears to be present on the cell surface in the basal state, and insulin has no further effect. Measurements of the kinetics of HA-GLUT4 endocytosis show that dynamin-K44A blocks internalization of the glucose transporters. In contrast, expression of dynamin wild type decreases the amount of cell surface HA-GLUT4 in both the basal and insulin-stimulated states. These data demonstrate that the endocytosis of GLUT4 is largely mediated by processes which require dynamin.
Al-Hasani
H
H
Experimental Diabetes, Metabolism, and Nutrition Section, Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA. hadi@helix.nih.gov
Hinck
C S
CS
Cushman
S W
SW
D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D034284:Dynamin I; D034281:Dynamins; D018274:Electroporation; D004705:Endocytosis; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D051275:Glucose Transporter Type 4; D006388:Hemagglutinins / Q000737:chemistry; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D009124:Muscle Proteins; D051381:Rats
D051275:Glucose Transporter Type 4; D006388:Hemagglutinins; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495694:Slc2a4 protein, rat; D034284:Dynamin I; D020558:GTP Phosphohydrolases; D034281:Dynamins