Glucose transporter 4 can be inserted in the membrane without exposing its catalytic site for photolabeling from the medium.
Insulin stimulates the production of PI(3,4,5)P(3) in muscle cells, and this is required to stimulate GLUT4 fusion with the plasma membrane. Introduction of exogenous PI(3,4,5)P(3) to muscle cells recapitulates insulin's effects on GLUT4 fusion with the plasma membrane, but not glucose uptake. This study aims to explore the mechanism behind this difference. In L6-GLUT4myc muscle cells, the availability of the GLUT4 intracellular C-terminus and extracellular myc epitopes for immunoreactivity on plasma membrane lawns was detected with the corresponding antibody. The availability of the active site of GLUT4 from extracellular medium was assessed by affinity photolabeling with the cell impermeant compound Bio-LC-ATB-BMPA. 100 nmol/L insulin and 10 mumol/L PI(3,4,5)P(3) caused myc signal gain on the plasma membrane lawns by 1.64-fold and 1.58-fold over basal, respectively. Insulin, but not PI(3,4,5)P(3), increased photolabeling of GLUT4 and immunolabeling with C-terminus antibody by 2.47-fold and 2.04-fold over basal, respectively. Upon insulin stimulation, the C-terminus signal gain was greater than myc signal gain (2.04-fold vs. 1.64-fold over basal, respectively) in plasma membrane lawns. These results indicate that (i) PI(3,4,5)P(3) does not make the active site of GLUT4 available from the extracellular surface despite causing GLUT4 fusion with the plasma membrane; (ii) the availability of the active site of GLUT4 from the extracellular medium and availability of the C-terminus from the cytosolic site are correlated; (iii) in addition to stimulating GLUT4 translocation, insulin stimulation displaces a protein which masks the GLUT4 C-terminus. We propose that a protein which masks the C-terminus also prevents the active site from being available for photolabelling and possibly glucose uptake after treatment with PI(3,4,5)P(3).
Niu
WenYan
W
Department of Immunology, Tianjin Medical University, Tianjin 300070, China. wniu@tijmu.edu.cn
Ishiki
Manabu
M
Bilan
Philip J
PJ
Yao
Zhi
Z
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation; D020134:Catalytic Domain; D002462:Cell Membrane / Q000378:metabolism; D051275:Glucose Transporter Type 4 / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology; D007739:L Cells; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D010716:Phosphatidylinositols / Q000494:pharmacology; D051381:Rats
D051275:Glucose Transporter Type 4; D007328:Insulin; D010446:Peptide Fragments; D010716:Phosphatidylinositols; C118303:phosphoinositide-3,4,5-triphosphate