Dietary glucose is taken up by skeletal muscle through GLUT4 (glucose transporter 4). We recently identified by MS proteins displaying insulin-dependent co-precipitation with Myc-tagged GLUT4 from L6 myotubes, including GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and HKII (hexokinase-II). In the present paper we explored whether GAPDH and HKII interact directly with cytoplasmic regions of GLUT4 and their possible inter-relationship. Endogenous and recombinant GAPDH and HKII bound to a chimeric protein linearly encoding all three cytosolic domains of GLUT4 [GST (glutathione-transferase)–GLUT4-cyto]. Both proteins bound to a lesser extent the middle cytosolic loop but not individual N- or C-terminal domains of GLUT4. Purified GAPDH and HKII competed for binding to GST–GLUT4-cyto; ATP increased GAPDH binding and decreased HKII binding to this construct. The physiological significance of the GAPDH–GLUT4 interaction was explored by siRNA (small interfering RNA)-mediated GAPDH knockdown. Reducing GAPDH expression by 70% increased HKII co-precipitation with GLUT4–Myc from L6 cell lysates. GAPDH knockdown had no effect on surface-exposed GLUT4–Myc in basal or insulin-stimulated cells, but markedly and selectively diminished insulin-stimulated 3-O-methyl glucose uptake and GLUT4–Myc photolabelling with ATB-BMPA {2-N-[4-(1-azitrifluoroethyl)benzoyl]-1,3-bis-(D-mannos-4-yloxy)-2-propylamine}, suggesting that the exofacial glucose-binding site was inaccessible. The results show that GAPDH and HKII reciprocally interact with GLUT4 and suggest that these interactions regulate GLUT4 intrinsic activity in response to insulin.

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