GAPDH binds GLUT4 reciprocally to hexokinase-II and regulates glucose transport activity.
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.
Zaid
Hilal
H
Program in Cell Biology, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8.
Talior-Volodarsky
Ilana
I
Antonescu
Costin
C
Liu
Zhi
Z
Klip
Amira
A
D000818:Animals; D001692:Biological Transport / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D005947:Glucose / Q000378:metabolism*; D051275:Glucose Transporter Type 4 / Q000235:genetics / Q000378:metabolism*; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics / Q000378:metabolism*; D006593:Hexokinase / Q000235:genetics / Q000378:metabolism*; D047468:Immunoprecipitation; D007328:Insulin / Q000378:metabolism; D051379:Mice; D016133:Polymerase Chain Reaction; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics / Q000502:physiology
D051275:Glucose Transporter Type 4; D007328:Insulin; D034741:RNA, Small Interfering; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D006593:Hexokinase; D005947:Glucose