Myosin Va mediates Rab8A-regulated GLUT4 vesicle exocytosis in insulin-stimulated muscle cells.
Rab-GTPases are important molecular switches regulating intracellular vesicle traffic, and we recently showed that Rab8A and Rab13 are activated by insulin in muscle to mobilize GLUT4-containing vesicles to the muscle cell surface. Here we show that the unconventional motor protein myosin Va (MyoVa) is an effector of Rab8A in this process. In CHO-IR cell lysates, a glutathione S-transferase chimera of the cargo-binding COOH tail (CT) of MyoVa binds Rab8A and the related Rab10, but not Rab13. Binding to Rab8A is stimulated by insulin in a phosphatidylinositol 3-kinase-dependent manner, whereas Rab10 binding is insulin insensitive. MyoVa-CT preferentially binds GTP-locked Rab8A. Full-length green fluorescent protein (GFP)-MyoVa colocalizes with mCherry-Rab8A in perinuclear small puncta, whereas GFP-MyoVa-CT collapses the GTPase into enlarged perinuclear depots. Further, GFP-MyoVa-CT blocks insulin-stimulated translocation of exofacially myc-tagged GLUT4 to the surface of muscle cells. Mutation of amino acids in MyoVa-CT predicted to bind Rab8A abrogates both interaction with Rab8A (not Rab10) and inhibition of insulin-stimulated GLUT4myc translocation. Of importance, small interfering RNA-mediated MyoVa silencing reduces insulin-stimulated GLUT4myc translocation. Rab8A colocalizes with GLUT4 in perinuclear but not submembrane regions visualized by confocal total internal reflection fluorescence microscopy. Hence insulin signaling to the molecular switch Rab8A connects with the motor protein MyoVa to mobilize GLUT4 vesicles toward the muscle cell plasma membrane.
Cell Biology Program, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada.
Yi
Y
Sun
Tim T
TT
Chiu
Kevin P
KP
Foley
Philip J
PJ
Bilan
Amira
A
Klip
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects*; D055785:Gene Knockdown Techniques; D020868:Gene Silencing / Q000187:drug effects; D051275:Glucose Transporter Type 4 / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D032342:Muscle Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D032446:Myoblasts / Q000187:drug effects / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000737:chemistry / Q000378:metabolism*; D024701:Myosin Type V / Q000737:chemistry / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D022142:Secretory Vesicles / Q000187:drug effects / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
D051275:Glucose Transporter Type 4; D006160:Guanosine Triphosphate; D007328:Insulin; D018995:Myosin Heavy Chains; D024701:Myosin Type V; D019869:Phosphatidylinositol 3-Kinases; D020691:rab GTP-Binding Proteins; C513081:Myo5a protein, rat; C495694:Slc2a4 protein, rat; C530052:Rab8a protein, rat