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THE CALMODULIN-BINDING DOMAIN OF AS160 REGULATES CONTRACTION- BUT NOT INSULIN-STIMULATED GLUCOSE UPTAKE IN SKELETAL MUSCLE

Henning F. Kramer^*, Eric B. Taylor^*, Carol A. Witczak^*, Nobuharu Fujii^*, Michael F. Hirshman^*, and Laurie J. Goodyear^*,,

*Joslin Diabetes Center Research Division, Metabolism, Boston, MA, USA
Brigham and Women's Hospital and Harvard Medical School, Medicine, Boston, MA, USA

Objective.: Insulin and contraction increase skeletal muscle glucose uptake through distinct and additive mechanisms. However, recent reports have demonstrated that both signals converge on AS160, a protein that regulates GLUT4 translocation. Although AS160 phosphorylation is believed to be the primary factor affecting its activity, AS160 also possesses a calmodulin-binding domain (CBD). This raises the possibility that contraction-stimulated increases in Ca²⁺/calmodulin could also modulate AS160 function.

Research Design and Methods.: To evaluate the AS160 CBD in skeletal muscle, empty-vector, wild-type or CBD-mutant AS160 cDNAs were injected into mouse muscles followed by in vivo electroporation. One-week later, AS160 was overexpressed by 14-fold over endogenous protein.

Results.: Immunoprecipitates of wild-type and CBD-mutant AS160 were incubated with biotinylated-calmodulin in the presence of Ca²⁺. Wild-type AS160, but not the CBD-mutant AS160, associated with calmodulin. Next, we measured insulin- and contraction-stimulated glucose uptake in vivo. Compared to empty-vector and wild-type AS160, insulin-stimulated glucose uptake were not altered in muscles expressing CBD-mutant AS160. In contrast, contraction-stimulated glucose uptake was significantly decreased in CBD-mutant expressing muscles. This inhibitory effect on glucose uptake was not associated with aberrant contraction-stimulated AS160 phosphorylation. Interestingly, AS160 expressing both calmodulin-binding and Rab-GAP domain point mutations (CBD+R/K) fully restored contraction-stimulated glucose uptake.

Conclusions.: Our results suggest that the AS160 CBD directly regulates contraction-induced glucose uptake in mouse muscle, and that calmodulin provides an additional means of modulating AS160 Rab-GAP function independent of phosphorylation. These findings define a novel AS160 signaling component, unique to contraction and not insulin, leading to glucose uptake in skeletal muscle.

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