RAD, A NOVEL RAS-RELATED GTPASE, INTERACTS WITH SKELETAL-MUSCLE BETA-TROPOMYOSIN

Rad, a prototypic member of a subfamily of Ras-related GTPases, is ove rexpressed in skeletal muscle of type II diabetic humans. By expressio n screening of mouse embryo and human skeletal muscle cDNA libraries, we found that Rad interacted with skeletal muscle beta-tropomyosin. In the mouse skeletal muscle cell line C2C12, this interaction was signi ficantly increased by the calcium ionophore A23187. A23187 also caused a time and concentration-dependent decrease in total cellular Rad wit h increased interaction between tropomyosin and Rad in the detergent-s oluble fraction and the appearance of Rad in the cytoskeleton. In C2C1 2 cells stably overexpressing a putative dominant negative mutant of R ad (S105N), there was an increase in the amount of tropomyosin in Rad immunoprecipitates. In cells overexpressing wild type Rad, much of Rad was associated with the cytoskeleton and was no longer responsive to A23187. In far-Western blotting and guanine nucleotide saturation stud ies, GDP-Rad bound to tropomyosin far better than GTP-Rad. We conclude that Rad interacts with skeletal muscle beta-tropomyosin and the cyto skeleton in a guanine nucleotide-dependent manner. These data suggest that Rad may be involved in skeletal muscle motor function and cytoske letal organization.