Rad, a novel Ras-related GTPase, interacts with skeletal muscle β-tropomyosin

Abstract

Rad, a prototypic member of a subfamily of Ras-related GTPases, is overexpressed in skeletal muscle of type II diabetic humans. By expression screening of mouse embryo and human skeletal muscle cDNA libraries, we found that Rad interacted with skeletal muscle β-tropomyosin. In the mouse skeletal muscle cell line C2C12, this interaction was significantly increased by the calcium ionophore A23187. A23187 also caused a time- and concentration-dependent decrease in total cellular Rad with increased interaction between tropomyosin and Rad in the detergent-soluble fraction and the appearance of Rad in the cytoskeleton. In C2C12 cells stably overexpressing a putative dominant negative mutant of Rad (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 studies, GDP-Rad bound to tropomyosin far better than GTP-Rad. We conclude that Rad interacts with skeletal muscle β-tropomyosin and the cytoskeleton in a guanine nucleotide-dependent manner. These data suggest that Rad may be involved in skeletal muscle motor function and cytoskeletal organization.