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Rad, a novel Ras-related GTPase, interacts with skeletal muscle beta-tropomyosin
1Research Division, Joslin Diabetes Center, Boston, Massachusetts, USA.
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 beta-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 beta-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.
Insights
Rad, a Ras-related GTPase, interacts with skeletal muscle beta-tropomyosin and the cytoskeleton. This interaction is regulated by guanine nucleotides and calcium, suggesting a role in muscle motor function and organization.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Rad, a Ras-related GTPase, is overexpressed in skeletal muscle of type II diabetic individuals.
- Understanding Rad's function is crucial for skeletal muscle biology and disease.
Purpose of the Study:
- To investigate the interaction of Rad with skeletal muscle beta-tropomyosin.
- To elucidate the regulatory mechanisms and functional implications of this interaction.
Main Methods:
- Expression screening of cDNA libraries (mouse embryo, human skeletal muscle).
- Cellular studies using C2C12 mouse skeletal muscle cell line.
- Biochemical assays including immunoprecipitation, far-Western blotting, and guanine nucleotide binding studies.
Main Results:
- Rad interacts with skeletal muscle beta-tropomyosin.
- Calcium ionophore A23187 increases Rad-tropomyosin interaction and Rad's association with the cytoskeleton.
- The interaction is dependent on the guanine nucleotide-bound state of Rad, with GDP-bound Rad showing higher affinity for tropomyosin.
- Overexpression of dominant-negative Rad mutant increases tropomyosin in Rad immunoprecipitates.
- Overexpression of wild-type Rad leads to constitutive association with the cytoskeleton.
Conclusions:
- Rad interacts with skeletal muscle beta-tropomyosin and the cytoskeleton in a guanine nucleotide-dependent manner.
- These findings suggest a potential role for Rad in skeletal muscle motor function and cytoskeletal organization.
- Rad's regulation by calcium and its nucleotide-bound state highlights its dynamic involvement in cellular processes.