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Identification of Rad's effector-binding domain, intracellular localization, and analysis of expression in Pima
M A Paulik1, L L Hamacher, D P Yarnall
1Department of Metabolic Diseases, Glaxo Wellcome Inc., Research Triangle Park, North Carolina 27709, USA.
Journal of Cellular Biochemistry
|June 15, 1997
Summary
Ras-related protein associated with diabetes (rad) is a 46 kDa muscle protein involved in myotube formation. Its expression does not correlate with diabetes in humans or rats, but specific amino acids may regulate its GTPase activity.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The ras-related protein associated with diabetes (rad) function and expression in muscle tissue are not fully understood.
- Investigating rad's role is crucial for understanding muscle development and potential links to metabolic diseases.
Purpose of the Study:
- To characterize the endogenous rad protein, including its expression, structure, and GTP-binding activity.
- To determine if rad expression is altered in diabetes in human and animal models.
- To identify regions of rad involved in its functional regulation.
Main Methods:
- Antibodies were generated against synthetic peptides of rad.
- Western blot analysis and immunolocalization were used to assess rad expression and localization in muscle.
- GTP-binding and GTPase activity assays were performed on purified rad.
- Muscle biopsies from diabetic and control Pima Indians and Zucker diabetic fatty rats were analyzed.
Main Results:
- Rad is a 46 kDa GTP-binding protein localized to thin filaments in skeletal muscle.
- Rad expression increases during myoblast fusion.
- No differences in rad protein or mRNA expression were found in diabetic versus control Pima Indians or Zucker diabetic fatty rats.
- Rad's GTP-binding activity is modulated by specific nucleotides, and amino acids 109-121 appear critical for regulating its GTPase activity.
Conclusions:
- Rad is a muscle-specific GTP-binding protein involved in myogenesis.
- Rad expression is not associated with diabetes in the studied populations or models.
- The N-terminal region (amino acids 109-121) of rad may play a regulatory role in its GTPase activity, potentially through interaction with cytosolic factors.