Identification of Rad's effector-binding domain, intracellular localization, and analysis of expression in Pima Indians

• Published in: Journal of cellular biochemistry Vol. 65; no. 4; pp. 527 - 541
• Main Authors: Paulik, Mark A., Hamacher, Lawrence L., Yarnall, David P., Simmons, Caroline J., Maianu, Lidia, Pratley, Richard E., Garvey, W. Timothy, Burns, Daniel K., Lenhard, James M.
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 15-06-1997
• Subjects: Actin Cytoskeleton - metabolism; Amino Acid Sequence; Animals; Arizona; Asian Continental Ancestry Group - genetics; Diabetes Mellitus, Type 2 - genetics; Epitope Mapping; GTP-binding protein; GTP-Binding Proteins - genetics; GTP-Binding Proteins - metabolism; Guanosine Triphosphate - metabolism; Humans; Indians, North American; insulin resistance; Molecular Sequence Data; Molecular Weight; Muscle, Skeletal - metabolism; Myocardium - metabolism; NIDDM; ras Proteins; Rats; Rats, Sprague-Dawley; Rats, Zucker; RNA, Messenger - metabolism; skeletal muscle; Structure-Activity Relationship; thin filaments; Tissue Distribution; Arizona
• ISSN: 0730-2312; 1097-4644
• DOI: 10.1002/(SICI)1097-4644(19970615)65:4<527::AID-JCB8>3.0.CO;2-Q

In order to characterize the endogenous gene product for rad (ras‐related protein associated with diabetes), we prepared antibodies to synthetic peptides that correspond to amino acids (109–121, 178–195, 254–271) within the protein. These antibodies were used to analyze the expression, structure, and function of rad. Western analysis with these antibodies revealed that rad was a 46 kDa protein which was expressed during myotube formation. Further, immunolocalization studies showed that rad localized to thin filamentous regions in skeletal muscle. Interestingly, when muscle biopsies from diabetic and control Pima Indians were compared, no differences in rad protein or mRNA expression were observed. Similarly, no differences were observed in protein expression in diabetic and control Zucker diabetic fatty (ZDF) rats. Functional analysis of muscle rad revealed that its GTP‐binding activity was inhibited by the addition of N‐ethylmaliemide, GTP, GTPγS, and GDPβS but not ATP or dithiothreitol. Moreover, cytosol‐dependent rad‐GTPase activity was stimulated by the peptide corresponding to amino acids 109–121. Antibodies corresponding to this epitope inhibited cytosol‐dependent rad‐GTPase activity. Taken together, the results indicate that 1) rad is a 46 kDa GTP‐binding protein localized to thin filaments in muscle and its expression increases during myoblast fusion, 2) expression of rad in Pima Indians and ZDF rats does not correlate with diabetes, and 3) the amino acids (109–121) may be involved in regulating rad‐GTPase activity, perhaps by interacting with a cytosolic factor(s) regulating nucleotide exchange and/or hydrolysis. J. Cell. Biochem. 65:527–541. © 1997 Wiley‐Liss Inc.