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

Academic Article

Abstract

  • In order to characterize the endogenous gene product for tad (ras- related protein associated with diabetes), we prepared antibodies to synthetic peptides that correspond to amine 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 tad 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 amine 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 amine 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.
  • Published In

    Author List

  • Paulik MA; Hamacher LL; Yarnall DP; Simmons CJ; Maianu L; Pratley RE; Garvey WT; Burns DK; Lenhard JM
  • Start Page

  • 527
  • End Page

  • 541
  • Volume

  • 65
  • Issue

  • 4