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Identification of Rad's effector‐binding domain, intracellular localization, and analysis of expression in Pima Indians
Author(s) -
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.
Publication year - 1997
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/(sici)1097-4644(19970615)65:4<527::aid-jcb8>3.0.co;2-q
Subject(s) - effector , identification (biology) , intracellular , microbiology and biotechnology , expression (computer science) , domain (mathematical analysis) , biology , chemistry , computational biology , computer science , mathematics , mathematical analysis , botany , programming language
In order to characterize the endogenous gene product for rad ( ras ‐related protein a ssociated with d iabetes), 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.