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

Abstract

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, GTP gamma S, and GDP beta S 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.

Overexpression of Rad in muscle worsens diet-induced insulin resistance and glucose intolerance and lowers plasma triglyceride level

“…Rad (Ras Associated with Diabetes) is a 35-kDa GTPase that was identified by subtractive cloning as overexpressed in skeletal muscle of patients with type 2 diabetes mellitus (1,2). Although some subsequent studies (3,4) did not confirm this direct association between the Rad mRNA level and diabetes, other studies have shown that Rad is an insulin regulated gene in muscle (5) and that Rad overexpression inhibits glucose transport in muscle cells in culture (6). There is also a correlation between the level of Rad expression and obesity (3), and genetic analysis studies of two different populations found a possible association between trinucleotide-repeat polymorphism of the Rad gene and type 2 diabetes (7,8).…”

The RGK family of GTP-binding proteins: Regulators of voltage-dependent calcium channels and cytoskeleton remodeling

“…Rem is expressed predominantly in cardiac muscle, but also at more modest levels in lung, kidney, and skeletal muscle [3]; Rem2 is highly expressed in the brain and kidney, but also in neuroendocrine tissues [4]; Rad is found in abundance in cardiac and skeletal muscle [6]; while Gem/Kir is found in a diverse set of tissues, including myeloid cells, kidney, liver, and lung [5]. Rad was initially identified as a gene overexpressed in the skeletal muscle of non-insulin dependent diabetics [6], though subsequent studies in Pima Indians and the Zucker diabetic rat model imply that this effect is not universal [39]. Regulation may result from the hyperinsulinemia associated with the disease [40] as insulin has been shown to increase Rad mRNA levels in muscle [25].…”

Rad GTPase Deficiency Leads to Cardiac Hypertrophy

“…32 Although an association between Rad upregulation and type 2 diabetes mellitus could not be confirmed, a correlation with obesity was reported. 33 It was also reported that Rad inhibits insulin-stimulated glucose uptake in myocyte and adipocyte cell lines. 7 A recent report revealed that Rad is highly expressed in a breast cancer cell line with high tumorigenic and metastatic potential, promoting growth and accelerating cell cycle transition.…”