Ras-Related Associated with Diabetes
Ras associated with diabetes (Rad) was identified in the beginning of the 1990s as a clone differentially expressed in two subtraction cDNA libraries, which were prepared from skeletal muscle of normal individuals and patients with type II (noninsulin-dependent) diabetes mellitus (Reynet and Kahn 1993). Analysis of the newly identified clone revealed about 50% identity at the nucleotide level with members of the Ras superfamily, which consists of more than a 100 low-molecular-weight guanine nucleotide–binding proteins, also referred to as small GTPases. In their seminal report, Reynet and Kahn observed that the expression of Rad is typically highest in skeletal muscle, cardiac muscle, and lung of normal individuals. They also found that Rad mRNA levels in type II diabetes muscle are higher than in type I diabetes and nondiabetic muscle. These findings provided the framework for follow-up studies on the comprehension of the biology of Rad in relation to muscle (patho)physiology and cancer.
Structure of Rad
The human Rad gene is localized in chromosome 16q22, spans 3.75 kb, and is composed of five exons and four introns. Translation presumably starts from an in-frame ATG codon in the second exon, which gives rise to a protein of 269 amino acids with a predicted molecular weight of 29,266 Da (Caldwell et al. 1996). Small GTPases are divided into six subfamilies: Ras, Rho, Arf, Rab, Ran, and RGK. Rad is the prototype member of the small GTPases in the RGK family, which also includes Rem, Rem2, and Gem/Kir. From a structural point of view, Rad possesses the five highly conserved GTPase domains G1 to G5 characteristic of the Ras-related proteins, but it also displays particular features distinct from that commonly observed in other small GTPases. First, Rad exhibits several nonconserved amino acids in G1, G2, and G3 domains that may affect its GTPase function. Second, Rad shows longer NH 2 and COOH termini of 88 and 31 amino acids, respectively. Third, The COOH terminus of Rad does not present the typical CAAX isoprenylation motif that usually facilitates the attachment to cell membranes (Reynet and Kahn 1993).
Regulation of the Activity of Rad
The major feature of Rad is its ability to cycle between a GDP-bound inactive and a GTP-bound active conformation. In addition, Rad interacts with a variety of proteins that can thereby determine its function and/or subcellular distribution. Overexpression of Rad in neuroblastoma cells triggers cellular flattening and neurite extension, very likely through binding of Rad to the Rho-associated protein kinase ROCK, which is known to regulate the shape and movement of cells by acting on the cytoskeleton. Such an interaction impedes ROCK activity, which in turn results in the inhibition of contraction and retraction (Ward et al. 2002). The subcellular distribution of Rad greatly depends on the interactions with the Ca2+-binding protein calmodulin (CaM) and the multifunctional regulatory protein 14-3-3. Lack of binding to CaM induces the accumulation of Rad in the nucleus, whereas the association with 14-3-3 maintains Rad within the cytoplasm (Mahalakshmi et al. 2007). Rad also presents several consensus phosphorylation sites in the extended COOH terminus, which are close to the CaM-binding domain. Rad can be phosphorylated on serine residues by Ca2+-/CaM-dependent protein kinase II (CaMKII), PKA, casein kinase II, and PKC. These posttranslational modifications do not seem to affect the ability of Rad to hydrolyze GTP but alter its affinity to bind CaM and 14-3-3, thus interfering with the subcellular localization of the protein (Moyers et al. 1998; Mahalakshmi et al. 2007).
Little is known about the mechanisms controlling the expression of Rad at the transcriptional level. Early studies localized Rad to thin filaments in skeletal muscle and observed that its expression increases during myoblast fusion (Paulik et al. 1997). In agreement with these findings, several transcription factors involved in myogenesis, including MEF2, MyoD, and Myf5, stimulate the activity of the Rad promoter (Hawke et al. 2006). The expression of Rad is also induced in experimental conditions characterized by an acute accumulation of toxic reactive oxygen species in skeletal muscle, such as hind limb ischemia–reperfusion and muscle denervation caused by sciatic nerve axotomy (Halter et al. 2010).
Role of Rad in Tissue Remodeling
The formation of vascular lesions in vascular proliferative diseases, such as atherosclerosis, mainly results from aberrant migration, attachment, and proliferation of vascular smooth muscle cells. Multiple factors contribute to (or attempt to attenuate) this pathological remodeling. Rad inhibits migration and attachment of vascular smooth muscle cells by a GTP-dependent mechanism. Indeed, Rad prevents the formation of stress fibers and focal contacts necessary to the remodeling process, by interfering with the Rho/ROCK signaling pathway (Fu et al. 2005). An additional mechanism by which Rad can reduce the formation of vascular lesions occurs, at least in part, via the direct upregulation of its expression by the transcriptional activator Brahma-related gene 1, which is known to inhibit proliferation and migration of smooth muscle cells in the aorta (Liao et al. 2015).
Tissue remodeling also happens in the heart in response to injury and stress. This reaction can lead to myocardial hypertrophy and fibrosis, and subsequent heart failure. The expression of Rad is normally high in the myocardium but decreases in diseased heart. Transgenic mice lacking the Rad gene are more prone to develop cardiac hypertrophy. It seems that Rad tends to lower this hypertrophy by decreasing the phosphorylation and activity of CaMKII (Chang et al. 2007). Rad also acts as a negative regulator of fibrosis in the heart, since Rad-deficient mice show severe myocardial fibrosis. In fact, Rad binds the transcription factor C/EBP-δ and impedes the expression of connective tissue growth factor, which is a key stimulator of the production of extracellular matrix leading to fibrosis (Zhang et al. 2011).
Role of Rad in Cancer
Initial reports postulated that Rad may be an oncogenic protein, since it increases the rate of growth of breast cancer cells in vitro. Moreover, the ability of these cells to form tumors in nude mice is exacerbated in the presence of Rad. The oncogenic potential of Rad seems to reside within both NH 2 and COOH termini without regard to its GTPase activity. However, nm23, which is a metastasis suppressor that stimulates GTP hydrolysis by Rad, reduces this tumor-promoting effect (Tseng et al. 2001).
In contrast to previous results, additional studies rather suggested that Rad is a tumor suppressor protein. Its expression is frequently inhibited in several types of tumor cells, and this inhibition occurs through promoter hypermethylation (Wang et al. 2014). As a result, the decrease in the expression of Rad stimulates glucose uptake and aerobic glycolysis, which in turn promotes tumor progression (Yan et al. 2016). Rad is also a direct transcriptional target of the tumor suppressor p53. Upon upregulation by this factor, Rad reduces migration and invasiveness of cancer cells by acting on cytoskeleton reorganization (Hsiao et al. 2011).
Role of Rad in Ca2+ Channel Activity
First studies provided evidence that Rad can inhibit Ca2+ currents through L-type voltage-dependent channels. This occurs via the interaction of Rad with auxiliary Ca Vβ subunits, which are involved in the trafficking of Ca2+ channels. This interaction leads to the sequestration of Ca Vβ subunits into the nucleus, thus preventing channel expression on the cell surface. Rad interaction depends on critical amino acid residues in the COOH terminus and is facilitated by the absence of binding to 14-3-3 and CaM, both of which allow relocalization of Rad within the cytoplasm (Finlin et al. 2003; Béguin et al. 2006).
Along with these findings, follow-up studies reported that decreased levels of Rad can also affect Ca2+ channel activity. Heart-specific overexpression of a dominant negative form of Rad, which binds GDP but not GTP, induces an increase in the number of L-type voltage-dependent Ca2+ channels on the cell surface and leads to subsequent cardiac arrhythmogenesis (Yada et al. 2007). Alternatively, a reduction of the interaction of Rad with PKA allows the activation of this kinase, which hyperphosphorylates the cardiac ryanodine receptor RyR2, thus triggering altered Ca2+ currents and aberrant excitability (Yamakawa et al. 2014).
Role of Rad in Muscle Metabolism, Development, and Disease
Rad was initially identified as being upregulated in a subset of diabetic patients. This finding was not confirmed in further studies, since Rad expression appears normal in other populations of diabetic patients and in the Zucker rat model of diabetes and obesity (Paulik et al. 1997). Of note, however, additional evidence from both in vitro and in vivo experiments strongly suggests the implication of Rad in glucose metabolism. First, overexpressing Rad in C2C12 and L6 myocyte cell lines reduces insulin-stimulated glucose uptake (Moyers et al. 1996). Second, the muscle-specific increase in the expression of Rad by genetic means acts synergistically with a high-fat diet to induce insulin resistance as observed in Type II diabetes (Ilany et al. 2006).
In addition to its implication in muscle metabolism, Rad is characteristically expressed during normal rat muscle development and in regenerating muscle in response to injury. This expression is mainly located in the myogenic progenitor cell population, as well as in the newly regenerated myofibers, as occurs in the mdx mouse model of Duchenne muscular dystrophy (Hawke et al. 2006). Extending these observations, Rad expression is upregulated in skeletal muscles affected by amyotrophic lateral sclerosis, which is a chronic neuromuscular degenerative condition in the elderly. In this disease, Rad is intimately associated with muscle atrophy, since its upregulation takes place within the myofibers that suffer from the degenerative process (Halter et al. 2010). In support of these findings, further studies showed that Rad triggers the apoptotic death of cardiomyocytes in vitro by inducing the phosphorylation of p38 MAPK and by decreasing the levels of the pro-survival molecule Bcl-xL (Sun et al. 2011).
- Liao WL, Tan MW, Yuan Y, Wang GK, Wang C, Tang H, et al. Brahma-related gene 1 inhibits proliferation and migration of human aortic smooth muscle cells by directly up-regulating Ras-related associated with diabetes in the pathophysiologic processes of aortic dissection. J Thorac Cardiovasc Surg. 2015;150:1292–301.PubMedCrossRefGoogle Scholar