The regulator of G protein signaling 10 (RGS10) protein is a regulatory molecule that belongs to a larger family of RGS proteins responsible for altering cellular signaling. Generally, RGS proteins are negative regulators of signal transduction pathways mediated by heterotrimeric G proteins. This function allows RGS proteins to control the duration and amplification of signaling activity, ultimately operating as shut-off switches. RGS proteins deactivate G protein subunits by serving as GTPase activating proteins (GAPs), which enhance the intrinsic GTPase activity of the active, GTP-bound Gα subunit and return G proteins to their inactive, GDP-bound form. The result is an acceleration of the deactivation of G protein signaling.
Protein Structure and Function
All RGS proteins share a conserved 120 amino acid helical RGS domain that is responsible for GAP activity. Based on sequence similarity of the RGS domain, RGS10 is grouped in the RGS 12 subfamily which also includes the proteins RGS12 and RGS14 (Alexander et al. 2015). Despite the similarity of their RGS domains, RGS10 (20 kDa) is much smaller than both RGS14 (61 kDa) and RGS12 (156 kDa), as RGS10 lacks the regulatory and multifunctional domains found in RGS12 and RGS14. Indeed, RGS10 is one of the smallest members of the entire RGS protein family. Aside from its RGS domain, RGS10 contains only short N-terminal and C-terminal extensions that lack any defined domain structure but may be involved in subcellular targeting (see below).
The structure of RGS10 and its interaction with Gαi subunits has been defined in detail (Soundararajan et al. 2008; Taylor et al. 2016). RGS10 contains the conserved RGS domain structure consisting of nine α helices, although Soundararajan and colleagues demonstrated that RGS10 possesses a shorter αVI helix and an extended αV–αVI loop compared to RGS domains of R4, R7, and RZ subfamilies. The RGS10-Gαi3 complex is generally consistent with G-protein complexes with other RGS subfamily members, but differences were again observed in the αVI helix. For example, a conserved RGS domain arginine residue in helix αVI that makes contacts with the Gα switch III region in complexes formed with members of the R4 family did not make contacts with Gαi3 in the RGS10 complex (Soundararajan et al. 2008). Finally, the GAP activity of RGS10 is under complex regulation by palmitoylation of a conserved cysteine in the RGS domain. Palmitoylation of this residue inhibits RGS10 single turnover GTPase activity but substantially potentiates steady-state GAP activity in proteoliposome reconstituted receptor-G protein complexes (Tu et al. 1997, 1999).
In spite of the plasma membrane localization of their canonical G protein targets, many RGS proteins are localized to the nucleus. This localization may serve as a cellular storage compartment to sequester RGS proteins and regulate their GAP activity, or may allow RGS proteins to regulate undefined nuclear targets (Hollinger and Hepler 2002). Several studies have demonstrated RGS10 localization to the nucleus and cytoplasm, with minimal plasma membrane localization (Chatterjee and Fisher 2000; Lee et al. 2008). Putative nuclear localization sequences have been identified in the RGS10 N- and C-termini but have not been functionally validated. Further, the nuclear localization of RGS10 is dynamically regulated by cellular activation (Lee et al. 2008) and through cyclic AMP-dependent protein kinase A mediated phosphorylation of RGS10 on serine 168 at its C terminus (Burgon et al. 2001) (Fig. 1b). Further implications and interpretations of this noncanonical localization by a G protein regulatory molecule suggest unknown and/or unappreciated roles for RGS10 outside of canonical understanding.
RGS10 is conserved in tetrapods including mammals, birds, and amphibians and is encoded by a single gene (Rgs10) on chromosome 10 in Homo sapiens (10q26.11). The mouse Rgs10 gene contains two transcriptional start sites, yielding two transcript isoforms with distinct first exons, and four common exons (Haller et al. 2002; Lee and Tansey 2015). Mouse transcripts mRgs10-1 and mRgs10-2 correspond to human transcripts with conserved exon structure and encoding highly conserved human RGS10 protein isoforms RGS10a (181 aa) and RGS10b (167 aa). A third human transcript (173 aa) has been proposed, resulting from an alternate transcriptional start site upstream of the first shared exon (Hunt et al. 1996), but the relevance of this transcript in mouse cells is unclear (Haller et al. 2002). Rgs10-1/RGS10a is the predominant form expressed in ovarian cancer cells, osteoclasts, microglia, and other immune cells (Yang and Li 2007; Yang et al. 2007; Ali et al. 2013; Lee and Tansey 2015).
Epigenetic Regulation of Expression
Cells control the expression of the Rgs10 gene epigenetically. In chemoresistant ovarian cancer cells, suppression of the RGS10 protein occurs due to both histone deacetylation and DNA hypermethylation, which are common chromatin modifications that synergistically regulate many genes. For example, knocking down the expression of either histone deacetylase 1 or DNA methyltransferase 1 significantly increases the expression of Rgs10 (Cacan et al. 2014). In addition, pharmacological inhibition of DNA methyltransferase activity increases the expression of Rgs10 transcription in neural progenitor cells (Tuggle et al. 2014). In contrast, in activated microglia Rgs10 is suppressed by histone deacetylation while DNA methylation does not have a significant effect. Thus, distinct cell types use distinct epigenetic mechanisms to suppress RGS10 levels, which will indirectly amplify the G protein signaling pathways that are targeted by RGS10.
Given that Rgs10 expression is dynamically regulated, Rgs10 expression levels or epigenetic marks may be useful biomarkers. A genome-wide study examining freely circulating DNA in the blood of patients with hepatocellular carcinoma using a technique called methylated CpG tandem amplification and sequencing (MCTA-Seq) found hypermethylated CpG islands of RGS10 in circulation. The study used an unbiased method to detect aberrant DNA methylation changes and showed that only 2.3% of hypermethylated CpG islands were sufficient biomarkers for detecting hepatocellular carcinoma in blood. Most importantly, RGS10 was among only four biomarkers that fit tumor-specific criteria used in this study (Wen et al. 2015) Thus, epigenetically modified RGS10 may have the potential to serve as a specific biomarker for the early stage detection of hepatocellular carcinoma.
Signaling in Ovarian Cancer Chemoresistance
Signaling in Platelets
Platelets respond to multiple GPCR agonists to regulate hemostasis and thrombogenesis. In platelets, RGS10 binds to a complex with the spinophilin scaffolding protein and the SHP-1 protein tyrosine phosphatase to negatively regulate platelet activation until further SHP-1 phosphorylation occurs, which causes the decay of this complex (Ma et al. 2012). In platelets derived from RGS10−/− mice, agonist-induced signaling was hyperresponsive and manifested by platelet aggregation and enhanced hemostasis. In these mice, there was also an observation of increased susceptibility to ischemia due to thrombosis (Hensch et al. 2016). Strikingly, loss of RGS10 expression amplified platelet aggregation in response to activation of receptors coupled to Gi, Gq, and G12 receptors, despite the clear Gi selectivity of RGS10 GAP activity. These studies suggest that RGS10 acts as an integral “braking system” that blunts platelet activation and thrombogenesis, but the mechanism remains undefined.
Signaling in Immune Cells
RGS10 is highly expressed in the brain and immune system, with particularly high expression in microglia, the resident macrophages of the central nervous system. Several studies have demonstrated that microglial RGS10 plays important anti-inflammatory and neuroprotective roles by inhibiting lipopolysaccharide-stimulated production of inflammatory cytokines including TNFα (Lee et al. 2008, 2011). Additional neuroprotective effects are mediated by neuronal RGS10, which increases cell survival and resistance to TNF-induced cytotoxicity (Lee et al. 2012). Together, these results suggest that RGS10 may be protective in neurodegenerative diseases with underlying neuroinflammation, such as Parkinson’s disease. In contrast, RGS10 is implicated in promoting progression of the autoimmune neurodegenerative disease multiple sclerosis (MS), and RGS10−/− display dramatically milder symptoms in a mouse model of MS (Lee et al. 2016). The effect of RGS10 in this model is attributed to its effects on peripheral T cells. RGS10 is also a critical regulator of peripheral macrophages, promoting the anti-inflammatory M2 alternative activation while suppressing the inflammatory M1 activation phenotype (Lee et al. 2013). Thus, RGS10 plays diverse roles in various cells of the immune system and may contribute to multiple neuroinflammatory and immune disorders. In particular, RGS10 may contribute to inflammatory disorders related to aging, as RGS10 expression changes significantly in various immune cells during aging (Kannarkat et al. 2015).
Signaling in Heart Failure
RGS10 protein levels are significantly lower in failing human hearts and in a mouse model of cardiac hypertrophy compared to normal controls, while overexpression of RGS10 suppresses cardiac hypertrophy in cell and animal models. Further, RGS10 inhibits signaling downstream of the Gαq coupled angiotensin II receptor, which induces cardiomyocyte hypertrophy. Thus, RGS10 negatively regulates signaling in this model and inhibits cardiac hypertrophy (Miao et al. 2016).
Signaling in Osteoclast Differentiation
RGS10 is dynamically expressed in bone resorbing osteoclast cells, and RGS10 plays an important role in regulating osteoclast function and differentiation. The RANKL-stimulated signaling pathway for differentiation of osteoclasts induces the production of RGS10. When RGS10 is suppressed in this model, intracellular Ca2+ oscillations are inhibited as well as expression of NFAT2 – the master switch for regulating terminal differentiation of osteoclasts. RGS10−/− mice display impaired osteoclast differentiation and osteopetrosis. This suggests that RGS10 is a key regulator that cooperates with other factors activated by RANKL and is an essential component of this signaling pathway in bone physiology (Yang and Li 2007; Yang et al. 2007).
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