Regulator of G-protein signaling (RGS) is a protein superfamily discovered in the mid-1990s (Druey et al. 1996; Watson et al. 1996; Hunt et al. 1996). Since that time, more than 30 members in the family have been identified. The major physiological function of RGS molecules is to negatively modulate G-protein-coupled receptor (GPCR)-mediated signaling and biology (Bansal et al. 2007; Neitzel and Hepler 2006; Thompson et al. 2008). RGS13 was first identified in 1996 and is one of the smallest molecules in the family (Druey et al. 1996). Full-length human RGS13 cDNA was cloned and deposited into gene bank in 1997 by Chatterjee and Fisher. In 2002, the first functional studies of human RGS13 were reported. RGS13 was shown to inhibit muscarinic M1- and M2 receptor-induced MAP kinase activation (Johnson and Druey 2002). Concurrently, a separate study characterized mouse RGS13, which was cloned from B cells. RGS13 expression was demonstrated in germinal center (GC) regions of mouse spleen and shown to inhibit chemokine receptor CXCR4 and CXCR5-mediated signaling pathways, suggesting a function of RGS13 in B cell migration and trafficking (Shi et al. 2002). More recently, detailed studies of RGS13-deficient mice and microarray analysis discovered potential roles of RGS13 in autoimmunity, lymphoid malignancy, and cancers (Wang et al. 2013; Sethakorn and Dulin 2013).
In additional to its canonical functions, GPCR-independent functions of RGS13 were also revealed. RGS13 suppressed IgE receptor-induced mast cell degranulation through interaction with the p85 subunit of phosphoinositide 3-kinase (Bansal et al. 2008b). Mice deficient in RGS13 have enhanced IgE-mediated anaphylaxis after antigen challenge. RGS13 was also shown to regulate cAMP-dependent pathways. RGS13 bound phosphorylated cAMP responsive element binding protein ( CREB) in the nucleus and inhibited CREB-mediated gene transcription (Xie et al. 2008), which resulted in the changes in the gene expression profiles of GC B cells and altered GC programming (Hwang et al. 2013; Wang et al. 2013)
RGS13 has very restricted tissue distribution, with expression mainly in B cells (Shi et al. 2002), mast cells (Bansal et al. 2008b), and enteroendocrine cells (Xie et al., unpublished observation). Central nervous system expression of Rgs13 mRNA has been observed in rat brain by in situ hybridization (Grafstein-Dunn et al. 2001), but not in the human brain (Larminie et al. 2004). The expression level of RGS13 may vary significantly under physiological or pathological conditions. For example, although RGS13 is highly expressed in GC B cells and Burkitt lymphoma cells, it is absent in mantle cell lymphoma (Islam et al. 2003). Upregulated Rgs13 mRNA has been reported in malignant T cells from acute T cell leukemia (ATL) patients by gene expression profiling (Pise-Masison et al. 2009); however, it was not detected in normal human tonsil T cells or in Jurkat and MOLT-4 T cell lines (Shi et al. 2002). In BXD2 autoimmune mice, increased IL-17 production is associated with elevated Rgs13 mRNA expression and the suppression of B cell chemotactic responses to the chemokine CXCL12 (Hsu et al. 2008). Recent analysis of gene expression patterns of the R4 subfamily of RGS proteins using the Oncomine database, which contains hundreds of microarray datasets from cancer studies, revealed that Rgs13 expression was altered in many solid tumors and various blood cancers (Sethakorn and Dulin 2013): Rgs13 expression was decreased in solid tumors of colon, lung, skin, and uterus, but increased in bladder carcinoma. The expression of Rgs13 was dramatically upregulated 14–34 fold in adult T cell leukemia/lymphoma, while downregulated 40–115 fold in chronic lymphocytic leukemia and mantle cell lymphoma. The restricted tissue distribution and apparent disease relevance make RGS13 an excellent target for drug development and/or as a diagnostic biomarker.
Regulation of RGS13 Expression
Similar to many RGS molecules, RGS13 expression is relatively low in quiescent cells, which may be necessary for allowing adequate GPCR signaling to maintain cell homeostatic functions. Incubation of bone marrow-derived mast cells (BMMCs) with IgE-antigen for 24 h results in a four- to five-fold increase in RGS13 mRNA and protein (Bansal et al. 2008a), which may have a negative feedback role to amplify the inhibitory effect of RGS13 on antigen-mediated mast cell degranulation. In other words, RGS13 may desensitize mast cells to antigen stimulation. Interestingly exposure of human LAD2 mast cells to cAMP decreases Rgs13 mRNA quantities (Xie et al. 2010), suggesting that RGS13 expression is differentially regulated by distinct ligands or signaling pathways. In human tonsillar B lymphocytes, anti-CD40 antibody augments Rgs13 mRNA expression (Shi et al. 2002), suggesting a role of RGS13 in B cell activation. Consistent with its expression in splenic GC B cells, human Burkitt lymphoma cell lines Ramos, HS-Sultan, and Raji express abundant RGS13, as do immunized mouse spleen B cells (Shi et al. 2002). Notably, the tumor suppressor p53 inhibits Rgs13 mRNA transcription in mast cells by binding to its promoter region (Iwaki et al. 2011).
RGS13 is also regulated at the posttranslational level. RGS13 protein undergoes proteasome-mediated degradation. Phosphorylation of RGS13 on Thr41 by protein kinase A (PKA) protects it from degradation, resulting in elevated steady-state RGS13 protein levels (Xie et al. 2010), which could promote the inhibitory function of RGS13 on transcription factor CREB.
RGS proteins interact with α subunit of the heterotrimeric G-protein in its activated (GTP-bound) state, leading to increased intrinsic GTPase activity of Gα. This GTPase activating protein (GAP) activity increases the rate of GTP hydrolysis, which hastens deactivation/termination of GPCR signaling and functions (Patel 2004). RGS13 interacts with the α subunit of Gi and Gq, but not Gs, accelerating the GTPase activity of Gα (7,8). MAP kinase Erk1/2 activation stimulated by Gq-coupled muscarinic M2 receptor is significantly inhibited by overexpression of RGS13 in human embryonic kidney (HEK) 293 T cells (Johnson and Druey 2002). Overexpression of RGS13 in Chinese Hamster Ovary (CHO) cells inhibited the Gi-coupled chemokine receptor CXCR4-mediated cell migration (Shi et al. 2002).
SiRNA-mediated knockdown of endogenous RGS13 in HS-Sultan cells results in enhanced Ca2+ flux and chemotaxis induced by the chemokines CXCL12 and CXCL13, which utilize Gi-coupled GPCRs CXCR4 and CXCR5, respectively (Han et al. 2006). Depletion of RGS13 in the human mast cell lines LAD2 and HMC-1 by shRNA increases degranulation evoked by the GPCR ligand sphingosine-1-phosphate (S1-P) and greater Ca2+ mobilization in response to several GPCR ligands including C5a, adenosine, and S1P (Bansal et al. 2008a).
By using Rgs13GFP KI mouse, where the Rgs13 coding region was replaced by green fluorescent protein (GFP), Dr. John Kehrl’s group demonstrated important roles of RGS13 in B cell functions and immune responses (Hwang et al. 2013). Following immunization, GFP expression was rapidly increased in activated B cells and persisted in GC B cells. In the absence of RGS13, enlarged GCs and increased GC numbers were observed in immunized spleen. The dark zone and light zone regions in Rgs13GFP KI mouse spleen were less distinct compared to those in WT mice. These data suggested that RGS13 helps organize GC morphology and limits the size of GCs.
Under pathological conditions, RGS13 may have distinct roles in B cell functions, GC formation, and immunity. A deficiency of RGS13 in autoimmune mice BXD2 significantly reduced the manifestations of lupus characteristic of BXD2-WT mice, including proteinuria, the deposition of IgG-containing immune complexes in the glomeruli (Wang et al. 2013). BXD2-Rgs13−/− mice exhibited smaller GCs and reduced serum IgG autoantibody levels.
RGS13 clearly plays crucial roles in GC B cell functions and adaptive immunity; however, whether these functions are solely due to its classical GAP activity is still unclear and needs to be determined.
In recent years, GPCR-independent cellular functions of RGS13 have been characterized, which do not involve classical RGS13 GAP activity. Cross-linking of the IgE receptor FcεRI by antigen on mast cells activates signaling molecules including Syk kinase, phospholipase Cγ, LAT, and PI3 Kinase, leading to mast cell degranulation and cytokine production (Gilfillan and Tkaczyk 2006). Mice lacking RGS13 displayed enhanced systemic and local cutaneous anaphylactic responses when challenged with IgE/antigen (Bansal et al. 2008b). BMMCs from Rgs13 −/− mice degranulated much more than those from wild-type littermates. Interestingly cytokine generation by BMMC after IgE/antigen stimulation were not affected by RGS13 protein deficiency, indicating that RGS13 specifically regulates IgE-mediated mast cell degranulation. Further analysis revealed that RGS13 binds to the p85 subunit of PI3 kinase enzyme, which is a critical downstream effector in the degranulation pathways. RGS13 inhibited formation of an FcεRI-associated p85-Gab2-Grb2 signaling complex, which is required for mast cell degranulation (Bansal et al. 2008b). Reconstitution of RGS13-deficient BMMC with a GAP-inactive RGS13 mutant suppressed antigen-stimulated degranulation similar to wild-type RGS13, indicating that RGS13 GAP activity is not required for its inhibition of mast cell function (Bansal et al. 2008b).
A separate set of studies led to the discovery that although RGS13 does not interact with Gαs, it inhibits Gs-mediated signaling through the β2-adrenergic receptor downstream of the G protein (Johnson and Druey 2002). Further studies revealed that PKA activation induced by Gs-coupled β2-adrenergic receptor stimulation leads to RGS13 accumulation in the nucleus. RGS13 binds to the phosphorylated transcription factor CREB in the presence of its co-activator CREB-binding protein (CBP). Binding of RGS13 to phosphorylated CREB eventually inhibited CREB transactivation, resulting in decreased CREB target gene expression (Xie et al. 2008).
The importance of nuclear repressor role of RGS13 on CREB-mediated gene expression has been demonstrated by recent studies of Rgs13 gene-deleted mice. The loss of Rgs13 in Rgs13GFP KI mice resulted in significantly increased expression of a number of CREB target genes as well as CREB itself and CREB co-activators including Crebbp, Crtc2, Ep300, Stk11, Smarca2, and Mta3 (Hwang et al. 2013). Mta3 encodes a protein that is instrumental in maintaining the GC B cell transcriptional program, which precludes premature plasma cell differentiation (Fujita et al. 2004, Cell). These data suggested that RGS13 might impact a genetic program that is known to control GC B cell proliferation, self-renewal, and differentiation.
Rgs13 deficiency in BDX2 autoimmune mice resulted in decreased GC program genes and increased plasma cell program genes including CREB target genes FosB and Obf1 (Wang et al. 2013). These mice exhibited smaller GCs in the spleen and reduced serum IgG autoantibody levels. However, whether the phenotypes were due to the changes in gene expression programs is unclear.
RGS13 acts as a signaling modulator, playing critical roles in both GPCR-dependent and GPCR-independent cellular processes. The high expression of RGS13 in murine GC B cells, its suppression of chemokine-induced B cell migration, and its impact on B cell gene expression suggest a potential role of RGS13 in adaptive immune responses. Altered expression of RGS13 in conditions of autoimmunity or malignancy suggests disease relevance. In the light of negative regulatory roles of RGS13 in mast cell functions, upregulated RGS13 expression in mast cells repeatedly exposed to antigen could increase inhibition of IgE/antigen-mediated mast cell degranulation and/or anaphylactic responses. Mast cell degranulation, which leads to release of granular contents such as histamine, plays a key role in many diseases including allergy, asthma, mastocytosis, and anaphylaxis. Therefore, RGS13 could be a potential therapeutic target, especially given its limited tissue distribution. Since most of the studies conducted thus far have been in mice or in murine cell lines, further investigations of human subjects, such as expression patterns of RGS13 in health and disease, will be of utmost importance.
This study was supported by the Intramural Research Program of the NIH, NIAID.
- Hsu HC, Yang P, Wang J, Wu Q, Myers R, Chen J, Yi J, Guentert T, Tousson A, Stanus AL, Le TV, Lorenz RG, Xu H, Kolls JK, Carter RH, Chaplin DD, Williams RW, Mountz JD. Interleukin 17-producing T helper cells and interleukin 17 orchestrate autoreactive germinal center development in autoimmune BXD2 mice. Nat Immunol. 2008;9:166–75.PubMedCrossRefGoogle Scholar
- Islam TC, Asplund AC, Lindvall JM, Nygren L, Liden J, Kimby E, Christensson B, Smith CI, Sander B. High level of cannabinoid receptor 1, absence of regulator of G protein signalling 13 and differential expression of Cyclin D1 in mantle cell lymphoma. Leukemia. 2003;17:1880–90.PubMedCrossRefGoogle Scholar
- Iwaki S, Lu Y, Xie Z, Druey KM. p53 negatively regulates RGS13 protein expression in immune cells. J Biol Chem. 2011; epublished ahead of print.Google Scholar
- Pise-Masison CA, Radonovich M, Dohoney K, Morris JC, O’Mahony D, Lee MJ, Trepel J, Waldmann TA, Janik JE, Brady JN. Gene expression profiling of ATL patients: compilation of disease-related genes and evidence for TCF4 involvement in BIRC5 gene expression and cell viability. Blood. 2009;113:4016–26.PubMedPubMedCentralCrossRefGoogle Scholar