Regulators of G-protein signaling (RGS) proteins are scaffolds that control diverse signaling pathways by modulating signalosome formation and by accelerating the GTPase activity of heterotrimeric G proteins. Although expression of many RGS proteins is relatively low in quiescent cells, transcriptional and post-translational responses to environmental cues regulate both their abundance and activity. We found previously that RGS13, one of the smallest RGS proteins in the family, inhibited cyclic AMP-dependent protein kinase (PKA)-induced gene expression through interactions with the transcription factor cAMP-response element-binding (CREB) protein. Here, we show that PKA activation also leads to increased steady-state RGS13 expression through RGS13 phosphorylation, which inhibits RGS13 protein degradation. RGS13 turnover was significantly reduced in cells stimulated with cAMP, which was reversed by expression of the PKA-specific inhibitory peptide PKI. RGS13 phosphorylation was diminished by mutation of an N-terminal Thr residue (T41) identified as a phosphorylation site by mass spectrometry. Mutation of Thr41 in RGS13 to Ala (T41A) reduced steady-state RGS13 levels and its ability to inhibit M2 muscarinic receptor-mediated Erk phosphorylation compared with wild-type RGS13 by attenuating the protective effect of cAMP on RGS13 degradation. RGS13 underwent ubiquitylation, indicating that it is a likely target of the proteasome. These studies are the first to demonstrate post-translational mechanisms controlling the expression of RGS13. Stabilization of RGS13 through PKA-mediated phosphorylation could enhance RGS13 functions, providing negative feedback regulation that promotes cellular desensitization.
RGS proteins; cAMP; protein kinase; phosphorylation
Regulator of G-protein signaling-10 (RGS10) is a GTPase activating protein (GAP) for Gαi/q/z subunits that is highly expressed in the immune system and in a broad range of brain regions including the hippocampus, striatum, dorsal raphe, and ventral midbrain. Previously, we reported that RGS10-null mice display increased vulnerability to chronic systemic inflammation-induced degeneration of nigral dopaminergic (DA) neurons. Given that RGS10 is expressed in DA neurons, we investigated the extent to which RGS10 regulates cell survival under conditions of inflammatory stress. Because of the inherent limitations associated with use of primary DA neurons for biochemical analyses, we employed a well-characterized ventral mesencephalon DA neuroblastoma cell line (MN9D) for our studies. We found that stable over-expression of RGS10 rendered them resistant to TNF-induced cytotoxicity; whereas MN9D cells expressing mutant RGS10-S168A (which is resistant to phosphorylation by protein kinase A (PKA) at a serine residue that promotes its nuclear translocation) showed similar sensitivity to TNF as the parental MN9D cells. Using biochemical and pharmacological approaches, we identified protein kinase A (PKA) and the downstream phospho-cAMP response element-binding (CREB) signaling pathway (and ruled out ERK 1/2, JNK, and NFkB) as key mediators of the neuroprotective effect of RGS10 against inflammatory stress.
RGS10; MN9D; dopaminergic; CREB; neuroinflammation; TNF
The Regulators of G protein signaling (RGS) protein superfamily negatively controls G-protein-coupled receptor (GPCR) signal transduction pathways. RGS16 is enriched in activated/effector T lymphocytes. Here, we show that RGS16 constrains pulmonary inflammation by regulating chemokine-induced T-cell trafficking in response to challenge with Schistosoma mansoni. Naïve Rgs16–/– mice were “primed” for inflammation by accumulation of CCR10+ T cells in the lung. Upon pathogen exposure, these mice developed more robust granulomatous lung fibrosis than wild-type (WT) counterparts. Distinct TH2 or putative TH17 subsets expressing CCR4 or CCR10 accumulated more rapidly in Rgs16–/– lungs following challenge and produced pro-inflammatory cytokines IL-13 and IL-17B. CCR4+ Rgs16–/– TH2 cells migrated excessively to CCL17 and localized aberrantly in challenged lungs. T lymphocytes were partially excluded from lung granulomas in Rgs16–/– mice, instead forming peribronchial/perivascular aggregates. Thus, RGS16-mediated confinement of T cells to Schistosome granulomas mitigates widespread cytokine-mediated pulmonary inflammation.
Chemokines; G proteins; RGS proteins; Schistosoma mansoni; fibrosis; TH2; TH17; cytokines
Sensitization to protease allergens, such as papain, or helminth infection is associated with basophil recruitment to draining lymph nodes. Basophils have the capacity to present antigen to naïve T cells and promote TH2 differentiation directly or indirectly through IL-4 production.
We studied how papain induces basophil migration to lymph nodes and the contribution of various leukocytes to papain-induced immune responses.
We immunized mice in the footpad with papain and studied leukocyte recruitment and inflammatory cytokine and chemokine production in the draining popliteal lymph nodes.
Papain directly activated naïve T cells through protease-activated receptor 2 (PAR2) to initiate a chemokine/cytokine program that includes CCL17, CCL22, and IL-4. Papain-triggered innate immune responses were dependent on both CD4 T cells and PAR2 and were strongly reduced in the absence of CCR4, the primary receptor for CCL17/22.
These results elucidate a novel innate allergen recognition pathway mediated by naïve T cells through PAR2, which provide an immediate source of chemokines and IL-4 upstream of basophils and antigen-restricted TH2 differentiation. PAR2 antagonism may thus hold promise for the treatment of allergic disease.
Chemokines; basophils; chemotaxis; TH2; allergens
The Regulators of G protein Signaling (RGS) proteins were initially characterized as inhibitors of signal transduction cascades initiated by G-protein-coupled receptors (GPCRs) because of their ability to increase the intrinsic GTPase activity of heterotrimeric G proteins. This GTPase accelerating (GAP) activity enhances G protein deactivation and promotes desensitization. However, in addition to this signature trait, emerging data have revealed an expanding network of proteins, lipids, and ions that interact with RGS proteins and confer additional regulatory functions. This review highlights recent advances in our understanding of the physiological functions of one subfamily of RGS proteins with a high degree of homology (B/R4) gleaned from recent studies of knockout mice or cells with reduced RGS expression. We also discuss some of the newly-appreciated interactions of RGS proteins with cellular factors that suggest RGS control of several components of G-protein-mediated pathways as well as a diverse array of non-GPCR-mediated biological responses.
Severe asthma is associated with fixed airway obstruction attributable to inflammation, copious luminal mucus, and increased airway smooth muscle (ASM) mass. Paradoxically, studies demonstrated that the hypertrophic and hyperplastic ASM characteristic of severe asthma has reduced contractile capacity. We compared the G-protein–coupled receptor (GPCR)–induced Ca2+ mobilization and expression of GPCRs and signaling proteins related to procontractile signaling in ASM derived postmortem from subjects who died of nonrespiratory causes, with cells from subjects who died of asthma. Despite the increased or comparable expression of contraction-promoting GPCRs (bradykinin B2 or histamine H1 and protease-activated receptor 1, respectively) in asthmatic ASM cells relative to cells from healthy donors, asthmatic ASM cells exhibited reduced histamine-induced Ca2+ mobilization and comparable responses to bradykinin and thrombin, suggesting a postreceptor signaling defect. Accordingly, the expression of regulator of G-protein signaling–5 (RGS5), an inhibitor of ASM contraction, was increased in cultured, asthmatic ASM cells and in bronchial smooth muscle bundles of both human subjects with asthma and allergen-challenged mice, relative to those of healthy human subjects or naive mice. The overexpression of RGS5 impaired the release of Ca2+ to thrombin, histamine, and carbachol, and reduced the contraction of precision-cut lung slices to carbachol. These results suggest that increased RGS5 expression contributes to decreased myocyte shortening in severe and fatal asthma.
asthma; bronchial smooth muscle; signal transduction; G-protein–coupled receptors
Diffuse myocardial fibrosis, and to a lesser extent global myocardial edema, are important processes in heart disease which are difficult to assess or quantify with cardiovascular magnetic resonance (CMR) using conventional late gadolinium enhancement (LGE) or T1-mapping. Measurement of the myocardial extracellular volume fraction (ECV) circumvents factors that confound T1-weighted images or T1-maps. We hypothesized that quantitative assessment of myocardial ECV would be clinically useful for detecting both focal and diffuse myocardial abnormalities in a variety of common and uncommon heart diseases.
A total of 156 subjects were imaged including 62 with normal findings, 33 patients with chronic myocardial infarction (MI), 33 with hypertrophic cardiomyopathy (HCM), 15 with non-ischemic dilated cardiomyopathy (DCM), 7 with acute myocarditis, 4 with cardiac amyloidosis, and 2 with systemic capillary leak syndrome (SCLS). Motion corrected ECV maps were generated automatically from T1-maps acquired pre- and post-contrast calibrated by blood hematocrit. Abnormally-elevated ECV was defined as >2SD from the mean ECV in individuals with normal findings. In HCM the size of regions of LGE was quantified as the region >2 SD from remote.
Mean ECV of 62 normal individuals was 25.4 ± 2.5% (m ± SD), normal range 20.4%-30.4%. Mean ECV within the core of chronic myocardial infarctions (without MVO) (N = 33) measured 68.5 ± 8.6% (p < 0.001 vs normal). In HCM, the extent of abnormally elevated ECV correlated to the extent of LGE (r = 0.72, p < 0.001) but had a systematically greater extent by ECV (mean difference 19 ± 7% of slice). Abnormally elevated ECV was identified in 4 of 16 patients with non-ischemic DCM (38.1 ± 1.9% (p < 0.001 vs normal) and LGE in the same slice appeared “normal” in 2 of these 4 patients. Mean ECV values in other disease entities ranged 32-60% for cardiac amyloidosis (N = 4), 40-41% for systemic capillary leak syndrome (N = 2), and 39-56% within abnormal regions affected by myocarditis (N = 7).
ECV mapping appears promising to complement LGE imaging in cases of more homogenously diffuse disease. The ability to display ECV maps in units that are physiologically intuitive and may be interpreted on an absolute scale offers the potential for detection of diffuse disease and measurement of the extent and severity of abnormal regions.
Fibrosis; Edema; Gadolinium; Myocardial infarction; Hypertrophic cardiomyopathy; Dilated cardiomyopathy; Myocarditis; Systemic capillary leak syndrome
In severe asthma, bronchodilator- and steroid-insensitive airflow obstruction develops through unknown mechanisms characterized by increased lung airway smooth muscle (ASM) mass and stiffness. We explored the role of a Regulator of G-protein Signaling protein (RGS4) in the ASM hyperplasia and reduced contractile capacity characteristic of advanced asthma. Using immunocytochemical staining, ASM expression of RGS4 was determined in endobronchial biopsies from healthy subjects and those from subjects with mild, moderate and severe asthma. Cell proliferation assays, agonist-induced calcium mobilization and bronchoconstriction were determined in cultured human ASM cells and in human precision cut lung slices. Using gain- and loss-of-function approaches, the precise role of RGS proteins was determined in stimulating human ASM proliferation and inhibiting bronchoconstriction. RGS4 expression was restricted to a subpopulation of ASM and was specifically upregulated by mitogens, which induced a hyperproliferative and hypocontractile ASM phenotype similar to that observed in recalcitrant asthma. RGS4 expression was markedly increased in bronchial smooth muscle of patients with severe asthma, and expression correlated significantly with reduced pulmonary function. Whereas RGS4 inhibited G protein-coupled receptor (GPCR)-mediated bronchoconstriction, unexpectedly RGS4 was required for PDGF-induced proliferation and sustained activation of PI3K, a mitogenic signaling molecule that regulates ASM proliferation. These studies indicate that increased RGS4 expression promotes a phenotypic switch of ASM, evoking irreversible airway obstruction in subjects with severe asthma.
In 1960, Dr. Bayard Clarkson described a patient experiencing sporadic episodes of hypovolemia, hypotension, and edema. Plasma during the acute attack induced a “shock”-like syndrome when given systemically in rats. The unusual and enigmatic “Systemic Capillary Leak Syndrome” (SCLS) named for Dr. Clarkson is of unknown etiology and is characterized by transient, severe, reversible hemoconcentration and hypoalbuminemia due to leakage of fluids and macromolecules (up to 900 kDa) into tissues (1). Fewer than 150 cases of SCLS have been reported since 1960, but the nonspecific presenting symptoms and signs and high mortality rate may have resulted in under-recognition of this disorder. Given the substantial overlap of SCLS with other “shock” syndromes, including sepsis, anaphylaxis, and angioedema, clinicians should consider this diagnosis in patients with unexplained edema, increased hematocrit, and hypotension.
Cyclic AMP-induced phosphorylation of the transcription factor CREB elicits expression of genes mediating diverse biological functions. In lymphoid organs, the neurotransmitter norepinephrine stimulates β2-adrenergic receptors on B lymphocytes to promote CREB-dependent expression of genes like the B-cell Oct 2 coactivator (OCA-B). Although CREB phosphorylation recruits co-factors such as CBP/p300 to stimulate transcription, bona fide endogenous inhibitors of CREB-coactivator or CREB-DNA interactions have not emerged. Here, we identified RGS13, a member of the Regulator of G protein Signaling (RGS) protein family, as a nuclear factor that suppresses CREB-mediated gene expression. cAMP or Ca2+ signaling promoted RGS13 accumulation in the nucleus, where it formed a complex with phosphorylated CREB and CBP/p300. RGS13 reduced the apparent affinity of pCREB for both the CRE and CBP. B lymphocytes from Rgs13−/− mice had more β2-agonist-induced OCA-B expression. Thus, RGS13 inhibits CREB-dependent transcription of target genes through disruption of complexes formed at the promoter.
IgE-mediated mast cell degranulation and release of vasoactive mediators induced by allergens elicits allergic responses. Although G-protein-coupled receptor (GPCR)-induced signals may amplify IgE-dependent degranulation, how GPCR signaling in mast cells is regulated remains incompletely defined. We investigated the role of Regulator of G protein signaling (RGS) proteins in the modulation of these pathways in human mast cells. Several RGS proteins were expressed in mast cells including RGS13, which we previously showed inhibited IgE-mediated mast cell degranulation and anaphylaxis in mice. To characterize how RGS13 affects GPCR-mediated functions of human mast cells, we analyzed human mast cell lines (HMC-1 or LAD2) depleted of RGS13 by specific siRNA or shRNA and HMC-1 cells overexpressing RGS13. Transient RGS13 knockdown in LAD2 cells led to increased degranulation to sphingosine-1-phosphate, but not to antigen/IgE or C3a. Relative to control cells, HMC-1 cells stably expressing an RGS13-specific shRNA had greater Ca2+ mobilization in response to several GPCR ligands such as adenosine, C5a, sphingosine-1-phosphate (S-1P), and CXCL12 than wild-type cells. Akt phosphorylation, chemotaxis and cytokine (interleukin 8, IL-8) secretion induced by CXCL12 were also greater in shRGS13-HMC-1 cells compared to control. RGS13 overexpression inhibited CXCL12-evoked Ca2+ mobilization, Akt phosphorylation and chemotaxis. These results suggest that RGS13 restricts certain GPCR-mediated biological responses of human mast cells.
Signal transduction; Chemokines; Mast cells/basophils
Abstract Allergic diseases such as asthma are elicited by maladaptive activation of immune cells such as mast cells and lymphocytes by otherwise innocuous allergens. The numerous mediators secreted by such cells promote both acute inflammation and, in many instances, chronic tissue remodeling. Most of these compounds exert their effects on end-organ targets such as epithelial and endothelial cells and airway smooth muscle by activating G-protein-coupled receptors (GPCRs), which are by far the most abundant type of cell surface receptor. Since GPCRs are also the most common target of allergy therapeutics, a better understanding of their intracellular signaling mechanisms is vital to improve the efficacy of such drugs or to develop new targets. In this review, we focus on some of the new regulatory elements that control the duration and amplitude of GPCR signal transduction pathways in immune effector cells and end-organ structural cells affected by allergic inflammation.
G proteins; RGS proteins; Signal transduction; Allergy; Asthma; Inflammation; Mast cells; Lymphocytes; Bronchial smooth muscle
Mast cells provoke allergic responses through degranulation and release of proinflammatory mediators after antigen crosslinking of the high affinity immunoglobulin E (IgE) receptor (FcεRI). Regulator of G protein Signaling (RGS) proteins negatively control G-protein-coupled receptor-mediated signaling through GTPase accelerating protein (GAP) activity. Here, we show that Rgs13 inhibits allergic responses by physically interacting with the regulatory p85α subunit of PI3K in mast cells and disrupting its association with an FcεRI-activated scaffold complex. Rgs13−/− mice exhibited increased IgE-mediated mast cell degranulation and anaphylaxis. Thus, apart from its regulation of GPCRs, Rgs13 inhibits immune receptor-induced signalosome assembly in MCs. Abnormal Rgs13 expression or function may underlie some cases of idiopathic anaphylaxis or disorders of amplified MC activity.
COPI, a protein complex consisting of coatomer and the small GTPase
ARF1, is an integral component of some intracellular transport
carriers. The association of COPI with secretory membranes has been
implicated in the maintenance of Golgi integrity and the normal
functioning of intracellular transport in eukaryotes. The regulator of
G protein signaling, RGS4, interacted with the COPI subunit β′-COP in
a yeast two-hybrid screen. Both recombinant RGS4 and RGS2 bound
purified recombinant β′-COP in vitro. Endogenous cytosolic RGS4 from
NG108 cells and RGS2 from HEK293T cells cofractionated with the
COPI complex by gel filtration. Binding of β′-COP to RGS4 occurred
through two dilysine motifs in RGS4, similar to those contained in some
aminoglycoside antibiotics that are known to bind coatomer. RGS4
inhibited COPI binding to Golgi membranes independently of its
GTPase-accelerating activity on Giα. In RGS4-transfected
LLC-PK1 cells, the amount of COPI in the Golgi region was considerably
reduced compared with that in wild-type cells, but there was no
detectable difference in the amount of either Golgi-associated ARF1 or
the integral Golgi membrane protein giantin, indicating that Golgi
integrity was preserved. In addition, RGS4 expression inhibited
trafficking of aquaporin 1 to the plasma membrane in LLC-PK1 cells and
impaired secretion of placental alkaline phosphatase from HEK293T
cells. The inhibitory effect of RGS4 in these assays was independent of
GTPase-accelerating activity but correlated with its ability to bind
COPI. Thus, these data support the hypothesis that these RGS proteins
sequester coatomer in the cytoplasm and inhibit its recruitment onto
Golgi membranes, which may in turn modulate Golgi–plasma membrane or