Regulators Of G Protein Signaling Proteins Research Articles

Basic Science of Cardiac Resynchronization Therapy

Electromechanical dyssynchrony can markedly worsen heart failure (HF) morbidity and mortality, independent of traditional risk factors.1–4 Depending on the metric used, current estimates of the prevalence of dyssynchrony vary from 25–30% in patients with HF (based on QRS widening) up to 60%, based on tissue Doppler or MRI measures of dyssynchronous contraction of the left ventricle (LV).5,6 Cardiac resynchronization therapy (CRT) or biventricular pacing has emerged as a promising option to treat patients with HF and dyssynchronous contraction.7–9 The past few decades have seen the rise of pharmacotherapy, primarily through agents that antagonize the effect of excessive concentrations of circulating neurohormones, yet, HF-related morbidity and mortality remain high.3–6 Biventricular stimulation has been demonstrated to improve contractile performance in patients with mechanical dyssynchrony acutely and chronically while also prolonging long-term survival—something not yet achieved by drug therapy.10 Although the clinical and mechanical effectiveness of CRT are well described, 30% of patients do not benefit from CRT and clinical criteria to identify CRT nonresponders remain elusive8,11,12 Currently, the most widely used predictor of reverse remodeling is the presence of marked mechanical dyssynchrony before CRT, as indexed by the width of the QRS.13 Mechanical dyssynchrony seems important, yet imaging-based measures have not predicted response well14 and even improvement in dyssynchrony after initiation of CRT only weakly predicts chronic response.15 Limited understanding of the molecular mechanisms underlying reverse cardiac remodeling induced by CRT has hampered the selection of potential responders. In this review, we focus on the electrophysiological aspects and molecular networks underlying the benefits of CRT. We will review how CRT homogenizes regional differences in stress kinase signaling and electric remodeling and then review its global effect on myocyte function and its …

Two Chimeric Regulators of G-protein Signaling (RGS) Proteins Differentially Modulate Soybean Heterotrimeric G-protein Cycle

Heterotrimeric G-proteins and the regulator of G-protein signaling (RGS) proteins, which accelerate the inherent GTPase activity of Gα proteins, are common in animals and encoded by large gene families; however, in plants G-protein signaling is thought to be more limited in scope. For example, Arabidopsis thaliana contains one Gα, one Gβ, three Gγ, and one RGS protein. Recent examination of the Glycine max (soybean) genome reveals a larger set of G-protein-related genes and raises the possibility of more intricate G-protein networks than previously observed in plants. Stopped-flow analysis of GTP-binding and GDP/GTP exchange for the four soybean Gα proteins (GmGα1-4) reveals differences in their kinetic properties. The soybean genome encodes two chimeric RGS proteins with an N-terminal seven transmembrane domain and a C-terminal RGS box. Both GmRGS interact with each of the four GmGα and regulate their GTPase activity. The GTPase-accelerating activities of GmRGS1 and -2 differ for each GmGα, suggesting more than one possible rate of the G-protein cycle initiated by each of the Gα proteins. The differential effects of GmRGS1 and GmRGS2 on GmGα1-4 result from a single valine versus alanine difference. The emerging picture suggests complex regulation of the G-protein cycle in soybean and in other plants with expanded G-protein networks.

A finer tuning of G-protein signaling through regulated control of RGS proteins

Regulators of G-protein signaling (RGS) proteins are GTPase-activating proteins (GAP) for various Gα subunits of heterotrimeric G proteins. Through this mechanism, RGS proteins regulate the magnitude and duration of G-protein-coupled receptor signaling and are often referred to as fine tuners of G-protein signaling. Increasing evidence suggests that RGS proteins themselves are regulated through multiple mechanisms, which may provide an even finer tuning of G-protein signaling and crosstalk between G-protein-coupled receptors and other signaling pathways. This review summarizes the current data on the control of RGS function through regulated expression, intracellular localization, and covalent modification of RGS proteins, as related to cell function and the pathogenesis of diseases.

Generation of Gαi2G184S conditional mutant mice to study regulator of G protein signaling (RGS) proteins

GTPase activity of G inhibitory proteins (Gαi) is strongly accelerated by RGS proteins. A mutant mouse (Gαi2 G184S) in which RGS‐mediated negative regulation is lost shows protection against ex vivo cardiac ischemia‐reperfusion injury, and had worsened outcomes in two in vivo models of heart failure. However, such mice have a complex phenotype and decreased viability. In order to better define the mechanisms mediating these cardiovascular effects, we have developed a novel Gαi2 G184S conditional knock‐in mouse model with a loxP‐flanked minigene encoding wild‐type exons 5–9 in front of the mutant exon 5. We show that floxed‐allele‐ carrying mice appear normal and fertile. Upon crossing the Gαi2 G184S conditional to the Cre‐ERT2 transgenic mice, the wildtype sequence is efficiently deleted by Cre‐mediated excision in vivo after tamoxifen treatment and Gαi2 mRNA contains mostly mutant sequence. Levels of Gαi2protein were somewhat reduced in several tissues, but responses to the stimulation of α2 and muscarinic M2 receptors were similar between Cre‐ERT2/Gαi2 +/+ and Cre‐ERT2/Gαi2 G184Scond/G184Scond mice before tamoxifen treatment. After tamoxifen treatment, Gαi2 mediated responses are enhanced in Cre‐ERT2/Gαi2 G184Scond/G184Scond mice. This mouse model will be a valuable tool to dissect temporal and tissue‐specific roles of GPCRs, Gαi2, and RGS proteins. (Supported by NIH R01 GM039561‐23)

Oxidative Stress Regulation of Regulator of G‐Protein Signaling 4 (RGS4)

Regulators of G‐protein signaling (RGS) proteins are a family of proteins that regulate G‐protein coupled receptors (GPCRs) by acting as GTPase accelerating proteins (GAPs). RGS proteins increase the rate of hydrolysis of GTP to GDP in select Gα subunits, including Gαo, Gαi, and Gαq but not Gαs. One of these proteins, RGS4, has been implicated in several neurological diseases. In this study, we analyze the effect of oxidative stress on RGS4 regulation in neuronal cells and the effect of lipid peroxidation products, such as 4‐hydroxy‐nonenal (4HNE), on RGS4 activity. Select RGS4 isoforms were induced during oxidative stress with H2O2. 4HNE forms covalent adducts on reactive amino acids such as cysteine. Previous research revealed that modification only occurs at select cysteine residues. Several mutants were made containing only one of the modified cysteines, either C71, C148, and, C183, and were treated with increasing amounts of 4HNE and then assayed for coupling to the native binding partner Gαo by ALPHA‐Screen. The wild type construct showed sensitivity to 4HNE but the cysteine null mutant did not. Of the single cysteine containing mutants, only C148 conferred sensitivity to RGS4. Research support provided by the Pharmacological Sciences Training Grant (NIH/NIGMST32GM067795).

Structural studies of RGS inhibitors

Regulators of G‐protein signaling (RGS) proteins regulate G‐protein coupled receptor (GPCR) signal transduction cascades by acting as GTPase accelerating proteins upon binding activated Gα subunits. RGS proteins express in cell‐type specific manners, couple to and regulate specific GPCRs, and are overexpressed in pathologies from cancer to Parkinson's disease, and as such, they are an attractive drug target. Small‐molecule RGS4 inhibitor screens have yielded a number of covalent thiol modifiers that act allosterically at Cys148, far from the RGS/Gα interface. Thus far, the structural basis of RGS4 inhibition is unknown, so in the current study we investigate the structural consequence of RGS4's covalent modification that leads to inhibition of Gα binding. To ensure structure homogeneity, we expressed in and purified from Escherichia coli recombinant RGS4 with cysteines removed, except Cys148, and obtained nuclear magnetic resonance spectra for free and modified RGS4. Overlaid 15N‐1H HSQC spectra show significant changes in RGS4 structure upon small molecule modification. These data provide further evidence for a region that may be amenable for rational drug design. Project funded by 5T32GM067795‐08.

Defective Retinal Depolarizing Bipolar Cells in Regulators of G Protein Signaling (RGS) 7 and 11 Double Null Mice

Two members of the R7 subfamily of regulators of G protein signaling, RGS7 and RGS11, are present at dendritic tips of retinal depolarizing bipolar cells (DBCs). Their involvement in the mGluR6/Gα(o)/TRPM1 pathway that mediates DBC light responses has been implicated. However, previous genetic studies employed an RGS7 mutant mouse that is hypomorphic, and hence the exact role of RGS7 in DBCs remains unclear. We have made a true RGS7-null mouse line with exons 6-8 deleted. The RGS7(-/-) mouse is viable and fertile but smaller in body size. Electroretinogram (ERG) b-wave implicit time in young RGS7(-/-) mice is prolonged at eye opening, but the phenotype disappears at 2 months of age. Expression levels of RGS6 and RGS11 are unchanged in RGS7(-/-) retina, but the Gβ5S level is significantly reduced. By characterizing a complete RGS7 and RGS11 double knock-out (711dKO) mouse line, we found that Gβ5S expression in the retinal outer plexiform layer is eliminated, as is the ERG b-wave. Ultrastructural defects akin to those of Gβ5(-/-) mice are evident in 711dKO mice. In retinas of mice lacking RGS6, RGS7, and RGS11, Gβ5S is undetectable, whereas levels of the photoreceptor-specific Gβ5L remain unchanged. Whereas RGS6 alone sustains a significant amount of Gβ5S expression in retina, the DBC-related defects in Gβ5(-/-) mice are caused solely by a combined loss of RGS7 and RGS11. Our data support the notion that the role of Gβ5 in the retina, and likely in the entire nervous system, is mediated exclusively by R7 RGS proteins.

Expression of the Gβ5/R7-RGS protein complex in pituitary and pancreatic islet cells

Heterotrimeric G protein subunit Gβ5 is expressed primarily in the nervous system and retina and, among Gβ isoforms, is unique in its ability to heterodimerize with regulator of G protein signaling (RGS) proteins of the R7 subfamily (R7-RGS) [see [1, 2] for recent reviews]. The R7-RGS subfamily consists of RGS proteins 6, 7, 9, and 11 which can form tight heterodimers with the G protein β5 subunit through a Gγ-like (GGL) domain [1, 2]. R7-RGS subfamily members are unstable in the absence of Gβ5 [3]. Previous study from our laboratory showed that in addition to its expression in brain and neural cell lines, Gβ5 was also expressed in the αT3-1 gonadotrophic pituitary cell line [4]. We show now that the Gβ5/R7-RGS complex is expressed in corticotroph-derived pituitary AtT-20 cells, pituitary gland and purified pancreatic islets. The expression of Gβ5 in various endocrine cell lines and native tissues raises the possibility that hormone secretion, and not just neurotransmission and phototransduction, may be regulated by the Gβ5/R7-RGS complex.

Fluorescence-based assay probing regulator of G protein signaling partner proteins

The regulator of G protein signaling (RGS) proteins are one of the essential modulators for the G protein system. Besides regulating G protein signaling by accelerating the GTPase activity of Gα subunits, RGS proteins are implicated in exerting other functions; they are also known to be involved in several diseases. Moreover, the existence of a single RGS protein in plants and its seven-transmembrane domain found in 2003 triggered efforts to unveil detailed structural and functional information of RGS proteins. We present a method for real-time examination of the protein–protein interactions between RGS and Gα subunits. AtRGS1 from plants and RGS4 from mammals were site-directedly labeled with the fluorescent probe Lucifer yellow on engineered cysteine residues and used to interact with different Gα subunits. The physical interactions can be revealed by monitoring the real-time fluorescence changes (8.6% fluorescence increase in mammals and 27.6% in plants); their correlations to functional exertion were shown with a GTPase accelerating activity assay and further confirmed by measurement of Kd. We validate the effectiveness of this method and suggest its application to the exploration of more RGS signaling partner proteins in physiological and pathological studies.

An RGS4-Mediated Phenotypic Switch of Bronchial Smooth Muscle Cells Promotes Fixed Airway Obstruction in Asthma

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.

RGS proteins in heart: brakes on the vagus

It has been nearly a century since Otto Loewi discovered that acetylcholine (ACh) release from the vagus produces bradycardia and reduced cardiac contractility. It is now known that parasympathetic control of the heart is mediated by ACh stimulation of Gi/o-coupled muscarinic M2 receptors, which directly activate G protein-coupled inwardly rectifying potassium (GIRK) channels via Gβγ resulting in membrane hyperpolarization and inhibition of action potential (AP) firing. However, expression of M2R–GIRK signaling components in heterologous systems failed to recapitulate native channel gating kinetics. The missing link was identified with the discovery of regulator of G protein signaling (RGS) proteins, which act as GTPase-activating proteins to accelerate the intrinsic GTPase activity of Gα resulting in termination of Gα- and Gβγ-mediated signaling to downstream effectors. Studies in mice expressing an RGS-insensitive Gαi2 mutant (G184S) implicated endogenous RGS proteins as key regulators of parasympathetic signaling in heart. Recently, two RGS proteins have been identified as critical regulators of M2R signaling in heart. RGS6 exhibits a uniquely robust expression in heart, especially in sinoatrial (SAN) and atrioventricular nodal regions. Mice lacking RGS6 exhibit increased bradycardia and inhibition of SAN AP firing in response to CCh as well as a loss of rapid activation and deactivation kinetics and current desensitization for ACh-induced GIRK current (IKACh). Similar findings were observed in mice lacking RGS4. Thus, dysregulation in RGS protein expression or function may contribute to pathologies involving aberrant electrical activity in cardiac pacemaker cells. Moreover, RGS6 expression was found to be up-regulated in heart under certain pathological conditions, including doxorubicin treatment, which is known to cause life-threatening cardiotoxicity and atrial fibrillation in cancer patients. On the other hand, increased vagal tone may be cardioprotective in heart failure where acetylcholinesterase inhibitors and vagal stimulation have been proposed as potential therapeutics. Together, these studies identify RGS proteins, especially RGS6, as new therapeutic targets for diseases such as sick sinus syndrome or other maladies involving abnormal autonomic control of the heart.

RGS Proteins Maintain Robustness of GPCR-GIRK Coupling

Regulators of G-protein signaling (RGS) are GTPase activating proteins (GAP) that reduce response amplitudes of activated G-protein coupled receptors (GPCRs). We discovered that, although RGS proteins drastically accelerate kinetics of GPCR-coupled K+ currents (GIRK), they actually increased amplitudes of inhibitory neurotransmitter-evoked GIRK currents. The RGS-Box domain alone is sufficient for stimulation of transmitter activation of K+ currents, but its membrane association enhances the efficiency of stimulation. Moreover, RGS4 mutants with compromised GAP activity still augment GPCR-GIRK coupling. Among the pertussis toxin sensitive G-proteins, we found that RGS4 selectively stimulates G alpha-o to maintain robustness of GPCR-GIRK coupling. Opposing actions of RGS proteins thus both stimulate and inhibit G-proteins to modulate ultimate amplitudes of transmitter-induced GIRK currents and to differentiate signal intensity coupled to various G-protein isoforms.

Controlling Parasympathetic Regulation of Heart Rate: A Gatekeeper Role for RGS Proteins in the Sinoatrial Node

Neurotransmitters released from sympathetic and parasympathetic nerve terminals in the sinoatrial node (SAN) exert their effects via G-protein-coupled receptors. Integration of these different G-protein signals within pacemaker cells of the SAN is critical for proper regulation of heart rate and function. For example, excessive parasympathetic signaling can be associated with sinus node dysfunction (SND) and supraventricular arrhythmias. Our previous work has shown that one member of the regulator of G-protein signaling (RGS) protein family, RGS4, is highly and selectively expressed in pacemaker cells of the SAN. Consistent with its role as an inhibitor of parasympathetic signaling, RGS4-knockout mice have reduced basal heart rates and enhanced negative chronotropic responses to parasympathetic agonists. Moreover, RGS4 appears to be an important part of SA nodal myocyte signaling pathways that mediate G-protein-coupled inwardly rectifying potassium channel (GIRK) channel activation/deactivation and desensitization. Since RGS4 acts immediately downstream of M2 muscarinic receptors, it is tempting to speculate that RGS4 functions as a master regulator of parasympathetic signaling upstream of GIRKs, HCNs, and L-type Ca2+ channels in the SAN. Thus, loss of RGS4 function may lead to increased susceptibility to conditions associated with increased parasympathetic signaling, including bradyarrhythmia, SND, and atrial fibrillation.

Small Molecule Inhibitors of Regulators of G Protein Signaling (RGS) Proteins

Recently regulators of G protein signalling (RGS) proteins have emerged as potential therapeutic targets since they provide an alternative method of modulating the activity of GPCRs, the target of so many drugs. Inhibitors of RGS proteins must block a protein-protein interaction (RGS-Gα), but also be cell and, depending on the therapeutic target, blood brain barrier permeable. A lead compound (1a) was identified as an inhibitor of RGS4 in a screening assay and this has now been optimised for activity, selectivity and solubility. The newly developed ligands (11b, 13) display substantial selectivity over the closely related RGS8 protein, lack the off-target calcium mobilisation activity of the lead 1a and have excellent aqueous solubility. They are currently being evaluated in vivo in rodent models of depression.

Structural Basis for the 14-3-3 Protein-dependent Inhibition of the Regulator of G Protein Signaling 3 (RGS3) Function

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins for the α-subunit of heterotrimeric G proteins. The function of certain RGS proteins is negatively regulated by 14-3-3 proteins, a family of highly conserved regulatory molecules expressed in all eukaryotes. In this study, we provide a structural mechanism for 14-3-3-dependent inhibition of RGS3-Gα interaction. We have used small angle x-ray scattering, hydrogen/deuterium exchange kinetics, and Förster resonance energy transfer measurements to determine the low-resolution solution structure of the 14-3-3ζ·RGS3 complex. The structure shows the RGS domain of RGS3 bound to the 14-3-3ζ dimer in an as-yet-unrecognized manner interacting with less conserved regions on the outer surface of the 14-3-3 dimer outside its central channel. Our results suggest that the 14-3-3 protein binding affects the structure of the Gα interaction portion of RGS3 as well as sterically blocks the interaction between the RGS domain and the Gα subunit of heterotrimeric G proteins.

G Protein-coupled Receptors and Resistance to Inhibitors of Cholinesterase-8A (Ric-8A) Both Regulate the Regulator of G Protein Signaling 14 (RGS14)·Gαi1 Complex in Live Cells

Regulator of G protein Signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates both conventional and unconventional G protein signaling pathways. Like other RGS (regulator of G protein signaling) proteins, RGS14 acts as a GTPase accelerating protein to terminate conventional Gα(i/o) signaling. However, unlike other RGS proteins, RGS14 also contains a G protein regulatory/GoLoco motif that specifically binds Gα(i1/3)-GDP in cells and in vitro. The non-receptor guanine nucleotide exchange factor Ric-8A can bind and act on the RGS14·Gα(i1)-GDP complex to play a role in unconventional G protein signaling independent of G protein-coupled receptors (GPCRs). Here we demonstrate that RGS14 forms a Gα(i/o)-dependent complex with a G(i)-linked GPCR and that this complex is regulated by receptor agonist and Ric-8A (resistance to inhibitors of cholinesterase-8A). Using live cell bioluminescence resonance energy transfer, we show that RGS14 functionally associates with the α(2A)-adrenergic receptor (α(2A)-AR) in a Gα(i/o)-dependent manner. This interaction is markedly disrupted after receptor stimulation by the specific agonist UK14304, suggesting complex dissociation or rearrangement. Agonist-mediated dissociation of the RGS14·α(2A)-AR complex occurs in the presence of Gα(i/o) but not Gα(s) or Gα(q). Unexpectedly, RGS14 does not dissociate from Gα(i1) in the presence of stimulated α(2A)-AR, suggesting preservation of RGS14·Gα(i1) complexes after receptor activation. However, Ric-8A facilitates dissociation of both the RGS14·Gα(i1) complex and the Gα(i1)-dependent RGS14·α(2A)-AR complex after receptor activation. Together, these findings indicate that RGS14 can form complexes with GPCRs in cells that are dependent on Gα(i/o) and that these RGS14·Gα(i1)·GPCR complexes may be substrates for other signaling partners such as Ric-8A.

The role of RGS protein in agonist-dependent relaxation of GIRK currents in Xenopus oocytes

G protein coupled inward rectifier K + channels (GIRK) are activated by the G βγ subunits of G proteins upon activation of G protein coupled receptors (GPCRs). Receptor-stimulated GIRK currents are known to possess a curious property, termed “agonist-dependent relaxation,” denoting a slow current increase upon stepping the membrane voltage from positive to negative potentials. Regulators of G protein signaling (RGS) proteins have earlier been implicated in this phenomenon since RGS coexpression was required for relaxation to be observed in heterologous expression systems. However, a recent study presented contrasting evidence that GIRK current relaxation reflects voltage sensitive agonist binding to the GPCR. The present study re-examined the role of RGS protein in agonist-dependent relaxation and found that RGS coexpression is not necessary for the relaxation phenomenon. However, RGS4 speeds up relaxation kinetics, allowing the phenomenon to be observed using shorter voltage steps. These findings resolve the controversy regarding the role of RGS protein vs. GPCR voltage sensitivity in mediating agonist-dependent relaxation of GIRK currents.

Integrating energy calculations with functional assays to decipher the specificity of G protein–RGS protein interactions

The diverse RGS protein family is responsible for the precise timing of G-protein signaling. To understand how RGS protein structure encodes their common ability to inactivate G-proteins and their selective G-protein recognition, we integrated structure-based energy calculations with biochemical measurements of RGS protein activity. We revealed that, in addition to previously identified conserved residues, RGS proteins contain another group of variable modulatory residues, which reside at the periphery of the RGS-domain–G-protein interface and fine-tune G-protein recognition. Mutations of modulatory residues in high-activity RGS proteins impaired RGS function, whereas redesign of low-activity RGS proteins in critical modulatory positions yielded complete gain-of-function. Therefore, RGS proteins combine a conserved core interface with peripheral modulatory residues to selectively optimize G-protein recognition and inactivation. Finally, we show that our quantitative framework for analyzing protein-protein interactions can be extended to analyze interaction specificity across other large protein families.

Type 5 G Protein β Subunit (Gβ5) Controls the Interaction of Regulator of G Protein Signaling 9 (RGS9) with Membrane Anchors

The R7 family of regulators of G protein signaling (RGS) proteins, comprising RGS6, RGS7, RGS9, and RGS11, regulate neuronal G protein signaling pathways. All members of the R7 RGS form trimeric complexes with the atypical G protein β subunit, Gβ5, and membrane anchor R7BP or R9AP. Association with Gβ5 and membrane anchors has been shown to be critical for maintaining proteolytic stability of the R7 RGS proteins. However, despite its functional importance, the mechanism of how R7 RGS forms complexes with Gβ5 and membrane anchors remains poorly understood. Here, we used protein-protein interaction, co-localization, and protein stability assays to show that association of RGS9 with membrane anchors requires Gβ5. We further establish that the recruitment of R7BP to the complex requires an intact interface between the N-terminal lobe of RGS9 and protein interaction surface of Gβ5. Site-directed mutational analysis reveals that distinct molecular determinants in the interface between Gβ5 and N-terminal Dishevelled, EGL-10, Pleckstrin/DEP Helical Extension (DEP/DHEY) domains are differentially involved in R7BP binding and proteolytic stabilization. On the basis of these findings, we conclude that Gβ5 contributes to the formation of the binding site to the membrane anchors and thus is playing a central role in the assembly of the proteolytically stable trimeric complex and its correct localization in the cell.

P53 Negatively Regulates RGS13 Protein Expression in Immune Cells

RGS13, a member of the regulator of G protein signaling (RGS) family, inhibits G protein-coupled receptor signaling in B cells and mast cells (MCs) and suppresses IgE-antigen-induced MC degranulation and anaphylaxis. Although RGS13 expression is induced by immune receptor and chemokine receptor stimulation, the molecular regulation of RGS13 transcription is unknown. Here, we investigated the role of two p53 response elements (REs) in the regulation of RGS13 promoter activity and expression. We found that a 1000-bp DNA fragment upstream of the ATG translation start site (TSS) had promoter activity in reporter gene assays, and deletion or mutation of a p53-binding motif nearest the TSS abolished promoter activity. Notably, p53 bound to both REs in the RGS13 promoter in vivo as assessed by chromatin immunoprecipitation, suggesting that the p53 RE most distal to the TSS is physiologically inactive. We detected reduced RGS13 expression in MCs exogenously expressing p53 or treated with doxorubicin, which induces genotoxic stress and leads to p53 accumulation. RNA silencing of p53 up-regulated RGS13 expression in B lymphocytes, and bone marrow-derived MCs from p53(-/-) mice had increased RGS13 expression. Finally, p53-depleted B cells with increased RGS13 expression had reduced Ca(2+) mobilization in response to sphingosine 1-phosphate. These studies indicate that p53 may modulate immune responses through suppression of RGS13 transcription in MCs and B cells.