Sequential Fractionation Strategy Identifies Three Missing Proteins in the Mitochondrial Proteome of Commonly Used Cell Lines.

Mitochondria are undeniably the cell powerhouse, directly affecting cell survival and fate. Growing evidence suggest that mitochondrial protein repertoire affects metabolic activity and plays an important role in determining cell proliferation/differentiation or quiescence shift. Consequently, the bioenergetic status of a cell is associated with the quality and abundance of the mitochondrial populations and proteomes. Mitochondrial morphology changes in the development of different cellular functions associated with metabolic switches. It is therefore reasonable to speculate that different cell lines do contain different mitochondrial-associated proteins, and the investigation of these pools may well represent a source for mining missing proteins (MPs). A very effective approach to increase the number of IDs through mass spectrometry consists of reducing the complexity of the biological samples by fractionation. The present study aims at investigating the mitochondrial proteome of five phenotypically different cell lines, possibly expressing some of the MPs, through an enrichment-fractionation approach at the organelle and protein level. We demonstrate a substantial increase in the proteome coverage, which, in turn, increases the likelihood of detecting low abundant proteins, often falling in the category of MPs, and resulting, for the present study, in the identification of METTL12, FAM163A, and RGS13. All MS data have been deposited to the MassIVE data repository ( https://massive.ucsd.edu ) with the data set identifier MSV000082409 and PXD010446.

Regulation of the regulator of G protein signaling 2 expression and cellular localization by PKA and PKC pathways in mouse granulosa cells.

G protein-coupled receptor (GPCR) activation-mediated PKA and PKC pathways have been recognized to be important in ovarian physiology. Expression of regulator of G-protein signaling 2 (RGS2) has been reported in ovarian granulosa cells. The detailed mechanisms in PKA- and PKC-regulated RGS2 expression and cellular translocation in granulosa cells remain mostly unclear. PKA activator 8-bromo-cAMP and PKC activator phorbol-12, 13-didecanoate appeared to rapidly elevate both protein and mRNA levels and promoter activation of RGS2 gene. Two consensus Sp1 elements within the shortest 78 bp fragment of RGS2 promoter sequence were essential for the full responsiveness to PKA and PKC. PKC activation appeared to increase the RGS2 translocation from nucleus to cytosol. PKA- and PKC-mediated RGS2 transcription in a Sp-1-dependent manner and a PKC-mediated RGS2 intracellular translocation were noted in granulosa cells.

Identification of extracellularly phosphorylated membrane proteins.

Ecto-protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC-MS/MS. A "false-positive" strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G-protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface-exposed peptides when extracellular adenosine-5'-triphosphate is elevated during purinergic signalling.

RGS9-2 modulates sensory and mood related symptoms of neuropathic pain.

The signal transduction modulator Rgs9-2 (Regulator of G protein signaling 9-2) plays a key role in dopaminergic and opioidergic transmission in the striatum. Rgs9-2 is a potent modulator of opiate reward and analgesia, but its role in chronic pain remains unknown. Here, we use the spared nerve injury model (SNI), to evaluate the influence of Rgs9-2 in sensory symptoms, as well as in anxiety and depression-like behaviors observed under neuropathic pain conditions. Our data demonstrate that knockout of the Rgs9 gene reduces the intensity of thermal hyperalgesia and mechanical allodynia the first few days after nerve injury. This small, but significant effect is only observed at early time points after nerve injury, whereas after the first week of SNI, Rgs9 knockout (Rgs9KO) and Rgs9 wildtype (Rgs9WT) mice show similar levels of mechanical allodynia and thermal hyperalgesia. Furthermore, Rgs9-2 deletion exacerbates anxiety and depression like behaviors several weeks after the emergence of the neuropathic pain symptoms. Our findings also reveal a temporal and regional regulation of Rgs9-2 protein expression by neuropathic pain, as Rgs9-2 levels are reduced in the spinal cord a few days after nerve injury, whereas decreased Rgs9-2 levels in the Nucleus Accumbens (NAc) are only observed several weeks after nerve injury. Thus, adaptations in Rgs9-2 activity in the spinal cord and in the NAc may contribute to sensory and affective components of neuropathic pain.

Regulator of g protein signaling 3 modulates wnt5b calcium dynamics and somite patterning.

Vertebrate development requires communication among cells of the embryo in order to define the body axis, and the Wnt-signaling network plays a key role in axis formation as well as in a vast array of other cellular processes. One arm of the Wnt-signaling network, the non-canonical Wnt pathway, mediates intracellular calcium release via activation of heterotrimeric G proteins. Regulator of G protein Signaling (RGS) proteins can accelerate inactivation of G proteins by acting as G protein GTPase-activating proteins (GAPs), however, the possible role of RGS proteins in non-canonical Wnt signaling and development is not known. Here, we identify rgs3 as having an overlapping expression pattern with wnt5b in zebrafish and reveal that individual knockdown of either rgs3 or wnt5b gene function produces similar somite patterning defects. Additionally, we describe endogenous calcium release dynamics in developing zebrafish somites and determine that both rgs3 and wnt5b function are required for appropriate frequency and amplitude of calcium release activity. Using rescue of gene knockdown and in vivo calcium imaging assays, we demonstrate that the activity of Rgs3 requires its ability to interact with Galpha subunits and function as a G protein GAP. Thus, Rgs3 function is necessary for appropriate frequency and amplitude of calcium release during somitogenesis and is downstream of Wnt5 activity. These results provide the first evidence for an essential developmental role of RGS proteins in modulating the duration of non-canonical Wnt signaling.

Regulator of G protein signaling 2 mediates cardiac compensation to pressure overload and antihypertrophic effects of PDE5 inhibition in mice.

The heart initially compensates for hypertension-mediated pressure overload by enhancing its contractile force and developing hypertrophy without dilation. Gq protein-coupled receptor pathways become activated and can depress function, leading to cardiac failure. Initial adaptation mechanisms to reduce cardiac damage during such stimulation remain largely unknown. Here we have shown that this initial adaptation requires regulator of G protein signaling 2 (RGS2). Mice lacking RGS2 had a normal basal cardiac phenotype, yet responded rapidly to pressure overload, with increased myocardial Gq signaling, marked cardiac hypertrophy and failure, and early mortality. Swimming exercise, which is not accompanied by Gq activation, induced a normal cardiac response, while Rgs2 deletion in Galphaq-overexpressing hearts exacerbated hypertrophy and dilation. In vascular smooth muscle, RGS2 is activated by cGMP-dependent protein kinase (PKG), suppressing Gq-stimulated vascular contraction. In normal mice, but not Rgs2-/- mice, PKG activation by the chronic inhibition of cGMP-selective phosphodiesterase 5 (PDE5) suppressed maladaptive cardiac hypertrophy, inhibiting Gq-coupled stimuli. Importantly, PKG was similarly activated by PDE5 inhibition in myocardium from both genotypes, but PKG plasma membrane translocation was more transient in Rgs2-/- myocytes than in controls and was unaffected by PDE5 inhibition. Thus, RGS2 is required for early myocardial compensation to pressure overload and mediates the initial antihypertrophic and cardioprotective effects of PDE5 inhibitors.

RGS9-2 rescues dopamine D2 receptor levels and signaling in DYT1 dystonia mouse models.

Dopamine D2 receptor signaling is central for striatal function and movement, while abnormal activity is associated with neurological disorders including the severe early-onset DYT1 dystonia. Nevertheless, the mechanisms that regulate D2 receptor signaling in health and disease remain poorly understood. Here, we identify a reduced D2 receptor binding, paralleled by an abrupt reduction in receptor protein level, in the striatum of juvenile Dyt1 mice. This occurs through increased lysosomal degradation, controlled by competition between ß-arrestin 2 and D2 receptor binding proteins. Accordingly, we found lower levels of striatal RGS9-2 and spinophilin. Further, we show that genetic depletion of RGS9-2 mimics the D2 receptor loss of DYT1 dystonia striatum, whereas RGS9-2 overexpression rescues both receptor levels and electrophysiological responses in Dyt1 striatal neurons. This work uncovers the molecular mechanism underlying D2 receptor downregulation in Dyt1 mice and in turn explains why dopaminergic drugs lack efficacy in DYT1 patients despite significant evidence for striatal D2 receptor dysfunction. Our data also open up novel avenues for disease-modifying therapeutics to this incurable neurological disorder.

RGS9 modulates dopamine signaling in the basal ganglia.

Regulators of G protein signaling (RGS) modulate heterotrimeric G proteins in part by serving as GTPase-activating proteins for Galpha subunits. We examined a role for RGS9-2, an RGS subtype highly enriched in striatum, in modulating dopamine D2 receptor function. Viral-mediated overexpression of RGS9-2 in rat nucleus accumbens (ventral striatum) reduced locomotor responses to cocaine (an indirect dopamine agonist) and to D2 but not to D1 receptor agonists. Conversely, RGS9 knockout mice showed heightened locomotor and rewarding responses to cocaine and related psychostimulants. In vitro expression of RGS9-2 in Xenopus oocytes accelerated the off-kinetics of D2 receptor-induced GIRK currents, consistent with the in vivo data. Finally, chronic cocaine exposure increased RGS9-2 levels in nucleus accumbens. Together, these data demonstrate a functional interaction between RGS9-2 and D2 receptor signaling and the behavioral actions of psychostimulants and suggest that psychostimulant induction of RGS9-2 represents a compensatory adaptation that diminishes drug responsiveness.

Microfabricated channel array electrophoresis for characterization and screening of enzymes using RGS-G protein interactions as a model system.

A microfluidic chip consisting of parallel channels designed for rapid electrophoretic enzyme assays was developed. Radial arrangement of channels and a common waste channel allowed chips with 16 and 36 electrophoresis units to be fabricated on a 7.62 x 7.62 cm(2) glass substrate. Fluorescence detection was achieved using a Xe arc lamp source and commercial charge-coupled device (CCD) camera to image migrating analyte zones in individual channels. Chip performance was evaluated by performing electrophoretic assays for G protein GTPase activity on chip using BODIPY-GTP as enzyme substrate. A 16-channel design proved to be useful in extracting kinetic information by allowing serial electrophoretic assays from 16 different enzyme reaction mixtures at 20 s intervals in parallel. This system was used to rapidly determine enzyme concentrations, optimal enzymatic reaction conditions, and Michaelis-Menten constants. A chip with 36 channels was used for screening for modulators of the G protein-RGS protein interaction by assaying the amount of product formed in enzyme reaction mixtures that contained test compounds. Thirty-six electrophoretic assays were performed in 30 s suggesting the potential throughput up to 4320 assays/h with appropriate sample handling procedures. Both designs showed excellent reproducibility of peak migration time and peak area. Relative standard deviations of normalized peak area of enzymatic product BODIPY-GDP were 5% and 11%, respectively, in the 16- and 36-channel designs.

RGS1 expression is associated with poor prognosis in multiple myeloma.

AIMS: Multiple myeloma (MM) is an invariably fatal disease with highly heterogeneous outcome. Because of this heterogeneity of MM, risk stratification is crucial for therapeutic decision-making. However, no immunohistochemical prognostic or predictive markers have been established yet. The expression of regulator of G-protein signalling (RGS) proteins, which desensitise G-protein-coupled receptor signalling, has been reported to be associated with the prognosis of various malignancies. Recently, our group demonstrated the importance of RGS1 in chemokine signalling in a human MM cell line and normal plasmablasts. In the present study, we explored the prognostic value of RGS1 expression in patients with MM using immunohistochemistry. METHODS: We evaluated RGS1 protein expression in 79 bone marrow biopsies obtained from patients with MM between 2008 and 2010 at Asan Medical Center. Correlations between RGS1 expression and clinicopathological factors were analysed. RESULTS: High RGS1 protein expression was significantly associated with poor overall survival (p=0.005). After an adjusted multivariable analysis, high RGS1 protein expression (p=0.010), high International Myeloma Working Group risk (p=0.003) and high serum lactate dehydrogenase levels (p=0.040) were significantly associated with poor outcomes. CONCLUSIONS: RGS1 expression may be a prognostic marker for risk stratification and a promising target for the development of a new MM therapy.

The pericyte antigen RGS5 in perivascular soft tissue tumors.

Perivascular soft tissue tumors are relatively uncommon neoplasms of unclear lineage of differentiation, although most are presumed to originate from or differentiate to pericytes or a modified perivascular cell. Among these, glomus tumor, myopericytoma, and angioleiomyoma share a spectrum of histologic findings and a perivascular growth pattern. In contrast, solitary fibrous tumor was once hypothesized to have pericytic differentiation--although little bona fide evidence of pericytic differentiation exists. Likewise the perivascular epithelioid cell tumor (PEComa) family shares a perivascular growth pattern, but with distinctive dual myoid-melanocytic differentiation. RGS5, regulator of G-protein signaling 5, is a novel pericyte antigen with increasing use in animal models. Here, we describe the immunohistochemical expression patterns of RGS5 across perivascular soft tissue tumors, including glomus tumor (n = 6), malignant glomus tumor (n = 4), myopericytoma (n = 3), angioleiomyoma (n = 9), myofibroma (n = 4), solitary fibrous tumor (n = 10), and PEComa (n = 19). Immunohistochemical staining and semi-quantification was performed, and compared to αSMA (smooth muscle actin) expression. Results showed that glomus tumor (including malignant glomus tumor), myopericytoma, and angioleiomyoma shared a similar diffuse immunoreactivity for RGS5 and αSMA across all tumors examined. In contrast, myofibroma, solitary fibrous tumor and PEComa showed predominantly focal to absent RGS5 immunoreactivity. These findings further support a common pericytic lineage of differentiation in glomus tumors, myopericytoma and angioleiomyoma. The pericyte marker RGS5 may be of future clinical utility for the evaluation of pericytic differentiation in soft tissue tumors.

Pericytic mimicry in well-differentiated liposarcoma/atypical lipomatous tumor.

Pericytes are modified smooth muscle cells that closely enwrap small blood vessels, regulating and supporting the microvasculature through direct endothelial contact. Pericytes demonstrate a distinct immunohistochemical profile, including expression of smooth muscle actin, CD146, platelet-derived growth factor receptor ß, and regulator of G-protein signaling 5. Previously, pericyte-related antigens have been observed to be present among a group of soft tissue tumors with a perivascular growth pattern, including glomus tumor, myopericytoma, and angioleiomyoma. Similarly, malignant tumor cells have been shown to have a pericyte-like immunoprofile when present in a perivascular location, seen in malignant melanoma, glioblastoma, and adenocarcinoma. Here, we examine well-differentiated liposarcoma specimens, which showed some element of perivascular areas with the appearance of smooth muscle (n = 7 tumors). Immunohistochemical staining was performed for pericyte antigens, including smooth muscle actin, CD146, platelet-derived growth factor receptor ß, and regulator of G-protein signaling 5. Results showed consistent pericytic marker expression among liposarcoma tumor cells within a perivascular distribution. MDM2 immunohistochemistry and fluorescence in situ hybridization for MDM2 revealed that these perivascular cells were of tumor origin (7/7 tumors), whereas double immunohistochemical detection for CD31/CD146 ruled out an endothelial cell contribution. These findings further support the concept of pericytic mimicry, already established in diverse malignancies, and its presence in well-differentiated liposarcoma. The extent to which pericytic mimicry has prognostic significance in liposarcoma is as yet unknown.

GTPase regulators and photoresponses in cones of the eastern chipmunk.

Vertebrate cone and rod photoreceptor cells use similar mechanisms to transduce light signals into electrical signals, but their responses to light differ in sensitivity and kinetics. To assess the role of G-protein GTP hydrolysis kinetics in mammalian cone photoresponses, we have characterized photoresponses and GTPase regulatory components of cones and rods from the cone-dominant retina of the eastern chipmunk. Sensitivity, based on the stimulus strength required for a half-maximum response, of the M-cone population was 38-fold lower than that of the rods. The relatively lower cone sensitivity could be attributed in part to lower amplification in the rising phase and in part to faster recovery kinetics. At a molecular level, cloning of chipmunk cDNA and expression of recombinant proteins provided standards for quantitative immunoblot analysis of proteins involved in GTPase acceleration. The ratio of the cGMP-phosphodiesterase inhibitory subunit gamma to cone pigment, 1:68, was similar to the levels observed for ratios to rhodopsin in bovine retina, 1:76, or mouse retina, 1:65. In contrast, the ratio to pigment of the GTPase-accelerating protein RGS9-1 was 1:62, more than 10 times higher than ratios observed in rod-dominant retinas. Immunoprecipitation experiments revealed that, in contrast to rods, RGS9-1 in chipmunk retina is associated with both the short and long isoforms of its partner subunit G(beta5). The much higher levels of the GTPase-accelerating protein complex in cones, compared with rods, suggest a role for GTPase acceleration in obtaining rapid photoresponse kinetics.

Second messengers regulate RGS2 expression which is targeted to the nucleus.

Regulators of G-protein Signaling (RGS) proteins attenuate signaling activities of G proteins, and modulation of expression appears to be a primary mechanism for regulating RGS proteins. In human astrocytoma 1321N1 cells RGS2 expression was increased by activation of muscarinic receptors coupled to phosphoinositide signaling with carbachol, or by increased cyclic AMP production, demonstrating that both signaling systems can increase the expression of a RGS family member in a single cell type. Carbachol-stimulated increases in endogenous RGS2 protein levels appeared by immunocytochemical analysis to be largely confined to the nucleus, and this localization was confirmed by Western blot analysis which showed increased nuclear, but not cytosolic, RGS2 after carbachol treatment. Additionally, transiently expressed green fluorescent protein (GFP)-tagged, 6xHis-tagged, or unmodified RGS2 resulted in a predominant nuclear localization, as well as a distinct accumulation of RGS2 along the plasma membrane. The intranuclear localization of GFP-RGS2 was confirmed with confocal microscopy. Thus, RGS2 expression is rapidly and transiently increased by phosphoinositide signaling and by cyclic AMP, and endogenous and transfected RGS2 is largely, although not entirely, localized in the nucleus.

Expression of ten RGS proteins in human myocardium: functional characterization of an upregulation of RGS4 in heart failure.

OBJECTIVE: RGS proteins (regulators of G protein signalling) negatively regulate G protein function as GTPase activating proteins. By controlling heterotrimeric G proteins they may regulate myocardial hypertrophy and contractility. We investigated the expression of RGS proteins in the human heart and whether they take part in the pathophysiological changes of heart failure. METHODS AND RESULTS: Using RNase protection assays (RPAs) RGS2, 3L, 3S, 4, 5 and 6 were identified in the myocardium from terminally failing human hearts with dilated (DCM, n=22) or ischemic (ICM, n=18) cardiomyopathy and from nonfailing donor hearts (NF, n=9). With reverse transcriptase polymerase chain reaction in addition mRNA of RGS1, 9, 12, 14 and 16 were detectable. Compared to NF in failing LV myocardium RGS4 mRNA and protein was upregulated 2-3-fold (mRNA, 10(-21) mol/microg+/-S.E.M.: NF: 22+/-5, DCM: 51+/-10*, ICM: 37+/-8; P<0.05 vs. DCM+ICM, *P<0.05 vs. NF, P<0.05 vs. DCM+ICM; protein, % of NF+/-S.E.M.: NF: 100+/-35, DCM 266+/-60*, ICM: 205+/-64, n=5, *P<0.05 vs. NF). In contrast, RGS2, 3L, 3S, 5, 6, and 16 protein and mRNA levels did not vary between failing and NF hearts. In order to investigate the impact of RGS4 on Gq/11 mediated signalling, PLC activity was measured in human LV membranes. Recombinant RGS4 blunted the endothelin-1 (ET-1) stimulated PLC activity. When overexpressed by adenoviral mediated gene transfer in rabbit ventricular myocytes RGS4 abolished the inotropic effect of ET-1. CONCLUSION: The upregulation of RGS4 in failing human myocardium diminishes Gq/11-mediated signalling and can be involved in the desensitization of Gq/11-mediated positive inotropic effects.

Cellular deficiency in the RGS10 protein facilitates chemoresistant ovarian cancer.

More than 30 regulators of G protein signaling (RGS) proteins encompass the RGS protein superfamily of critical regulators essential to cellular homeostasis. There is enormous structural and functional diversity among the RGS superfamily, and as such they serve a wide range of functions in regulating cell biology and physiology. Recent evidence has suggested roles for multiple RGS proteins in cancer initiation and progression, which has prompted research toward the potential modulation of these proteins as a new approach in cancer therapy. This article will discuss basic RGS molecular pharmacology, summarize the cellular functions and epigenetic regulation of RGS10, review ovarian cancer chemotherapy and describe the role of RGS10 in ovarian cancer survival signaling.

Elevated levels of DeltaFosB and RGS9 in striatum in Parkinson's disease.

INTRODUCTION: In the present study, we determined whether certain proteins known to mediate dopamine signaling in striatum show abnormal levels in Parkinson's disease. METHODS: Protein levels were assayed by western blotting in samples of caudate nucleus and putamen obtained at autopsy from patients with Parkinson's disease and from control subjects. Levels of several markers of dopaminergic function were also assayed. RESULTS: Levels of the transcription factor DeltaFosB and of the G protein modulatory protein RGS9 were both increased in caudate and putamen from patients with Parkinson's disease. Levels of several other proteins were not affected. Interestingly, levels of both DeltaFosB and RGS9 correlated inversely with putamen levels of dopamine, dopamine metabolites, and the dopamine transporter. CONCLUSIONS: These findings are consistent with observations in laboratory animals, which have demonstrated elevated levels of DeltaFosB in striatum after denervation of the midbrain dopamine system, and confirm that similar adaptations in DeltaFosB and RGS9 occur in humans with Parkinson's disease. Knowledge of these adaptations can help us understand the changes in striatal function associated with Parkinson's disease and assist in the development of novel treatments.

Structure, alternative splicing, and expression of the human RGS9 gene.

An isoform of RGS9 was recently identified as the GTPase activating protein in bovine and mouse rod and cone photoreceptors. To explore the potential role of the RGS9 gene in human retinal disease, we determined its exon/intron arrangement, and investigated its expression in human retina. The results show that the gene, located on 17q24, consists of 19 exons and spans more than 75kb of genomic DNA. The entire gene was found to be contained on a single BAC clone with an insert size of 170kb. The major transcripts of the gene are alternatively spliced into a 9.5kb retina-specific transcript (RGS9-1) and a brain specific 2.5kb transcript (RGS9-2). Exons 1-16 are constitutive and present in both variants. Exon 17 contains the 3' end of the open reading frame and the 3'-UTR of the RGS9-1 variant. In RGS9-2, exon 17 is alternatively spliced and joined to exons 18 and 19 that are not present in the retina variant. Immunolocalization with a monoclonal antibody recognizing the retina and brain variants shows abundant expression in photoreceptors and possibly very low levels in cell types of the inner retina. Owing to the specific expression of RGS9-1 in photoreceptors the RGS9 gene is a candidate gene for RP17, a form of autosomal retinitis pigmentosa, located on the long arm of chromosome 17.

Quantitative real-time polymerase chain reaction measurement of regulators of G-protein signaling mRNA levels in mouse tissues.

Regulators of G-protein signaling (RGS) play a critical role in G-protein-coupled receptor signaling in mammalian cells. RGS proteins are GTPase-accelerating proteins (GAPs) for alpha subunits of heterotrimeric G proteins of the Gi and Gq class. RGS GAPs can modulate the frequency and duration of G-protein signaling and may constitute a new family of therapeutic targets. Identifying the tissue distribution and cellular localization of RGS proteins has been hindered by the lack of effective antibodies for immunodetection. The measurement of mRNA levels for RGS proteins, however, can provide insight into their tissue specificity and regulation. This article describes the use of a highly sensitive and rapid method for measuring RGS mRNA in mouse tissues. This quantitative real-time polymerase chain reaction method is established for the 19 reported mouse RGS genes and is used to study the tissue distribution of the R4 family of RGS genes and the diurnal regulation of RGS16 in mouse liver.

The GABAB receptor associates with regulators of G-protein signaling 4 protein in the mouse prefrontal cortex and hypothalamus.

Regulators of G-protein signaling (RGS) proteins regulate certain G-protein-coupled receptor (GPCR)-mediated signaling pathways. The GABA(B) receptor (GABA(B)R) is a GPCR that plays a role in the stress response. Previous studies indicate that acute immobilization stress (AIS) decreases RGS4 in the prefrontal cortex (PFC) and hypothalamus (HY) and suggest the possibility of a signal complex composed of RGS4 and GABA(B)R. Therefore, in the present study, we tested whether RGS4 associates with GABA(B)R in these brain regions. We found the co-localization of RGS4 and GABA(B)R subtypes in the PFC and HY using double immunohistochemistry and confirmed a direct association between GABA(B2)R and RGS4 proteins using co-immunoprecipitation. Furthermore, we found that AIS decreased the amount of RGS4 bound to GABA(B2)R and the number of double-positive cells. These results indicate that GABA(B)R forms a signal complex with RGS4 and suggests that RGS4 is a regulator of GABA(B)R.