Genome-wide screening for the G-protein-coupled receptor (GPCR) pathway-related therapeutic gene RGS19 (regulator of G protein signaling 19) in bladder cancer.

Bladder cancer is one of the most severe genitourinary cancers, causing high morbidity worldwide. However, the underlying molecular mechanism is not clear, and it is urgent to find target genes for treatment. G-protein-coupled receptors are currently a target of high interest for drug design. Thus, we aimed to identify a target gene-related to G-protein-coupled receptors for therapy. We used The Cancer Genome Atlas (TCGA) and DepMap databases to obtain the expression and clinical data of RGS19. The results showed that RGS19 was overexpressed in a wide range of tumor, especially bladder cancer. We also explored its effect on various types of cancer. High expression of RGS19 was also shown to be significantly associated with poor prognosis. Cell models were constructed for cell cycle detection. shRGS19 can halt the cell cycle at a polyploid point. RGS19 is a G-protein-coupled receptor signaling pathway-related gene with a significant effect on survival. We chose RGS19 as a therapeutic target gene in bladder cancer. The drug GSK1070916 was found to inhibit the effect of RGS19 via cell rescue experiments in vitro.

Regulator of G protein signaling 17 represents a novel target for treating cisplatin induced hearing loss.

Regulators of G protein signaling (RGS) accelerate the GTPase activity of G proteins to enable rapid termination of the signals triggered by G protein-coupled receptors (GPCRs). Activation of several GPCRs, including cannabinoid receptor 2 (CB2R) and adenosine A1 receptor (A1AR), protects against noise and drug-induced ototoxicity. One such drug, cisplatin, an anticancer agent used to treat various solid tumors, produces permanent hearing loss in experimental animals and in a high percentage of cancer patients who undergo treatments. In this study we show that cisplatin induces the expression of the RGS17 gene and increases the levels of RGS17 protein which contributes to a significant proportion of the hearing loss. Knockdown of RGS17 suppressed cisplatin-induced hearing loss in male Wistar rats, while overexpression of RGS17 alone produced hearing loss in vivo. Furthermore, RGS17 and CB2R negatively regulate the expression of each other. These data suggest that RGS17 mediates cisplatin ototoxicity by uncoupling cytoprotective GPCRs from their normal G protein interactions, thereby mitigating the otoprotective contributions of endogenous ligands of these receptors. Thus, RGS17 represents a novel mediator of cisplatin ototoxicity and a potential therapeutic target for treating hearing loss.

The Antipsychotic Drug Clozapine Suppresses the RGS4 Polyubiquitylation and Proteasomal Degradation Mediated by the Arg/N-Degron Pathway.

Although diverse antipsychotic drugs have been developed for the treatment of schizophrenia, most of their mechanisms of action remain elusive. Regulator of G-protein signaling 4 (RGS4) has been reported to be linked, both genetically and functionally, with schizophrenia and is a physiological substrate of the arginylation branch of the N-degron pathway (Arg/N-degron pathway). Here, we show that the atypical antipsychotic drug clozapine significantly inhibits proteasomal degradation of RGS4 proteins without affecting their transcriptional expression. In addition, the levels of Arg- and Phe-GFP (artificial substrates of the Arg/N-degron pathway) were significantly elevated by clozapine treatment. In silico computational model suggested that clozapine may interact with active sites of N-recognin E3 ubiquitin ligases. Accordingly, treatment with clozapine resulted in reduced polyubiquitylation of RGS4 and Arg-GFP in the test tube and in cultured cells. Clozapine attenuated the activation of downstream effectors of G protein-coupled receptor signaling, such as MEK1 and ERK1, in HEK293 and SH-SY5Y cells. Furthermore, intraperitoneal injection of clozapine into rats significantly stabilized the endogenous RGS4 protein in the prefrontal cortex. Overall, these results reveal an additional therapeutic mechanism of action of clozapine: this drug posttranslationally inhibits the degradation of Arg/N-degron substrates, including RGS4. These findings imply that modulation of protein post-translational modifications, in particular the Arg/N-degron pathway, may be a novel molecular therapeutic strategy against schizophrenia.

Fragment-Based Nuclear Magnetic Resonance Screen against a Regulator of G Protein Signaling Identifies a Binding "Hot Spot".

Regulator of G protein signaling (RGS) proteins have attracted attention as a result of their primary role in directing the specificity as well as the temporal and spatial aspects of G protein-coupled receptor signaling. In addition, alterations in RGS protein expression have been observed in a number of disease states, including certain cancers. In this area, RGS17 is of particular interest. It has been demonstrated that, while RGS17 is expressed primarily in the central nervous system, it has been found to be inappropriately expressed in lung, prostate, breast, cervical, and hepatocellular carcinomas. Overexpression of RGS17 leads to dysfunction in inhibitory G protein signaling and an overproduction of the intracellular second messenger cAMP, which in turn alters the transcription patterns of proteins known to promote various cancer types. Suppressing RGS17 expression with RNA interference (RNAi) has been found to decrease tumorigenesis and sufficiently prevents cancer cell migration, leading to the hypothesis that pharmacological blocking of RGS17 function could be useful in anticancer therapies. We have identified small-molecule fragments capable of binding the RGS homology (RH) domain of RGS17 by using a nuclear magnetic resonance fragment-based screening approach. By chemical shift mapping of the two-dimensional 15 N,1 H heteronuclear single quantum coherence (HSQC) spectra of the backbone-assigned 15 N-labeled RGS17-RH, we determined the fragment binding sites to be distant from the Gα interface. Thus, our study identifies a putative fragment binding site on RGS17 that was previously unknown.

Regulator of G Protein Signalling 4 (RGS4) as a Novel Target for the Treatment of Sensorineural Hearing Loss.

We and others have previously identified signalling pathways associated with the adenosine A1 receptor (A1R) as important regulators of cellular responses to injury in the cochlea. We have shown that the "post-exposure" treatment with adenosine A1R agonists confers partial protection against acoustic trauma and other forms of sensorineural hearing loss (SNHL). The aim of this study was to determine if increasing A1R responsiveness to endogenous adenosine would have the same otoprotective effect. This was achieved by pharmacological targeting of the Regulator of G protein Signalling 4 (RGS4). RGS proteins inhibit signal transduction pathways initiated by G protein-coupled receptors (GPCR) by enhancing GPCR deactivation and receptor desensitisation. A molecular complex between RGS4 and neurabin, an intracellular scaffolding protein expressed in neural and cochlear tissues, is the key negative regulator of A1R activity in the brain. In this study, Wistar rats (6-8 weeks) were exposed to traumatic noise (110 dBSPL, 8-16 kHz) for 2 h and a small molecule RGS4 inhibitor CCG-4986 was delivered intratympanically in a Poloxamer-407 gel formulation for sustained drug release 24 or 48 h after noise exposure. Intratympanic administration of CCG-4986 48 h after noise exposure attenuated noise-induced permanent auditory threshold shifts by up to 19 dB, whilst the earlier drug administration (24 h) led to even better preservation of auditory thresholds (up to 32 dB). Significant improvement of auditory thresholds and suprathreshold responses was linked to improved survival of sensorineural tissues and afferent synapses in the cochlea. Our studies thus demonstrate that intratympanic administration of CCG-4986 can rescue cochlear injury and hearing loss induced by acoustic overexposure. This research represents a novel paradigm for the treatment of various forms of SNHL based on regulation of GPCR.

The endomorphin-1/2 and dynorphin-B peptides display biased agonism at the mu opioid receptor.

BACKGROUND: Opioid agonist activation at the mu opioid receptor (MOR) can lead to a wide variety of physiological responses. Many opioid agonists share the ability to selectively and preferentially activate specific signaling pathways, a term called biased agonism. Biased opioid ligands can theoretically induce specific physiological responses and might enable the generation of drugs with improved side effect profiles. METHODS: Dynorphins, enkephalins, and endomorphins are endogenous opioid agonist peptides that may possess distinct bias profiles; biased agonism of endogenous peptides could explain the selective roles of these ligands in vivo. Our purpose in the present study was to investigate biased signaling and potential underlying molecular mechanisms of bias using 35S-GTPγS and cAMP assays, specifically focusing on the role of adenylyl cyclases (ACs) and regulators of G-protein signaling proteins (RGSs) in CHO, N2a, and SH-SY5Y cell lines, all expressing the human MOR. RESULTS: We found that endomorphin-1/2 preferentially activated cAMP signaling, while dynorphin-B preferentially activated 35S-GTPγS signaling in most cell lines. Experiments carried out in the presence of an isoform selective RGS-4 inhibitor, and siRNA knockdown of AC6 in N2a cells did not significantly affect the bias properties of endomorphins, suggesting that these proteins may not play a role in endomorphin bias. CONCLUSION: We found that endomorphin-1/2 and dynorphin-B displayed contrasting bias profiles at the MOR, and ruled out potential AC6 and RGS4 mechanisms in this bias. This identified signaling bias could be involved in specifying endogenous peptide roles in vivo, where these peptides have low selectivity between opioid receptor family members.

An Interhelical Salt Bridge Controls Flexibility and Inhibitor Potency for Regulators of G-protein Signaling Proteins 4, 8, and 19.

Regulators of G-protein signaling (RGS) proteins modulate receptor signaling by binding to activated G-protein α-subunits, accelerating GTP hydrolysis. Selective inhibition of RGS proteins increases G-protein activity and may provide unique tissue specificity. Thiadiazolidinones (TDZDs) are covalent inhibitors that act on cysteine residues to inhibit RGS4, RGS8, and RGS19. There is a correlation between protein flexibility and potency of inhibition by the TDZD 4-[(4- fluorophenyl)methyl]-2-(4-methylphenyl)-1,2,4-thiadiazolidine-3,5-dione (CCG-50014). In the context of a single conserved cysteine residue on the α 4 helix, RGS19 is the most flexible and most potently inhibited by CCG-50014, followed by RGS4 and RGS8. In this work, we identify residues responsible for differences in both flexibility and potency of inhibition among RGS isoforms. RGS19 lacks a charged residue on the α 4 helix that is present in RGS4 and RGS8. Introducing a negative charge at this position (L118D) increased the thermal stability of RGS19 and decreased the potency of inhibition of CCG-50014 by 8-fold. Mutations eliminating salt bridge formation in RGS8 and RGS4 decreased thermal stability in RGS8 and increased potency of inhibition of both RGS4 and RGS8 by 4- and 2-fold, respectively. Molecular dynamics simulations with an added salt bridge in RGS19 (L118D) showed reduced RGS19 flexibility. Hydrogen-deuterium exchange studies showed striking differences in flexibility in the α 4 helix of RGS4, 8, and 19 with salt bridge-modifying mutations. These results show that the α 4 salt bridge-forming residue controls flexibility in several RGS isoforms and supports a causal relationship between RGS flexibility and the potency of TDZD inhibitors. SIGNIFICANCE STATEMENT: Inhibitor potency is often viewed in relation to the static structure of a target protein binding pocket. Using both experimental and computation studies we assess determinants of dynamics and inhibitor potency for three different RGS proteins. A single salt bridge-forming residue determines differences in flexibility between RGS isoforms; mutations either increase or decrease protein motion with correlated alterations in inhibitor potency. This strongly suggests a causal relationship between RGS protein flexibility and covalent inhibitor potency.

Differential Protein Dynamics of Regulators of G-Protein Signaling: Role in Specificity of Small-Molecule Inhibitors.

Small-molecule inhibitor selectivity may be influenced by variation in dynamics among members of a protein family. Regulator of G-protein Signaling (RGS) proteins are a family that plays a key role in G-Protein Coupled Receptor (GPCR) signaling by binding to active Gα subunits and accelerating GTP hydrolysis, thereby terminating activity. Thiadiazolidinones (TDZDs) inhibit the RGS-Gα interaction by covalent modification of cysteine residues in RGS proteins. Some differences in specificity may be explained by differences in the complement of cysteines among RGS proteins. However, key cysteines shared by RGS proteins inhibited by TDZDs are not exposed on the protein surface, and differences in potency exist among RGS proteins containing only buried cysteines. We hypothesize that differential exposure of buried cysteine residues among RGS proteins partially drives TDZD selectivity. Hydrogen-deuterium exchange (HDX) studies and molecular dynamics (MD) simulations were used to probe the dynamics of RGS4, RGS8, and RGS19, three RGS proteins inhibited at a range of potencies by TDZDs. When these proteins were mutated to contain a single, shared cysteine, RGS19 was found to be most potently inhibited. HDX studies revealed differences in α4 and α6 helix flexibility among RGS isoforms, with particularly high flexibility in RGS19. This could cause differences in cysteine exposure and lead to differences in potency of TDZD inhibition. MD simulations of RGS proteins revealed motions that correspond to solvent exposure observed in HDX, providing further evidence for a role of protein dynamics in TDZD selectivity.

Screen Targeting Lung and Prostate Cancer Oncogene Identifies Novel Inhibitors of RGS17 and Problematic Chemical Substructures.

Regulator of G protein signaling (RGS) proteins temporally regulate heterotrimeric G protein signaling cascades elicited by G protein-coupled receptor activation and thus are essential for cell homeostasis. The dysregulation of RGS protein expression has been linked to several pathologies, spurring discovery efforts to identify small-molecule inhibitors of these proteins. Presented here are the results of a high-throughput screening (HTS) campaign targeting RGS17, an RGS protein reported to be inappropriately upregulated in several cancers. A screen of over 60,000 small molecules led to the identification of five hit compounds that inhibit the RGS17-Gαo protein-protein interaction. Chemical and biochemical characterization demonstrated that three of these hits inhibited the interaction through the decomposition of parent compound into reactive products under normal chemical library storage/usage conditions. Compound substructures susceptible to decomposition are reported and the decomposition process characterized, adding to the armamentarium of tools available to the screening field, allowing for the conservation of resources in follow-up efforts and more efficient identification of potentially decomposed compounds. Finally, analogues of one hit compound were tested, and the results establish the first ever structure-activity relationship (SAR) profile for a small-molecule inhibitor of RGS17.

Regulating the regulators: Epigenetic, transcriptional, and post-translational regulation of RGS proteins.

Regulators of G protein signaling (RGS) are a family of proteins classically known to accelerate the intrinsic GTPase activity of G proteins, which results in accelerated inactivation of heterotrimeric G proteins and inhibition of G protein coupled receptor signaling. RGS proteins play major roles in essential cellular processes, and dysregulation of RGS protein expression is implicated in multiple diseases, including cancer, cardiovascular and neurodegenerative diseases. The expression of RGS proteins is highly dynamic and is regulated by epigenetic, transcriptional and post-translational mechanisms. This review summarizes studies that report dysregulation of RGS protein expression in disease states, and presents examples of drugs that regulate RGS protein expression. Additionally, this review discusses, in detail, the transcriptional and post-transcriptional mechanisms regulating RGS protein expression, and further assesses the therapeutic potential of targeting these mechanisms. Understanding the molecular mechanisms controlling the expression of RGS proteins is essential for the development of therapeutics that indirectly modulate G protein signaling by regulating expression of RGS proteins.

Evaluation of the Selectivity and Cysteine Dependence of Inhibitors across the Regulator of G Protein-Signaling Family.

Since their discovery more than 20 years ago, regulators of G protein-signaling (RGS) proteins have received considerable attention as potential drug targets because of their ability to modulate Gα activity. Efforts to identify small molecules capable of inhibiting the protein-protein interactions between activated Gα subunits and RGS proteins have yielded a substantial number of inhibitors, especially toward the well studied RGS4. These efforts also determined that many of these small molecules inhibit the protein-protein interactions through covalent modification of cysteine residues within the RGS domain that are located distal to the Gα-binding interface. As some of these cysteine residues are highly conserved within the RGS family, many of these inhibitors display activity toward multiple RGS family members. In this work, we sought to determine the selectivity of these small-molecule inhibitors against 12 RGS proteins, as well as against the cysteine-null mutants for 10 of these proteins. Using both biochemical and cell-based methods to assess Gα-RGS complex formation and Gα enzymatic activity, we found that several previously identified RGS4 inhibitors were active against other RGS members, such as RGS14, with comparable or greater potency. Additionally, for every compound tested, activity was dependent on the presence of cysteine residues. This work defines the selectivity of commercially available RGS inhibitors and provides insight into the RGS family members for which drug discovery efforts may be most likely to succeed.

Epigenetic regulation of RGS2 (Regulator of G-protein signaling 2) in chemoresistant ovarian cancer cells.

Regulator of G-protein signaling 2 (RGS2) is a GTPase-activating protein functioning as an inhibitor of G-protein coupled receptors (GPCRs). RGS2 dysregulation was implicated in solid tumour development and RGS2 downregulation has been reported in prostate and ovarian cancer progression. However, the molecular mechanism by which RGS2 expression is suppressed in ovarian cancer remains unknown. The expression and epigenetic regulation of RGS2 in chemosensitive and chemoresistant ovarian cancer cells were determined by qRT-PCR and chromatin immunoprecipitation assays, respectively. In the present study, the molecular mechanisms contributing to the loss of RGS2 expression were determined in ovarian cancer. The data indicated that suppression of RGS2 gene in chemoresistant ovarian cancer cells, in part, due to accumulation of histone deacetylases (HDACs) and DNA methyltransferase I (DNMT1) at the promoter region of RGS2. Inhibition of HDACs or DNMTs significantly increases RGS2 expression. These results suggest that epigenetic changes in histone modifications and DNA methylation may contribute to the loss of RGS2 expression in chemoresistant ovarian cancer cells. The results further suggest that class I HDACs and DNMT1 contribute to the suppression of RGS2 during acquired chemoresistance and support growing evidence that inhibition of HDACs/DNMTs represents novel therapeutic approaches to overcome ovarian cancer chemoresistance.

A new model of nerve injury in the rat reveals a role of Regulator of G protein Signaling 4 in tactile hypersensitivity.

Tactile hypersensitivity is one of the most debilitating symptoms of neuropathic pain syndromes. Clinical studies have suggested that its presence at early postoperative stages may predict chronic (neuropathic) pain after surgery. Currently available animal models are typically associated with consistent tactile hypersensitivity and are therefore limited to distinguish between mechanisms that underlie tactile hypersensitivity as opposed to mechanisms that protect against it. In this study we have modified the rat model of spared nerve injury, restricting the surgical lesion to a single peripheral branch of the sciatic nerve. This modification reduced the prevalence of tactile hypersensitivity from nearly 100% to approximately 50%. With this model, we here also demonstrated that the Regulator of G protein Signaling 4 (RGS4) was specifically up-regulated in the lumbar dorsal root ganglia and dorsal horn of rats developing tactile hypersensitivity. Intrathecal delivery of the RGS4 inhibitor CCG63802 was found to reverse tactile hypersensitivity for a 1h period. Moreover, tactile hypersensitivity after modified spared nerve injury was most frequently persistent for at least four weeks and associated with higher reactivity of glial cells in the lumbar dorsal horn. Based on these data we suggest that this new animal model of nerve injury represents an asset in understanding divergent neuropathic pain outcomes, so far unravelling a role of RGS4 in tactile hypersensitivity. Whether this model also holds promise in the study of the transition from acute to chronic pain will have to be seen in future investigations.

RGS6 as a Novel Therapeutic Target in CNS Diseases and Cancer.

Regulator of G protein signaling (RGS) proteins are gatekeepers regulating the cellular responses induced by G protein-coupled receptor (GPCR)-mediated activation of heterotrimeric G proteins. Specifically, RGS proteins determine the magnitude and duration of GPCR signaling by acting as a GTPase-activating protein for Gα subunits, an activity facilitated by their semiconserved RGS domain. The R7 subfamily of RGS proteins is distinguished by two unique domains, DEP/DHEX and GGL, which mediate membrane targeting and stability of these proteins. RGS6, a member of the R7 subfamily, has been shown to specifically modulate Gαi/o protein activity which is critically important in the central nervous system (CNS) for neuronal responses to a wide array of neurotransmitters. As such, RGS6 has been implicated in several CNS pathologies associated with altered neurotransmission, including the following: alcoholism, anxiety/depression, and Parkinson's disease. In addition, unlike other members of the R7 subfamily, RGS6 has been shown to regulate G protein-independent signaling mechanisms which appear to promote both apoptotic and growth-suppressive pathways that are important in its tumor suppressor function in breast and possibly other tissues. Further highlighting the importance of RGS6 as a target in cancer, RGS6 mediates the chemotherapeutic actions of doxorubicin and blocks reticular activating system (Ras)-induced cellular transformation by promoting degradation of DNA (cytosine-5)-methyltransferase 1 (DNMT1) to prevent its silencing of pro-apoptotic and tumor suppressor genes. Together, these findings demonstrate the critical role of RGS6 in regulating both G protein-dependent CNS pathology and G protein-independent cancer pathology implicating RGS6 as a novel therapeutic target.

Regulator of G Protein Signaling 17 as a Negative Modulator of GPCR Signaling in Multiple Human Cancers.

Regulators of G protein signaling (RGS) proteins modulate G protein-coupled receptor (GPCR) signaling networks by terminating signals produced by active Gα subunits. RGS17, a member of the RZ subfamily of RGS proteins, is typically only expressed in appreciable amounts in the human central nervous system, but previous works have shown that RGS17 expression is selectively upregulated in a number of malignancies, including lung, breast, prostate, and hepatocellular carcinoma. In addition, this upregulation of RGS17 is associated with a more aggressive cancer phenotype, as increased proliferation, migration, and invasion are observed. Conversely, decreased RGS17 expression diminishes the response of ovarian cancer cells to agents commonly used during chemotherapy. These somewhat contradictory roles of RGS17 in cancer highlight the need for selective, high-affinity inhibitors of RGS17 to use as chemical probes to further the understanding of RGS17 biology. Based on current evidence, these compounds could potentially have clinical utility as novel chemotherapeutics in the treatment of lung, prostate, breast, and liver cancers. Recent advances in screening technologies to identify potential inhibitors coupled with increasing knowledge of the structural requirements of RGS-Gα protein-protein interaction inhibitors make the future of drug discovery efforts targeting RGS17 promising. This review highlights recent findings related to RGS17 as both a canonical and atypical RGS protein, its role in various human disease states, and offers insights on small molecule inhibition of RGS17.

RGS proteins as targets in the treatment of intestinal inflammation and visceral pain: New insights and future perspectives.

Regulators of G protein signaling (RGS) proteins provide timely termination of G protein-coupled receptor (GPCR) responses. Serving as a central control point in GPCR signaling cascades, RGS proteins are promising targets for drug development. In this review, we discuss the involvement of RGS proteins in the pathophysiology of the gastrointestinal inflammation and their potential to become a target for anti-inflammatory drugs. Specifically, we evaluate the emerging evidence for modulation of selected receptor families: opioid, cannabinoid and serotonin by RGS proteins. We discuss how the regulation of RGS protein level and activity may modulate immunological pathways involved in the development of intestinal inflammation. Finally, we propose that RGS proteins may serve as a prognostic factor for survival rate in colorectal cancer. The ideas introduced in this review set a novel conceptual framework for the utilization of RGS proteins in the treatment of gastrointestinal inflammation, a growing major concern worldwide.

Inhibition of the regulator of G protein signalling RGS4 in the spinal cord decreases neuropathic hyperalgesia and restores cannabinoid CB1 receptor signalling.

BACKGROUND AND PURPOSE: Regulators of G protein signalling (RGS) are major determinants of metabotropic receptor activity, reducing the lifespan of the GTP-bound state of G proteins. Because the reduced potency of analgesic agents in neuropathic pain may reflect alterations in RGS, we assessed the effects of CCG 63802, a specific RGS4 inhibitor, on pain hypersensitivity and signalling through cannabinoid receptors, in a model of neuropathic pain. EXPERIMENTAL APPROACH: The partial sciatic nerve ligation (PSNL) model in male Sprague Dawley rats was used to measure paw withdrawal thresholds to mechanical (von Frey hairs) or thermal (Hargreaves method) stimuli, during and after intrathecal injection of CCG 63802. HEK293 cells expressing CB1 receptors and conditional expression of RGS4 were used to correlate cAMP production and ERK phosphorylation with receptor activation and RGS4 action. KEY RESULTS: Treatment of PSNL rats with CCG 63802, twice daily for 7 days after nerve injury, attenuated thermal hyperalgesia during treatment. Spinal levels of anandamide were higher in PSNL animals, irrespective of the treatment. Although expression of CB1 receptors was unaffected, HU210-induced CB1 receptor signalling was inhibited in PSNL rats and restored after intrathecal CCG 63802. In transfected HEK cells expressing CB1 receptors and RGS4, inhibition of cAMP production, a downstream effect of CB1 receptor signalling, was blunted after RGS4 overexpression. RGS4 expression also attenuated the CB1 receptor-controlled activation of ERK1/2. CONCLUSIONS AND IMPLICATIONS: Inhibition of spinal RGS4 restored endogenous analgesic signalling pathways and mitigated neuropathic pain. Signalling through CB1 receptors may be involved in this beneficial effect.

Regulator of G protein signaling 1 suppresses CXCL12-mediated migration and AKT activation in RPMI 8226 human plasmacytoma cells and plasmablasts.

Migration of plasma cells to the bone marrow is critical factor to humoral immunity and controlled by chemokines. Regulator of G protein signaling 1 (RGS1) is a GTPase-activating protein that controls various crucial functions such as migration. Here, we show that RGS1 controls the chemotactic migration of RPMI 8226 human plasmacytoma cells and human plasmablasts. LPS strongly increased RGS1 expression and retarded the migration of RPMI 8226 cells by suppressing CXCL12-mediated AKT activation. RGS1 knockdown by siRNA abolished the retardation of migration and AKT suppression by LPS. RGS1-dependent regulation of migration via AKT is also observed in cultured plasmablasts. We propose novel functions of RGS1 that suppress AKT activation and the migration of RPMI 8226 cells and plasmablasts in CXCL12-mediated chemotaxis.

Selectivity and anti-Parkinson's potential of thiadiazolidinone RGS4 inhibitors.

Many current therapies target G protein coupled receptors (GPCR), transporters, or ion channels. In addition to directly targeting these proteins, disrupting the protein-protein interactions that localize or regulate their function could enhance selectivity and provide unique pharmacologic actions. Regulators of G protein signaling (RGS) proteins, especially RGS4, play significant roles in epilepsy and Parkinson's disease. Thiadiazolidinone (TDZD) inhibitors of RGS4 are nanomolar potency blockers of the biochemical actions of RGS4 in vitro. Here, we demonstrate the substantial selectivity (8- to >5000-fold) of CCG-203769 for RGS4 over other RGS proteins. It is also 300-fold selective for RGS4 over GSK-3ß, another target of this class of chemical scaffolds. It does not inhibit the cysteine protease papain at 100 µM. CCG-203769 enhances Gαq-dependent cellular Ca(2+) signaling in an RGS4-dependent manner. TDZD inhibitors also enhance Gαi-dependent δ-OR inhibition of cAMP production in SH-SY-5Y cells, which express endogenous receptors and RGS4. Importantly, CCG-203769 potentiates the known RGS4 mechanism of Gαi-dependent muscarinic bradycardia in vivo. Furthermore, it reverses raclopride-induced akinesia and bradykinesia in mice, a model of some aspects of the movement disorder in Parkinson's disease. A broad assessment of compound effects revealed minimal off-target effects at concentrations necessary for cellular RGS4 inhibition. These results expand our understanding of the mechanism and specificity of TDZD RGS inhibitors and support the potential for therapeutic targeting of RGS proteins in Parkinson's disease and other neural disorders.

5-HT1A receptor-mediated phosphorylation of extracellular signal-regulated kinases (ERK1/2) is modulated by regulator of G protein signaling protein 19.

The 5-HT1A receptor is a G protein coupled receptor (GPCR) that activates G proteins of the Gαi/o family. 5-HT1A receptors expressed in the raphe, hippocampus and prefrontal cortex are implicated in the control of mood and are targets for anti-depressant drugs. Regulators of G protein signaling (RGS) proteins are members of a large family that play important roles in signal transduction downstream of G protein coupled receptors (GPCRs). The main role of RGS proteins is to act as GTPase accelerating proteins (GAPs) to dampen or negatively regulate GPCR-mediated signaling. We have shown that a mouse expressing Gαi2 that is insensitive to all RGS protein GAP activity has an anti-depressant-like phenotype due to increased signaling of postsynaptic 5-HT1A receptors, thus implicating the 5-HT1A receptor-Gαi2 complex as an important target. Here we confirm that RGS proteins act as GAPs to regulate signaling to adenylate cyclase and the mitogen-activated protein kinase (MAPK) pathway downstream of the 5-HT1A receptor, using RGS-insensitive Gαi2 protein expressed in C6 cells. We go on to use short hairpin RNA (shRNA) to show that RGS19 is responsible for the GAP activity in C6 cells and also that RGS19 acts as a GAP for 5-HT1A receptor signaling in human neuroblastoma SH-SY5Y cells and primary hippocampal neurons. In addition, in both cell types the synergy between 5-HT1A receptor and the fibroblast growth factor receptor 1 in stimulating the MAPK pathway is enhanced following shRNA reduction of RGS19 expression. Thus RGS19 may be a viable new target for anti-depressant medications.