Identification of peptides that inhibit regulator of G protein signaling 4 function.

Regulators of G protein signaling (RGS) are a family of GTPase-activating proteins (GAP) that interact with heterotrimeric G proteins in the negative regulation of G-protein-coupled receptor (GPCR) signaling. RGS4, the first identified mammalian member of the RGS family, has been implicated in many GPCR signaling pathways involved in disease states. We report herein the identification of a 16-amino-acid peptide (P17) as an inhibitor of RGS4. The peptide was found by screening a random peptide library using RGS4 as 'bait' in a yeast two-hybrid system. This peptide inhibited RGS4 GAP activity on Galpha(i1)in a GTPase assay, and blocked the interaction between RGS4 and Galpha(i1)in a pull-down assay. The peptide displayed dose-dependent inhibition of RGS4 and Galpha-interacting protein (GAIP) GAP activities, yet showed no substantial effect on RGS7. Electrophysiological studies in Xenopus oocytes demonstrated that P17 attenuates RGS4 modulation of M(2) muscarinic receptor stimulation of GIRK (G-protein-mediated inwardly rectifying potassium) channels. Deletion of an arginine at the N terminus of P17 abolished its ability to inhibit RGS4 GAP activity, as did deletions of C-terminal residues. The P17 peptide showed no similarity to any known peptide sequence. Further investigation and optimization of the peptide may provide unique information for the development of RGS4 inhibitors for future therapeutic application.

Activation of serum response element by D2 dopamine receptor is governed by Gbetagamma-mediated MAPK and Rho pathways and regulated by RGS proteins.

In this study, we investigated the activation of the serum response element (SRE) by the D2 dopamine receptor (D2R) agonist quinpirole. Stimulation of CHO cells expressing the D2R by quinpirol evoked a dose-dependent SRE activation, which was completely blocked by overnight treatment of pertussis toxin or by co-expression of the beta-adrenergic receptor kinase C-terminus, implicating the involvement of Galpha(i )and Gbetagamma in the signal transduction. Furthermore, using MEK inhibitors and dominant negative mutants of RhoA, Rac1, and Cdc42, we showed that the Gbetagamma-mediated activation of the SRE in CHO cells utilizes both MAPK and Rho pathways. Expression of either regulator of G protein signaling 2 or 4 (RGS2 or RGS4) proteins significantly attenuated the quinpirole-induced SRE activation. These results delineate the signaling pathways which couple D2 receptor to the transcriptional activation of SRE and demonstrate a modulatory role for RGS proteins in these processes.

RGSZ1 interacts with protein kinase C interacting protein PKCI-1 and modulates mu opioid receptor signaling.

Protein kinase C interacting protein (PKCI-1) was identified among the potential interactors from a yeast two hybrid screen of human brain library using N terminal of RGSZ1 as a bait. The cysteine string region, unique to the RZ subfamily, contributes to the observed interaction because PKCI-1 interacted with N-terminus of RGS17 and GAIP, but not with that of RGS2 or RGS7 where cysteine string motif is absent. The interaction between RGSZ1 and PKCI-1 was confirmed by coimmunoprecipitation and immunofluorescence. PKCI-1 and RGSZ1 could be detected by coimmunoprecipitation using 14-3-3 antibody in cells transfected with PKCI-1 or RGSZ1 respectively, but when transfected with PKCI-1 and RGSZ1 together, only RGSZ1 could be detected. Phosphorylation of Galphaz by protein kinase C (PKC) reduces the ability of the RGS to effectively function as GTPase accelerating protein for Galphaz, and interferes with ability of Galphaz to interact with betagamma complex. We investigated the roles of 14-3-3 and PKCI-1 in phosphorylation of Galphaz. Phosphorylation of Galphaz by PKC was inhibited by 14-3-3 and the presence of PKCI-1 did not provide any further inhibition. PKCI-1 interacts with mu opioid receptor and suppresses receptor desensitization and PKC related mu opioid receptor phosphorylation [W. Guang, H. Wang, T. Su, I.B. Weinstein, J.B. Wang, Mol. Pharmacol. 66 (2004) 1285.]. Previous studies have also shown that mu opioid receptor co-precipitates with RGSZ1 and influence mu receptor signaling by acting as effector antagonists [J. Garzon, M. Rodriguez-Munoz, P. Sanchez-Blazquez, Neuropharmacology 48 (2005) 853., J. Garzon, M. Rodriguez-Munoz, A. Lopez-Fando, P. Sanchez-Blazquez Neuropsychopharmacology 30 (2005) 1632.]. Inhibition of cAMP by mu opioid receptor was significantly reduced by RGSZ1 and this effect was enhanced in combination with PKCI-1. Our studies thus provide a link between the previous observations mentioned above and indicate that the major function of PKCI-1 is to modulate mu opioid receptor signaling pathway along with RGSZ1, rather than directly mediating the Galphaz RGSZ1 interaction.

Alterations in 5-HT2A receptor signaling in male and female transgenic rats over-expressing either Gq or RGS-insensitive Gq protein.

Serotonin 2A (5-HT2A) receptors are coupled to Galphaq and Galpha11 proteins to activate phospholipase C (PLC). Regulators of G-protein signaling proteins (RGS) modulate G-protein signaling by accelerating the intrinsic GTPase activity of Galphaq and Galpha11. This study investigated the effects of over-expression of wild-type Galphaq proteins (Gq-Tg) and over-expression of RGS-insensitive Galphaq proteins (G188S, RGSi-Tg) on 5-HT2A receptor mediated signaling in transgenic rats. Over-expression of wild-type Galphaq and RGS insensitive mutant Galphaq did not produce significant alterations in the levels of Galpha11, RGS2, RGS4, RGS7, RGS16 or 5-HT2A proteins. RGSi-Tg rats had higher oxytocin and corticosterone responses to (-)DOI, a 5-HT2A/2C receptor agonist, compared to Gq-Tg rats. RGSi-Tg and Gq-Tg rats had higher ACTH responses to (-)DOI compared to control rats. Similarly, 5-HT-stimulated PLC activity in the frontal cortex was higher in RGSi-Tg and Gq-Tg rats compared to control rats. In contrast, GTPgammaS-stimulated PLC activity was higher in Gq-Tg rats but not in RGSi-Tg rats compared to control rats. There was a small but statistically significant increase in the affinity of [125I]-DOI labeled 5-HT2A receptors in RGSi-Tg rats and Gq-Tg rats compared to controls. There were no significant differences in Bmax and Kd of [3H] ketanserin labeled 5-HT2A receptors among the three groups. These data suggest that the effect of RGS proteins on 5-HT2A receptor signaling is cell type specific. In transgenic rats over-expressing Galphaq, endogenous RGS proteins have a negative effect on 5-HT2A receptor-mediated oxytocin release. In contrast, endogenous RGS protein had no impact on 5-HT2A receptor-mediated ACTH release in transgenic rats.

Analysis of chimeric RGS proteins in yeast for the functional evaluation of protein domains and their potential use in drug target validation.

For the identification of regulators of G-protein signaling (RGS) modulators, previously, we developed a luciferase based yeast pheromone response (YPhR) assay to functionally investigate RGS4 (K.H. Young, Y. Wang, C. Bender, S. Ajit, F. Ramirez, A. Gilbert, B.W. Nieuwenhuijsen, in: D.P. Siderovski (Ed.), Meth. Enzymol. 389 Regulators of G_protein Signaling, Part A, 2004.). To extend the diversity of this assay, additional RGS proteins were evaluated for functional complementation in a RGS (sst2Delta) knockout yeast strain. For RGS proteins that did not function in their native form, a series of chimeric constructs were generated with the N terminus of RGS4 fused in frame with the partial or full-length RGS cDNA of interest. RGS4 N terminus fused to either full-length or the C terminus of RGS7 successfully complemented sst2Delta. On the contrary, the RGS7N/RGS4C chimera (N terminus of RGS7 in frame with RGS domain of RGS4) was not effective, showing that N terminus of RGS4 helps in targeting. RGS10 exists as two splice variants, differing only by 8 amino acids (aa) in the N terminus, being either 168 aa (RGS10S), or 174 aa (RGS10). While RGS10 was functional in yeast, RGS10S required the presence of the N terminus of RGS4 for its activity. Although the same RGS4 N terminus domain was present in chimeras generated, the GTPase accelerating protein (GAP) function observed was not similar, suggesting differences in the RGS domain function. In conclusion, the use of RGS4 N terminus chimeric constructs enabled us to develop a selectivity assay for different RGS proteins.

Enhancement of pheromone response by RGS9 and Gbeta5 in yeast.

The G-protein gamma-subunit-like (GGL) domain present within a subfamily of RGS proteins binds specifically to Gbeta5. This interaction and resulting biological effect impacts the standard model of heterotrimeric G-protein signaling. It has been hypothesized that the RGS/Gbeta5 may potentially substitute for Gbetagamma in the heterotrimeric complex. Saccharomyces cerevisiae pheromone responsive mating signaling pathway is primarily driven by Gbetagamma. We evaluated GGL containing RGS9 and RGS7 for functional complementation in a RGS (sst2Delta) knockout yeast strain. The potential of Gbeta5 to augment the function of these RGS proteins was also evaluated. While Gbeta5 had no effect on RGS7, coexpression of Gbeta5 with RGS9 enhanced cell cycle arrest, suggesting that under certain conditions, RGS9 and Gbeta5 may possibly function as betagamma dimer. Furthermore, we demonstrate that Gbeta5 can complement a ste4Delta, the yeast beta-subunit, thus providing the first evidence of functional complementation of a mammalian Gbeta.

Analysis of RGSZ1 protein interaction with Galphai subunits.

RGSZ1 has been reported to interact with G-protein subunits of the Galphai family and function as a GTPase-accelerating protein on intrinsic Galphai GTPase activity. This article describes several experimental approaches and assays used to investigate the effect of RGSZ1 on Galphai subunits. The formats described here include physical and functional interaction assays by which the association of RGSZ1 with Galphai is explored both in vitro and in vivo. The methods analyzing physical interaction include pull-down and coimmunoprecipitation assays. We also apply yeast two-hybrid techniques to detect RGSZ1 protein interaction with Galpha subunits. Additionally, we developed several functional assay systems to identify the functional relationship between RGSZ1 and Galphai, such as the single turnover GTPase assay, yeast pheromone response assay, mitogen-activated protein kinase assay, and serum response element reporter assay.

Regulators of G-protein signaling 4: modulation of 5-HT1A-mediated neurotransmitter release in vivo.

Regulators of G-protein signaling (RGS) play a key role in the signal transduction of G-protein-coupled receptors (GPCRs). Specifically, RGS proteins function as GTPase accelerating proteins (GAPs) to dampen or "negatively regulate" GPCR-mediated signaling. Our group recently showed that RGS4 effectively GAPs Galpha(i)-mediated signaling in CHO cells expressing the serotonin-1A (5-HT(1A)) receptor. However, whether a similar relationship exists in vivo has yet to be identified. In present studies, a replication-deficient herpes simplex virus (HSV) was used to elevate RGS4 mRNA in the rat dorsal raphe nuclei (DRN) while extracellular levels of 5-HT in the striatum were monitored by in vivo microdialysis. Initial experiments conducted with noninfected rats showed that acute administration of 8-OH-DPAT (0.01-0.3 mg/kg, subcutaneous [s.c.]) dose dependently decreased striatal levels of 5-HT, an effect postulated to result from activation of somatodendritic 5-HT(1A) autoreceptors in the DRN. In control rats receiving a single intra-DRN infusion of HSV-LacZ, 8-OH-DPAT (0.03 mg/kg, s.c.) decreased 5-HT levels to an extent similar to that observed in noninfected animals. Conversely, rats infected with HSV-RGS4 in the DRN showed a blunted neurochemical response to 8-OH-DPAT (0.03 mg/kg, s.c.); however, increasing the dose to 0.3 mg/kg reversed this effect. Together, these findings represent the first in vivo evidence demonstrating that RGS4 functions to GAP Galpha(i)-coupled receptors and suggest that drug discovery efforts targeting RGS proteins may represent a novel mechanism to manipulate 5-HT(1A)-mediated neurotransmitter release.

Yeast-based screening for inhibitors of RGS proteins.

This article provides information on two screening platforms for the identification of regulators of G-protein signaling (RGS) protein modulators. Utilization of the yeast pheromone response pathway enabled the creation of a functional screen for RGS4 modulators. The RGSZ1-focused screen employs advances in yeast two-hybrid screening technologies and targets the protein-protein interface of the RGS domain/Galpha interaction. Moreover, the RGSZ1 screen provides the opportunity to multiplex the screening of two targets of interest, given the development of two different luciferase reporter genes that enabled sequential determination and intraassay controls. The screen formats were validated, implemented, and conducted as automated 384-well, liquid-based, high-throughput small molecule screens. Primary "hits" were confirmed using benchtop 96-well formats of these assays and advanced to in vitro functional evaluation assays. The yeast-based assay platforms provide robust cellular assays that result in the identification of small molecule modulators for both RGS targets. These molecules can serve both as tools with which to probe biological implications of RGS proteins and as potential starting points toward the development of novel modulators of G-protein signaling pathways. Such modulators may show potential for controlling and treating diseases resulting from inappropriate activity of G-protein signaling pathways.

Differential effects of regulator of G protein signaling (RGS) proteins on serotonin 5-HT1A, 5-HT2A, and dopamine D2 receptor-mediated signaling and adenylyl cyclase activity.

Regulator of G protein signaling (RGS) proteins function as GTPase accelerating proteins (GAP) for Galpha subunits, attenuating G-protein-coupled receptor signal transduction. The present study tested the ability of members of different subfamilies of RGS proteins to modulate both G-protein-dependent and -independent signaling in mammalian cells. RGS4, RGS10, and RGSZ1 significantly attenuated Galphai-mediated signaling by 5-HT1A, but not by dopamine D2, receptor-expressing cells. Additionally, RGS4 and RGS10 significantly inhibited forskolin-stimulated cAMP production in both cell lines. In contrast, RGS2, RGS7, and RGSZ1 had no effect on forskolin-stimulated cAMP production in these cells. RGS2 and RGS7 significantly decreased Galphaq-mediated signaling by 5-HT2A receptors, confirming that the RGS4 and RGS10 effects on forskolin-stimulated cAMP production were specific, and not simply due to overexpression. Interestingly, similar expression levels of RGS4 protein resulted in greater inhibition of G-protein-independent cAMP production compared to G-protein-dependent GAP activity. Our results suggest specificity and selectivity of RGS proteins on G-protein-dependent and -independent signaling in mammalian cells.

Mutation of cysteine 214 in Gi1 alpha subunit abolishes its endogenous GTPase activity.

The functional consequences of the mutation of a conserved Cys-214 in Galpha(i1) have been investigated. We reported herein that substitutions of Cys-214 of Galpha(i1) to either alanine or tryptophan abolished the intrinsic GTPase activity. Free phosphate release from [32P]GTP-bound Galpha(i1) C214A or [32P]GTP-bound Galpha(i1) C214W was at least 30-fold lower than that of the wild-type Galpha(i1) in single-turnover GTPase assays. Consistently, tryptic proteolysis of C214A and C214W proteins showed that they were partially protected by GTP, further confirming that the GTPase activity in both mutant proteins was impaired. Expression of C214A or C214W mutants in Chinese hamster ovary K1 cells caused significant inhibition of forskolin-stimulated adenylate cyclase activity. However, the mutations did not significantly affect the GTP[S] (guanosine 5'-[gamma-[35S]thio]triphosphate)-binding activity. Both C214A and C214W mutants serve as good substrates for pertussis toxin-catalysed ADP ribosylation, indicating that they interact well with betagamma subunits. Moreover, RGS4 protein, a GTPase-activating protein for Galpha(i1), cannot interact with Cys-214 mutants even in the presence of AlF4-, which induces the transition state of Galpha. In summary, our findings suggest that C214A or C214W are GTPase-deficient mutants and can functionally serve as constitutively active forms of Galpha(i1) in cells.

Snapin interacts with the N-terminus of regulator of G protein signaling 7.

The N-terminus of regulator of G protein signaling 7 (RGS7) contains a dishevelled/egl-10/pleckstrin (DEP) domain of unknown function. To gain insight into its function, we used yeast two-hybrid analysis to screen a human whole brain cDNA library in order to identify proteins that interact specifically with the N-terminus of human RGS7 (amino acid residues 1-248). From this analysis, we identified snapin, a protein associated with the SNARE complex in neurons, as an interactor with the N-terminus of RGS7. Deletion mutation analysis in yeast demonstrated that the interaction between RGS7 and snapin is specific and is mediated primarily by amino acid residues 1-69 of RGS7 (which contains the proximal portion of the DEP domain). The interaction between RGS7 and snapin was also demonstrated in mammalian cells by coimmunoprecipitation and pull-down assays. Our results suggest that RGS7 could play a role in synaptic vesicle exocytosis through its interaction with snapin.

A dual luciferase multiplexed high-throughput screening platform for protein-protein interactions.

To study the biology of regulators of G-protein signaling (RGS) proteins and to facilitate the identification of small molecule modulators of RGS proteins, the authors recently developed an advanced yeast 2-hybrid (YTH) assay format for GalphaZ and RGS-Z1. Moreover, they describe the development of a multiplexed luciferase-based assay that has been successfully adapted to screen large numbers of small molecule modulators of protein-protein interactions. They generated and evaluated 2 different luciferase reporter gene systems for YTH interactions, a Gal4 responsive firefly luciferase reporter gene and a Gal4 responsive Renilla luciferase reporter gene. Both the firefly and Renilla luciferase reporter genes demonstrated a 40- to 50-fold increase in luminescence in strains expressing interacting YTH fusion proteins versus negative control strains. Because the firefly and Renilla luciferase proteins have different substrate specificity, the assays were multiplexed. The multiplexed luciferase-based YTH platform adds speed, sensitivity, simplicity, quantification, and efficiency to YTH high-throughput applications and therefore greatly facilitates the identification of small molecule modulators of protein-protein interactions as tools or potential leads for drug discovery efforts.

Regulator of G protein signaling Z1 (RGSZ1) interacts with Galpha i subunits and regulates Galpha i-mediated cell signaling.

Regulator of G protein signaling (RGS) proteins constitute a family of over 20 proteins that negatively regulate heterotrimeric G protein-coupled receptor signaling pathways by enhancing endogenous GTPase activities of G protein alpha subunits. RGSZ1, one of the RGS proteins specifically localized to the brain, has been cloned previously and described as a selective GTPase accelerating protein for Galpha(z) subunit. Here, we employed several methods to provide new evidence that RGSZ1 interacts not only with Galpha(z,) but also with Galpha(i), as supported by in vitro binding assays and functional studies. Using glutathione S-transferase fusion protein pull-down assays, glutathione S-transferase-RGSZ1 protein was shown to bind (35)S-labeled Galpha(i1) protein in an AlF(4)(-)dependent manner. The interaction between RGSZ1 and Galpha(i) was confirmed further by co-immunoprecipitation studies and yeast two-hybrid experiments using a quantitative luciferase reporter gene. Extending these observations to functional studies, RGSZ1 accelerated endogenous GTPase activity of Galpha(i1) in single-turnover GTPase assays. Human RGSZ1 functionally regulated GPA1 (a yeast Galpha(i)-like protein)-mediated yeast pheromone response when expressed in a SST2 (yeast RGS protein) knockout strain. In PC12 cells, transfected RGSZ1 blocked mitogen-activated protein kinase activity induced by UK14304, an alpha(2)-adrenergic receptor agonist. Furthermore, RGSZ1 attenuated D2 dopamine receptor agonist-induced serum response element reporter gene activity in Chinese hamster ovary cells. In summary, these data suggest that RGSZ1 serves as a GTPase accelerating protein for Galpha(i) and regulates Galpha(i)-mediated signaling, thus expanding the potential role of RGSZ1 in G protein-mediated cellular activities.

Regulation of stress response signaling by the N-terminal dishevelled/EGL-10/pleckstrin domain of Sst2, a regulator of G protein signaling in Saccharomyces cerevisiae.

All members of the regulator of G protein signaling (RGS) family contain a conserved core domain that can accelerate G protein GTPase activity. The RGS in yeast, Sst2, can inhibit a G protein signal leading to mating. In addition, some RGS proteins contain an N-terminal domain of unknown function. Here we use complementary whole genome analysis methods to investigate the function of the N-terminal Sst2 domain. To identify a signaling pathway regulated by N-Sst2, we performed genome-wide transcription profiling of cells expressing this fragment alone and found differences in 53 transcripts. Of these, 40 are induced by N-Sst2, and nearly all contain a stress response element (STRE) in the promoter region. To identify components of a signaling pathway leading from N-Sst2 to STREs, we performed a genome-wide two-hybrid analysis using N-Sst2 as bait and found 17 interacting proteins. To identify the functionally relevant interacting proteins, we analyzed all of the available gene deletion mutants and found three (vps36 Delta, pep12 Delta, and tlg2 Delta) that induce STRE and also repress pheromone-dependent transcription. We selected VPS36 for further characterization. A vps36 Delta mutation diminishes signaling by pheromone as well as by downstream components including the G protein, effector kinase (Ste11), and transcription factor (Ste12). Conversely, overexpression of Vps36 enhances the pheromone response in sst2 Delta cells but not in wild type. These findings indicate that Vps36 and Sst2 have opposite and opposing effects on the pheromone and stress response pathways, with Vps36 acting downstream of the G protein and independently of Sst2 RGS activity.