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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.