📋 Browse Articles

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. Show less

Dysregulation of dopamine (DA) receptor signalling induces specific changes in behaviour, neuronal circuitry and gene expression in the mammalian forebrain. In order to better understand signalling adaptations at the molecular level, we used high-density oligonucleotide microarrays (Codelink Mouse 20K) to define alterations in the expression of transcripts encoding regulator of G-protein coupled receptor signalling in dopamine D1 receptor knockout mice (Drd1a-KO). Regulator of G-protein signalling (Rgs) 2, Rgs4, and Rgs9 were significantly decreased in the striatum (STR) of Drd1a-KO mice. These changes were confirmed by in situ hybridization, and were also observed in the nucleus accumbens (NAc). In contrast, analysis of the medial frontal cortex (MFC) revealed a significant decrease in Rgs17 expression exclusively, and a modest up-regulation of Rgs5 transcript. The expression of these gene products were not significantly altered in the dopamine-poor visual cortex (VC). The Drd1a-KO mouse, and a rabbit model of in utero cocaine exposure, in which D1R signalling is permanently reduced, possess analogous morphological and functional alterations in dopamine-modulated brain circuits; thus we also examined long-lasting changes in RGS transcript expression following prenatal exposure to cocaine. In sharp contrast to the Drd1a-KO, Rgs2 and Rgs4 were unchanged, and Rgs9 and Rgs17 transcripts were increased in prenatal cocaine-exposed progeny. These data suggest that an absolute absence of D1R signalling (Drd1a-KO) and hypomorphic D1R signalling (prenatal cocaine) produce common alterations in neuronal morphology, but distinct outcomes in molecular neuroadaptations. Show less

Regulators of G-protein signaling (RGS proteins) comprise over 20 different proteins that have been classified into subfamilies on the basis of structural homology. The RZ/A family includes RGSZ2/RGS17 (the most recently discovered member of this family), GAIP/RGS19, RGSZ1/RGS20, and the RGSZ1 variant Ret-RGS. The RGS proteins are GTPase activating proteins (GAPs) that turn off G-proteins and thus negatively regulate the signaling of G-protein coupled receptors (GPCRs). In addition, some RZ/A family RGS proteins are able to modify signaling through interactions with adapter proteins (such as GIPC and GIPN). The RZ/A proteins have a simple structure that includes a conserved amino-terminal cysteine string motif, RGS box and short carboxyl-terminal, which confer GAP activity (RGS box) and the ability to undergo covalent modification and interact with other proteins (amino-terminal). This review focuses on RGS17 and its RZ/A sibling proteins and discusses the similarities and differences among these proteins in terms of their palmitoylation, phosphorylation, intracellular localization and interactions with GPCRs and adapter proteins. The specificity of these RGS protein for different Galpha proteins and receptors, and the consequences for signaling are discussed. The tissue and brain distribution, and the evolving understanding of the roles of this family of RGS proteins in receptor signaling and brain function are highlighted. Show less

The regulator of G-protein signaling RGS17(Z2) is a member of the RGS-Rz subfamily of GTPase-activating proteins (GAP) that efficiently deactivate GalphazGTP subunits. We have found that in the central nervous system (CNS), the levels of RGSZ2 mRNA and protein are elevated in the hypothalamus, midbrain, and pons-medulla, and that RGSZ2 is glycosylated in synaptosomal membranes isolated from CNS tissue. In analyzing the function of RGSZ2 in the CNS, we found that when the expression of RGSZ2 was impaired, the antinociceptive response to morphine and [D-Ala2, N-MePhe4, Gly-ol5]-enkephalin (DAMGO) augmented. This potentiation involved mu-opioid receptors and increased tolerance to further doses of these agonists administered 24 h later. High doses of morphine promoted agonist desensitization even within the analgesia time-course, a phenomenon that appears to be related to the great capacity of morphine to activate Gz proteins. In contrast, the knockdown of RGSZ2 proteins did not affect the activity of delta receptor agonists, [D-Pen2,5]-enkephalin (DPDPE), and [D-Ala2] deltorphin II. In membranes from periaqueductal gray matter (PAG), both RGSZ2 and the related RGS20(Z1) co-precipitated with mu-opioid receptors. While a morphine challenge reduced the association of Gi/o/z with mu receptors, it increased their association with the RGSZ2 and RGSZ1 proteins. However, only Galphaz subunits co-precipitated with RGSZ2. Doses of morphine that produced acute tolerance maintained the association of Galpha subunits with RGSZ proteins even after the analgesic effects had ceased. These results indicate that both RGSZ1 and RGSZ2 proteins influence mu receptor signaling by sequestering Galpha subunits, therefore behaving as effector antagonists. Show less

Chromosomal rearrangements resulting in gene fusions are frequently involved in carcinogenesis. Here, we describe a semiautomatic procedure for identifying fusion gene transcripts by using publicly available mRNA and EST databases. With this procedure, we have identified 96 transcript sequences that are derived from 60 known fusion genes. Also, 47 or more additional sequences appear to be derived from 20 or more previously unknown putative fusion genes. We have experimentally verified the presence of a previously unknown IRA1/RGS17 fusion in the breast cancer cell line MCF7. The fusion gene encodes the full-length RGS17 protein, a regulator of G protein-coupled signaling, under the control of the IRA1 gene promoter. This study demonstrates that databases of ESTs can be used to discover fusion genes resulting from structural rearrangement of chromosomes. Show less

To identify novel regulators of Galpha(o), the most abundant G-protein in brain, we used yeast two-hybrid screening with constitutively active Galpha(o) as bait and identified a new regulator of G-protein signaling (RGS) protein, RGS17 (RGSZ2), as a novel human member of the RZ (or A) subfamily of RGS proteins. RGS17 contains an amino-terminal cysteine-rich motif and a carboxyl-terminal RGS domain with highest homology to hRGSZ1- and hRGS-Galpha-interacting protein. RGS17 RNA was strongly expressed as multiple species in cerebellum and other brain regions. The interactions between hRGS17 and active forms of Galpha(i1-3), Galpha(o), Galpha(z), or Galpha(q) but not Galpha(s) were detected by yeast two-hybrid assay, in vitro pull-down assay, and co-immunoprecipitation studies. Recombinant RGS17 acted as a GTPase-activating protein (GAP) on free Galpha(i2) and Galpha(o) under pre-steady-state conditions, and on M2-muscarinic receptor-activated Galpha(i1), Galpha(i2), Galpha(i3), Galpha(z), and Galpha(o) in steady-state GTPase assays in vitro. Unlike RGSZ1, which is highly selective for G(z), RGS17 exhibited limited selectivity for G(o) among G(i)/G(o) proteins. All RZ family members reduced dopamine-D2/Galpha(i)-mediated inhibition of cAMP formation and abolished thyrotropin-releasing hormone receptor/Galpha(q)-mediated calcium mobilization. RGS17 is a new RZ member that preferentially inhibits receptor signaling via G(i/o), G(z), and G(q) over G(s) to enhance cAMP-dependent signaling and inhibit calcium signaling. Differences observed between in vitro GAP assays and whole-cell signaling suggest additional determinants of the G-protein specificity of RGS GAP effects that could include receptors and effectors. Show less

The human tissue distribution of the nineteen known human regulators of G-protein signaling (RGS) is described. Measurement of RGS mRNA levels in human brain and in nine peripheral tissues revealed striking tissue preferences in gene expression. Five RGS members were identified with enriched expression in brain. RGS4, RGS7, RGS8, RGS11 and RGS17 were all significantly expressed in striatal regions including the nucleus accumbens and putamen. RGS4 had the highest measured levels of mRNA expression and was highly enriched in the gyrus of the cortex and in the parahippocampus. RGS7 and RGS17 had overlapping distribution profiles and were both noticeably enriched in the cerebellum. Several RGS family members showed high expression in peripheral tissues. RGS5 was preferentially expressed in heart, and RGS1, RGS13, RGS18 and GAIP were predominately expressed in lymphocytes. RGS1 was also highly enriched in the lung, as was RGS2 and RGS16. Five family members, RGS3, RGS9, RGS10, RGS 12 and RGS14 had a broad and overlapping mRNA distribution. These results suggest roles of the individual RGS members in a diversity of functions in humans and support a role of several RGS members in the regulation of central nervous system function via modulation of signaling by G-protein coupled receptors. Show less

We used the yeast two-hybrid system to identify proteins that interact directly with Galpha(o). Mutant-activated Galpha(o) was used as the bait to screen a cDNA library from chick dorsal root ganglion neurons. We found that Galpha(o) interacted with several proteins including Gz-GTPase-activating protein (Gz-GAP), a new RGS protein (RGS-17), a novel protein of unknown function (IP6), and Rap1GAP. This study focuses on Rap1GAP, which selectively interacts with Galpha(o) and Galpha(i) but not with Galpha(s) or Galpha(q). Rap1GAP interacts more avidly with the unactivated Galpha(o) as compared with the mutant (Q205L)-activated Galpha(o). When expressed in HEK-293 cells, unactivated Galpha(o) co-immunoprecipitates with the Rap1GAP. Expression of chick Rap1GAP in PC-12 cells inhibited activation of Rap1 by forskolin. When unactivated Galpha(o) was expressed, the amount of activated Rap1 was greatly increased. This effect was not observed with the Q205L-Galpha(o). Expression of unactivated Galpha(o) stimulated MAP-kinase (MAPK1/2) activity in a Rap1GAP-dependent manner. These results identify a novel function of Galpha(o), which in its resting state can sequester Rap1GAP thereby regulating Rap1 activity and consequently gating signal flow from Rap1 to MAPK1/2. Thus, activation of G(o) could modulate the Rap1 effects on a variety of cellular functions. Show less