RGS4 Research Articles

Brain-Specific Regulator of G-Protein Signaling 9-2 Selectively Interacts with α-Actinin-2 to Regulate Calcium-Dependent Inactivation of NMDA Receptors

Regulator of G-protein signaling 9-1 (RGS9-1) and RGS9-2 are highly related RGS proteins with distinctive C termini arising from alternative splicing of RGS9 gene transcripts. RGS9-1 is expressed in photoreceptors where it functions as a regulator of transducin. In contrast, RGS9-2 is abundantly expressed in the brain, especially in basal ganglia, where its specific function remains poorly understood. To gain insight into the function of RGS9-2, we screened a human cDNA library for potential interacting proteins. This screen identified a strong interaction between RGS9-2 and alpha-actinin-2, suggesting a possible functional relationship between these proteins. Consistent with this idea, RGS9-2 and alpha-actinin-2 coimmunoprecipitated after coexpression in human embryonic kidney 293 (HEK-293) cells. Furthermore, endogenous RGS9-2 and alpha-actinin-2 could also be coimmunoprecipitated from extracts of rat striatum, an area highly enriched in both these proteins. These results supported the idea that RGS9-2 and alpha-actinin-2 could act in concert in central neurons. Like alpha-actinin-2, RGS9-2 coimmunoprecipitated NMDA receptors from striatal extracts, suggesting an interaction between RGS9-2, alpha-actinin-2, and NMDA receptors. Previous studies have shown that alpha-actinin mediates calcium-dependent inactivation of NMDA receptors. In HEK-293 cells expressing NMDA receptors, expression of RGS9-2 significantly modulated this form of NMDA receptor inactivation. Furthermore, this modulation showed remarkable preference for NMDA receptor inactivation mediated by alpha-actinin-2. Using a series of deletion constructs, we localized this effect to the RGS domain of the protein. These results identify an unexpected functional interaction between RGS9-2 and alpha-actinin-2 and suggest a potential novel role for RGS9-2 in the regulation of NMDA receptor function.

Essential role for RGS9 in opiate action.

Regulators of G protein signaling (RGS) are a family of proteins known to accelerate termination of effector stimulation after G protein receptor activation. RGS9-2, a brain-specific splice variant of the RGS9 gene, is highly enriched in striatum and also expressed at much lower levels in periaqueductal gray and spinal cord, structures known to mediate various actions of morphine and other opiates. Morphine exerts its acute rewarding and analgesic effects by activation of inhibitory guanine nucleotide-binding regulatory protein-coupled opioid receptors, whereas chronic morphine causes addiction, tolerance to its acute analgesic effects, and profound physical dependence by sustained activation of these receptors. We show here that acute morphine administration increases expression of RGS9-2 in NAc and the other CNS regions, whereas chronic exposure decreases RGS9-2 levels. Mice lacking RGS9 show enhanced behavioral responses to acute and chronic morphine, including a dramatic increase in morphine reward, increased morphine analgesia with delayed tolerance, and exacerbated morphine physical dependence and withdrawal. These findings establish RGS9 as a potent negative modulator of opiate action in vivo, and suggest that opiate-induced changes in RGS9 levels contribute to the behavioral and neural plasticity associated with chronic opiate administration.

Human RGS6 Gene Structure, Complex Alternative Splicing, and Role of N Terminus and G Protein γ-Subunit-like (GGL) Domain in Subcellular Localization of RGS6 Splice Variants

RGS proteins are defined by the presence of a semiconserved RGS domain that confers the GTPase-activating activity of these proteins toward certain G alpha subunits. RGS6 is a member of a subfamily of RGS proteins distinguished by the presence of DEP and GGL domains, the latter a G beta 5-interacting domain. Here we report identification of 36 distinct transcripts of human RGS6 that arise by unusually complex processing of the RGS6 gene, which spans 630 kilobase pairs of genomic DNA in human chromosome 14 and is interrupted by 19 introns. These transcripts arise by use of two alternative transcription sites and complex alternative splicing mechanisms and encode proteins with long or short N-terminal domains, complete or incomplete GGL domains, 7 distinct C-terminal domains and a common internal domain where the RGS domain is found. The role of structural diversity in the N-terminal and GGL domains of RGS6 splice variants in their interaction with G beta 5 and subcellular localization and of G beta 5 on RGS6 protein localization was examined in COS-7 cells expressing various RGS6 splice variant proteins. RGS6 splice variants with complete GGL domains interacted with G beta 5, irrespective of the type of N-terminal domain, while those lacking a complete GGL domain did not. RGS6 protein variants displayed subcellular distribution patterns ranging from an exclusive cytoplasmic to exclusive nuclear/nucleolar localization, and co-expression of G beta 5 promoted nuclear localization of RGS6 proteins. Analysis of our results show that the long N-terminal and GGL domain sequences of RGS6 proteins function as cytoplasmic retention sequences to prevent their nuclear/nucleolar accumulation. These findings provide the first evidence for G beta 5-independent functions of the GGL domain and for a role of G beta 5 in RGS protein localization. This study reveals extraordinary complexity in processing of the human RGS6 gene and provides new insights into how structural diversity in the RGS6 protein family is involved in their localization and likely function(s) in cells.

Mild Heat and Proteotoxic Stress Promote Unique Subcellular Trafficking and Nucleolar Accumulation of RGS6 and Other RGS Proteins: ROLE OF THE RGS DOMAIN IN STRESS-INDUCED TRAFFICKING OF RGS PROTEINS

RGS proteins comprise a large family of proteins named for their ability to negatively regulate heterotrimeric G protein signaling. RGS6 is a member of the R7 subfamily of RGS proteins possessing DEP (disheveled/Egl-10/pleckstrin) homology and GGL (G protein gamma-subunit-like) domains in addition to the semiconserved RGS domain. Our previous study documented unusual complexity in splicing of the human RGS6 gene, and we demonstrated localization of various RGS6 splice forms at sites other than the plasma membrane, including the cytoplasm and nucleus, where G proteins are not localized (Chatterjee, T. K., Liu, Z., and Fisher, R. A. (2003) J. Biol. Chem. 278, 30261-30271). Here we provide new evidence that mild heat stress, proteasome-mediated proteotoxic stress, and HSF1 expression induces dramatic relocalization of RGS6 proteins from such sites to nucleoli. This response was observed in COS-7 cells expressing various splice forms of RGS6, was not elicited by other forms of cellular stress and was observed in cells treated with various protein kinase inhibitors or co-expressing a dominant-negative kinase inactive SAPK. The RGS domain of RGS6 was identified as a primary structural module providing support for its stress-induced nucleolar trafficking and various other RGS proteins or their isolated RGS domains similarly undergo nucleolar migration in response to heat or proteotoxic stress or during co-expression of HSF1. The atypical RGS domains of axin and AKAP10 also underwent stress-induced nucleolar trafficking while structural domains outside of the RGS domain of some RGS proteins can override nucleolar trafficking in response to stress. Inhibition of rDNA transcription also promoted nucleolar migration of RGS6, a response previously observed in a subset of nucleolar proteins. The DEP domain of RGS6, but not its RGS domain, conferred structural support for its transcription-linked nucleolar migration. RGS6 exhibited trafficking from subnuclear dots to nucleoli in response to heat-, proteotoxic- or transcription-linked stress. These results provide new evidence that mammalian RGS proteins undergo unique subcellular trafficking in response to specific forms of cellular stress and implicate the RGS family of proteins in cellular stress signaling pathways.

RGS2: a “turn-off” in hypertension

RGS2: a “turn-off” in hypertension

Regulation of RGS3 and RGS10 Palmitoylation by GnRH

Regulation of RGS3 and RGS10 Palmitoylation by GnRH

Lack of RGS2 Leaves Mice Anxious

The intrinsic guanosine triphosphatase (GTPase) activity of heterotrimeric G proteins is increased by RGS (regulators of G protein signaling) proteins. Oliveira-dos-Santos et al. identified a role for RGS2 in T cell activation. In response to T cell receptor (TCR) engagement, amounts of rgs2 mRNA and protein increase; however, in rgs2-/- T cells, proliferation and IL-2 production are decreased. rgs2-/- T cells treated with phorbol myristate acetate (PMA) and ionomycin also exhibited defective proliferation, indicating that in rgs2-/- animals the defect lies distal to TCR activation. Defective T cell proliferation was manifested by reduced immunity to viral infection in rgs2-/- mice. Male, but not female, rgs2-deficient mice also exhibited decreased aggression and increased anxiety. Motor skills and cognition appeared unaffected. Gross examination of hippocampi revealed that CA1 neurons from rgs2-/- mice had reduced numbers of apical and basal spines (and possibly fewer synapses), and reduced electrical activity. Thus, further research into the signaling pathway(s) mediated by RGS2 in the brain may lead to the development of neuropsychiatric agents that control anxiety and aggression.Oliveira-dos-Santos, A.J., Matsumoto, G., Snow, B.E., Bai, D., Houston, F.P., Whishaw, I.Q., Mariathasan, S., Sasaki, T., Wakeham, A., Ohashi, P.S., Roder, J.C., Barnes, C.A., Siderovski, D.P., and Penninger, J.M. (2000) Regulation of T cell activation, anxiety, and male aggression by RGS2. Proc. Natl. Acad. Sci. U.S.A. 97: 12272-12277. [Abstract] [Full Text]

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.

Cloning and Characterization of RGS9-2: A Striatal-Enriched Alternatively Spliced Product of the RGS9 Gene

Regulators of G-protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) for alpha subunits of heterotrimeric G-proteins. Previous in situ hybridization analysis of mRNAs encoding RGS3-RGS11 revealed region-specific expression patterns in rat brain. RGS9 showed a particularly striking pattern of almost exclusive enrichment in striatum. In a parallel study, RGS9 cDNA, here referred to as RGS9-1, was cloned from retinal cDNA libraries, and the encoded protein was identified as a GAP for transducin (Galphat) in rod outer segments. In the present study we identify a novel splice variant of RGS9, RGS9-2, cloned from a mouse forebrain cDNA library, which encodes a striatal-specific isoform of the protein. RGS9-2 is 191 amino acids longer than the retinal isoform, has a unique 3' untranslated region, and is highly enriched in striatum, with much lower levels seen in other brain regions and no expression detectable in retina. Immunohistochemistry showed that RGS9-2 protein is restricted to striatal neuropil and absent in striatal terminal fields. The functional activity of RGS9-2 is supported by the finding that it, but not RGS9-1, dampens the Gi/o-coupled mu-opioid receptor response in vitro. Characterization of a bacterial artificial chromosome genomic clone of approximately 200 kb indicates that these isoforms represent alternatively spliced mRNAs from a single gene and that the RGS domain, conserved among all known RGS members, is encoded over three distinct exons. The distinct C-terminal domains of RGS9-2 and RGS9-1 presumably contribute to unique regulatory properties in the neural and retinal cells in which these proteins are selectively expressed.

The human regulator of G-protein signaling protein 6 gene (RGS6) maps between markers WI-5202 and D14S277 on chromosome 14q24.3.

The recently discovered regulators of G-protein signaling proteins, termed the RGS family, have been shown to modulate the functioning of G-proteins by activating the intrinsic guanosine triphosphatase (GTPase) activity of the alpha subunits. Here, we report the chromosomal location and tissue expression of the human regulator of RGS6 gene. The messenger RNA was ubiquitously expressed in various tissues. Polymerase chain reaction (PCR)-based analysis with a human/rodent monochromosomal hybrid panel and a radiation hybrid panel indicated that the gene was mapped between genetic markers WI-5202 and D14S277 on chromosome 14q24.3 region.