A novel member of the recently identified family of regulators of heterotrimeric G protein signalling (RGS) in the yeast Saccharomyces cerevisiae has been characterized. The YOR107w/RGS2 gene was isolated as a multi-copy suppressor of glucose-induced loss of heat resistance in stationary phase cells. The N-terminal half of the Rgs2 protein consists of a typical RGS domain. Deletion of Rgs2 enhances, while its overexpression reduces, the glucose-induced accumulation of cAMP. Overexpression of RGS2 generates phenotypes consistent with low activity of cAMP-dependent protein kinase A (PKA), such as enhanced accumulation of trehalose and glycogen, enhanced heat resistance and elevated expression of STRE-controlled genes. Deletion of RGS2 causes the opposite phenotypes. Rgs2 functions as a negative regulator of glucose-induced cAMP signaling through direct GTPase activation of the Gs-alpha protein Gpa2. Rgs2 and Gpa2 constitute the second cognate RGS-G-alpha protein pair identified in yeast, in addition to the mating pheromone pathway regulators Sst2 and Gpa1. Moreover, Rgs2 and Sst2 exert specific, non-overlapping functions, and deletion mutants in Rgs2 and Sst2 are complemented to some extent by different mammalian RGS proteins (Versele, 1999).

The frequencies of certain periodic behaviors of the nematode C. elegans are regulated in a dose-dependent manner by the activity of the gene egl-10, a regulator of G protein signaling. These behaviors are modulated oppositely by the activity of the G protein alpha subunit gene goa-1, suggesting that egl-10 may regulate a G protein signaling pathway in a dose-dependent fashion. Loss-of-function alleles of goa-1 strongly increase the frequency of egg-laying behavior and also increase the frequencies of locomotory body bends and other behaviors. Loss-of-function alleles of egl-1 severely reduce or abolish egg-laying behavior and initiate body bends less frequently. egl-10 encodes a protein similar to Sst2p, a negative regulator of G protein signaling in yeast. EGL-10 protein is localized in neural processes, where it may function in neurotransmitter signaling. Two previously known and 13 newly identified mammalian genes have similarity to egl-10 and SST2; it is proposed that members of this family regulate many G protein signaling pathways (Koelle, 1996).

Asymmetric divisions are crucial for generating cell diversity; they rely on coupling between polarity cues and spindle positioning, but how this coupling is achieved is poorly understood. In one-cell stage Caenorhabditis elegans embryos, polarity cues set by the PAR proteins mediate asymmetric spindle positioning by governing an imbalance of net pulling forces acting on spindle poles. The GoLoco-containing proteins GPR-1 and GPR-2, as well as the Galpha subunits GOA-1 and GPA-16, are essential for generation of proper pulling forces. GPR-1/2 interacts with guanosine diphosphate-bound GOA-1 and were enriched on the posterior cortex in a par-3- and par-2-dependent manner. Thus, the extent of net pulling forces may depend on cortical Galpha activity, which is regulated by anterior-posterior polarity cues through GPR-1/2 (Colombo, 2003).

A Drosophila gene encoding a homolog of the regulator of G-protein signaling (RGS) protein family has been identified. This gene (dRGS7) is expressed in neurons of the embryo and adult fly and is predicted to encode a 428-amino acid protein with >55% overall amino acid sequence identity with the vertebrate protein RGS7 and the C. elegans EGL-10. The dRGS7 protein is 50% conserved in the C-terminal RGS domain with RGS7 and EGL-10 but, remarkably, displays much greater conservation with the N-terminal regions of these proteins. This finding implies a conserved function for these homologs from divergent species involving domains outside the RGS domain. The dRGS7 protein also has a domain of similarity with Dishevelled and pleckstrin, raising the possibility that these proteins interact with common signaling components (Elmore, 1998).

To elucidate the cellular role of the heterotrimeric G protein G_o, a molecular genetic approach has been taken in Caenorhabditis elegans. A screen was carried out for suppressors of activated GOA-1 (G_oalpha) that do not simply decrease GOA-1 expression. Mutations were found in only two genes, sag-1 and eat-16. Animals defective in either gene display a hyperactive phenotype similar to that of goa-1 loss-of-function mutants. Double-mutant analysis indicates that both sag-1 and eat-16 act downstream of, or parallel to, G_oalpha and negatively regulate EGL-30 (G_qalpha) signaling. eat-16 encodes a regulator of G protein signaling (RGS) most similar to the mammalian RGS7 and RGS9 proteins and can inhibit endogenous mammalian G_q/G₁₁ in COS-7 cells. Animals defective in both sag-1 and eat-16 are not viable, but reducing function in egl-30 restores viability, indicating that the lethality of the eat-16; sag-1 double mutant is due to excessive G_qalpha activity. Analysis of these mutations indicates that the G_o and G_q pathways function antagonistically in C. elegans, and that G_oalpha negatively regulates the G_q pathway, possibly via EAT-16 or SAG-1. It is proposed that a major cellular role of G_o is to antagonize signaling by G_q (Hajdu-Cronin, 1999).

These results are consistent with a model in which a network of G protein pathways within cells can affect behavior by both positive and negative cross talk. Although synergistic effects between G_i/o and G_q pathways are known, the results presented here indicate negative regulation of G_qalpha or its downstream targets by G_oalpha. That G_o and G_q function antagonistically in some way was implied from the opposite phenotypes of goa-1 and egl-30 mutations (Brundage, 1996). The isolation and analysis of GOA-1 suppressors involved in G_qalpha signaling support the model that G_oalpha functions to modulate behavior by down-regulating the G_q pathway in C. elegans and perhaps in other species as well. These results are analogous to the stimulatory and inhibitory effects of G_s and G_i on adenylyl cyclase, raising the possibility that G protein subunits that act antagonistically are more universal than previously thought (Hajdu-Cronin, 1999 and references).

Heterotrimeric G proteins promote microtubule forces that position mitotic spindles during asymmetric cell division in C. elegans embryos. While all previously studied G protein functions require activation by seven-transmembrane receptors, this function appears to be receptor independent. Mutating a regulator of G protein signaling, RGS-7, results in hyperasymmetric spindle movements due to decreased force on one spindle pole. RGS-7 is localized at the cell cortex, and its effects require two redundant Galpha_o-related G proteins and their nonreceptor activators RIC-8 and GPR-1/2. Using recombinant proteins, it was found that RIC-8 stimulates GTP binding by Galpha_o and that the RGS domain of RGS-7 stimulates GTP hydrolysis by Galpha_o, demonstrating that Galpha_o passes through the GTP bound state during its activity cycle. While GTPase activators typically inactivate G proteins, RGS-7 instead appears to promote G protein function asymmetrically in the cell, perhaps acting as a G protein effector (Hess, 2004).

The heterotrimeric G proteins that control C. elegans spindle movements operate via an activation/inactivation cycle different from the signal transduction G protein cycle. Two redundant Gα_o-related Gα proteins, GOA-1 and GPA-16, along with the Gβ subunit GPB-1 and the Gγ subunit GPC-2, are required for proper spindle movements in C. elegans embryos. Activation of these G proteins is thought to be receptor independent, since (1) it occurs in the one-cell C. elegans zygote, which is encased by an impermeable egg shell, so that no source of an extracellular ligand is obvious, and (2) a set of nontransmembrane proteins have been identified that appear to activate the G proteins in lieu of transmembrane receptor(s). Removal of any of these activators results in spindle movement defects similar to those in embryos lacking the Gα proteins. The activators include the 97% identical GPR-1 and GPR-2 proteins, which contain a GPR/GoLoco motif that binds GOA-1 in its GDP bound form. The involvement of Gα_o and GPR/GoLoco proteins in mitotic spindle control appears to be evolutionarily conserved, since the GPR/GoLoco motif protein PINS acts with a Gα_i/o protein to control asymmetric neuroblast divisions in Drosophila, the mammalian GPR/GoLoco protein LGN regulates mitotic spindle organization, and the mammalian Gα_o protein is found associated with the mitotic spindle in cultured cells. In C. elegans, GPR-1/2 proteins form a complex with the coiled-coil protein LIN-5, which localizes GPR-1/2 to the cell cortex and mitotic spindle. An additional nonreceptor activator that controls C. elegans centrosome movements is RIC-8, whose mammalian ortholog Ric-8A was recently shown to act in vitro as a guanine nucleotide exchange factor for G proteins including Gα_o (Hess, 2004).

Fundamental issues regarding the mechanism of asymmetric spindle positioning remain unresolved: (1) all models propose that asymmetric microtubule forces are generated by greater G protein activity at the posterior than at the anterior pole of the zygote, but it remains unclear how such asymmetric G protein activity is generated; (2) alternative models have been proposed in which either a Gα·GDP/GPR complex or Gα·GTP is the active G protein species that promotes microtubule forces, but it remains to be established which of these species are actually generated and active; (3) the mechanism by which an active G protein controls microtubule forces is unknown (Hess, 2004).

This study shows that an RGS protein, RGS-7, controls asymmetric movements of the mitotic spindle. RGS-7 affects force on the anterior but not the posterior spindle pole, suggesting that it is a source of asymmetric G protein function. In vitro, RIC-8 promotes GTP binding by Gα_o, while RGS-7 acts as a Gα_o GTPase activator, demonstrating that Gα_o is present in its GTP bound form as part of its receptor-independent activity cycle. While GTPase activators typically inactivate G proteins, RGS-7 apparently promotes G protein function. RGS-7 could serve dual roles as both a Gα_o inactivator and a Gα_o effector so that its net function is to promote microtubule force (Hess, 2004).

Signal transduction through heterotrimeric G proteins is critical for sensory response across species. Regulator of G protein signaling (RGS) proteins are negative regulators of signal transduction. This study describes a role for C. elegans RGS-3 in the regulation of sensory behaviors. rgs-3 mutant animals fail to respond to intense sensory stimuli but respond normally to low concentrations of specific odorants. Loss of RGS-3 leads to aberrantly increased G protein-coupled calcium signaling but decreased synaptic output, ultimately leading to behavioral defects. Thus, rgs-3 responses are restored by decreasing G protein-coupled signal transduction, either genetically or by exogenous dopamine, by expressing a calcium-binding protein to buffer calcium levels in sensory neurons or by enhancing glutamatergic synaptic transmission from sensory neurons. Therefore, while RGS proteins generally act to downregulate signaling, loss of a specific RGS protein in sensory neurons can lead to defective responses to external stimuli (Ferkey, 2007).

Two novel rat regulators of G-protein signaling (RGS) cDNAs were cloned using a degenerate PCR strategy. The rRgs12 and rRgs14 cDNAs encode predicted polypeptides of 1387 and 544 amino acids, respectively. The human ortholog of rRgs12 was identified by alignment of cosmid sequences in the database that map the human RGS12 gene to chromosome 4p16.3. Furthermore, human ESTs with high homology to rRgs14 that map to human chromosome 5qter were identified. Northern blot analysis indicates that rRgs14 is expressed at high levels in brain, lung, and spleen, whereas rRgs12 is expressed at high levels in brain and lung and lower levels in testis, heart, and spleen. Analysis of the predicted rRGS12 and rRGS14 polypeptides indicates that they are closely related and possess regions of homology outside of the conserved RGS domain. Conserved regions in RGS12 were identified which are similar to protein domains found in mouse rhophilin and coiled-coil proteins, suggesting possible interactions with ras-like G-proteins (Snow, 1997).

Using the yeast two-hybrid system, a human protein GAIP (G Alpha Interacting Protein) has been isolated that specifically interacts with the heterotrimeric GTP-binding protein G alpha i3. Interaction was verified by specific binding of in vitro-translated G alpha i3 with a GAIP-glutathione S-transferase fusion protein. GAIP is a small protein (217 amino acids, 24 kDa) that contains two potential phosphorylation sites for protein kinase C and seven for casein kinase 2. GAIP shows high homology to two previously identified human proteins, GOS8 and 1R20; two Caenorhabditis elegans proteins, CO5B5.7 and C29H12.3, and the FLBA gene product in Aspergillus nidulans -- all of unknown function. Significant homology was also found to the SST2 gene product in Saccharomyces cerevisiae that is known to interact with a yeast G alpha subunit (Gpa1). A highly conserved core domain of 125 amino acids characterizes this family of proteins. Analysis of deletion mutants demonstrates that the core domain is the site of GAIP's interaction with G alpha i3. GAIP is likely to be an early inducible phosphoprotein, because in its 3'-untranslated region its cDNA contains the TTTTGT sequence characteristic of early response genes. By Northern analysis GAIP's 1.6-kb mRNA is most abundant in lung, heart, placenta, and liver and is very low in brain, skeletal muscle, pancreas, and kidney. GAIP appears to interact exclusively with G alpha i3, since it did not interact with G alpha i2 and G alpha q. The fact that GAIP and Sst2 interact with G alpha subunits and share a common domain suggests that other members of the GAIP family also interact with G alpha subunits through the 125-amino-acid core domain (De Vries, 1995).

Palmitoylation inhibits by more than 90 percent the response of the alpha subunit of the guanine nucleotide-binding protein Gz to the guanosine triphosphatase (GTPase)-accelerating activity of Gz GAP, a Gz-selective member of the regulators of G-protein signaling (RGS) protein family of GTPase-activating proteins (GAPs). Palmitoylation both decreases the affinity of Gz GAP for the GTP-bound form of Galphaz by at least 90 percent and decreases the maximum rate of GTP hydrolysis. Inhibition is reversed by removal of the palmitoyl group. Palmitoylation of Galphaz also inhibits its response to the GAP activity of Galpha-interacting protein (GAIP), another RGS protein, and palmitoylation of Galphai1 inhibits its response to RGS4. The extent of inhibition of Gz GAP, GAIP, RGS4, and RGS10 correlates roughly with their intrinsic GAP activities for the Galpha target used in the assay. Reversible palmitoylation is thus a major determinant of Gz deactivation after its stimulation by receptors, and may be a general mechanism for prolonging or potentiating G-protein signaling (Tu, 1997).

Protein regulators of G protein signaling (RGS proteins) were discovered as negative regulators of heterotrimeric G protein-mediated signal transduction in yeast and worms. Experiments with purified recombinant proteins in vitro have established that RGS proteins accelerate the GTPase activity of certain G protein alpha subunits (the reaction responsible for their deactivation); these subunits can also act as effector antagonists. Either of two such RGS proteins, RGS4 or GAIP, attenuate signal transduction mediated by endogenous receptors, G proteins, and effectors when stably expressed as tagged proteins in transfected mammalian cells. The pattern of selectivity observed in vivo is similar to that seen in vitro. RGS4 and GAIP both attenuate Gi-mediated inhibition of cAMP synthesis. RGS4 is more effective than GAIP in blocking Gq-mediated activation of phospholipase Cbeta (C. Huang, 1997).

Recombinant regulators of G protein-signaling (RGS) proteins stimulate hydrolysis of GTP by alpha subunits of the Gi family but have not been reported to regulate other G protein alpha subunits. Expression of recombinant RGS proteins in cultured cells inhibits Gi-mediated hormonal signals, probably by acting as GTPase-activating proteins for Galphai subunits. To find out whether an RGS protein can also regulate cellular responses mediated by G proteins in the Gq/11 family, a comparison was undertaken of the activation of mitogen-activated protein kinase (MAPK) by (1) a Gq/11-coupled receptor [the bombesin receptor (BR)], and (2) a Gi-coupled receptor (the D2 dopamine receptor). RGS and these receptors were transiently co-expressed with or without recombinant RGS4 in COS-7 cells. Pertussis toxin, which uncouples Gi from receptors, blocks MAPK activation by the D2 dopamine receptor but not by the BR. Co-expression of RGS4, however, inhibits activation of MAPK by both receptors, causing a rightward shift of the concentration-effect curve for both receptor agonists. RGS4 also inhibits BR-stimulated synthesis of inositol phosphates by an effector target of Gq/11, phospholipase C. Moreover, RGS4 inhibits inositol phosphate synthesis activated by the addition of AlF4- to cells overexpressing recombinant alphaq, probably by binding to alphaq.GDP.AlF4-. These results demonstrate that RGS4 can regulate Gq/11-mediated cellular signals by competing for effector binding as well as by acting as a GTPase-activating protein (Yan, 1997).

Regulators of heterotrimeric G protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) that accelerate GTP hydrolysis by Gq and Gi alpha subunits, thus attenuating signaling. Mechanisms that provide more precise regulatory specificity have been elusive. An N-terminal domain of RGS4 discriminates among receptor signaling complexes coupled via Gq. Accordingly, deletion of the N-terminal domain of RGS4 eliminates receptor selectivity and reduces potency by 10(4)-fold. Receptor selectivity and potency of inhibition are partially restored when the RGS4 box is added together with an N-terminal peptide. In vitro reconstitution experiments also indicate that sequences flanking the RGS4 box are essential for high potency GAP activity. Thus, RGS4 regulates Gq class signaling by the combined action of two domains: 1) the RGS box accelerates GTP hydrolysis by Galphaq and 2) the N terminus conveys high affinity and receptor-selective inhibition. These activities are each required for receptor selectivity and high potency inhibition of receptor-coupled Gq signaling (Zeng, 1998).

An investigation was carried out of the function and the mechanism of action of RGS3. An 80-kDa protein has been identified as RGS3 by immunoprecipitation and immunoblotting with anti-RGS3 antibodies in a human mesangial cell line (HMC) stably transfected with RGS3 cDNA. RGS3 binds to aluminum fluoride-activated Galpha11 and to a lesser extent to Galphai3. This binding is mediated by the RGS domain of RGS3. A role of RGS3 in postreceptor signaling was demonstrated by decreased calcium responses and mitogen-activated protein (MAP) kinase activity induced by endothelin-1 in HMC stably overexpressing RGS3. Depletion of endogenous RGS3 by transfection of antisense RGS3 cDNA in NIH 3T3 cells results in enhanced MAP kinase activation induced by endothelin-1. RGS3 has a unique cytosolic localization. Activation of G proteins by AlF4-, NaF, or endothelin-1 results in redistribution of RGS3 from cytosol to the plasma membrane. Agonist-induced translocation of RGS3 occurs by a dual mechanism involving both C-terminal (RGS domain) and N-terminal regions of RGS3. Thus, coexpression of RGS3 with a constitutively active mutant of Galpha11 (Galpha11-QL) results in the binding of RGS3 (but not of its N-terminal fragment) to the membrane fraction and in its interaction with Galpha11-QL in vitro without any stimuli. However, both full-length RGS3 and its N-terminal domain translocate to the plasma membrane upon stimulation of intact cells with endothelin-1. The effect of endothelin-1 is mimicked by calcium ionophore A23187, suggesting the importance of Ca2+ in the mechanism of redistribution of RGS3. These data indicate that RGS3 inhibits G protein-coupled receptor signaling by a complex mechanism involving its translocation to the membrane in addition to its established function as a GTPase-activating protein (Dulin, 1999).

Regulators of G protein signaling (RGS) modulate G protein activity by functioning as GTPase-activating proteins (GAPs) for alpha-subunits of heterotrimeric G proteins. RGS14 regulates G protein nucleotide exchange and hydrolysis by acting as a GAP through its RGS domain and as a guanine nucleotide dissociation inhibitor (GDI) through its GoLoco motif. RGS14 exerts GDI activity on Galpha_i1, but not Galpha_o. Selective interactions are mediated by contacts between the alphaA and alphaB helices of the Galpha_i1 helical domain and the GoLoco C terminus. Three isoforms of Galpha_i exist in mammalian cells. This study asked whether all three isoforms were subject to RGS14 GDI activity. RGS14 inhibits guanine nucleotide exchange on Galpha_i1 and Galpha_i3, but not Galpha_i2. Galpha_i2 could be rendered sensitive to RGS14 GDI activity by replacement of residues within the alpha-helical domain. In addition to the contact residues in the alphaA and alphaB helices previously identified, it was found that the alphaA/alphaB and alphaB/alphaC loops are important determinants of Galpha_i selectivity. The striking selectivity observed for RGS14 GDI activity in vitro points to Galpha_i1 and Galpha_i3 as the likely targets of RGS14-GoLoco regulation in vivo (Mittal, 2004).

RGS proteins stimulate the intrinsic GTPase activity of the a subunits of heterotrimeric G-proteins, and thereby negatively regulate G-protein-coupled receptor signalling. RGS14 stimulates the GTPase activities of Ga_o and Ga_i subunits through its N-terminal RGS domain, and down-modulates signalling from receptors coupled to G_i. It also contains a central domain that binds active Rap proteins, as well as a C-terminal GoLoco/G-protein regulatory motif that has been shown to act in vitro as a GDP-dissociation inhibitor for Ga_i. In order to elucidate the respective contributions of the three functional domains of RGS14 to its ability to regulate G_i signalling, RGS14 mutants invalidated in each of its domains, as well as truncated molecules, were generated and their effects on G_i signalling were assessed via the bg pathway in a stable cell line ectopically expressing the G_i-coupled M2 muscarinic acetylcholine receptor (HEK-m2). The RGS and GoLoco domains of RGS14 are independently able to inhibit signalling downstream of G_i. Targeting of the isolated GoLoco domain to membranes, by myristoylation/palmitoylation or Rap binding, enhances its inhibitory activity on G_i signalling. Finally, in the context of the full RGS14 molecule, the RGS and GoLoco domains co-operate to confer maximal activity on RGS14. It is therefore proposed that RGS14 combines the inhibition of G_i activation or coupling to receptors via its GoLoco domain with stimulation of the GTPase activity of Ga_i-GTP via its RGS domain to negatively regulate signalling downstream of G_i (Traver, 2004).

Heterotrimeric G-proteins bind to cell-surface receptors and are integral in transmission of signals from outside the cell. Upon activation of the Galpha subunit by binding of GTP, the Galpha and Gbetagamma subunits dissociate and interact with effector proteins for signal transduction. Regulatory proteins with the 19-amino-acid GoLoco motif can bind to Galpha subunits and maintain G-protein subunit dissociation in the absence of Galpha activation. This study describes the structural determinants of GoLoco activity as revealed by the crystal structure of Galpha(i1) GDP bound to the GoLoco region of the RGS protein RGS14. Key contacts are described between the GoLoco motif and Galpha protein, including the extension of GoLoco's highly conserved Asp/Glu-Gln-Arg triad into the nucleotide-binding pocket of Galpha to make direct contact with the GDP alpha- and beta-phosphates. The structural organization of the GoLoco Galpha(i1) complex, when combined with supporting data from domain-swapping experiments, suggests that the Galpha all-helical domain and GoLoco-region carboxy-terminal residues control the specificity of GoLoco Galpha interactions (Kimple, 2002).

Regulators of G protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) toward the alpha subunits of heterotrimeric, signal-transducing G proteins. RGS11 contains a G protein gamma subunit-like (GGL) domain between its Dishevelled/Egl-10/Pleckstrin and RGS domains. GGL domains are also found in RGS6, RGS7, RGS9, and the Caenorhabditis elegans protein EGL-10. Coexpression of RGS11 with different Gbeta subunits reveals specific interaction between RGS11 and Gbeta5. The expression of mRNA for RGS11 and Gbeta5 in human tissues overlaps. The Gbeta5/RGS11 heterodimer acts as a GAP on Galphao, apparently selectively. RGS proteins that contain GGL domains appear to act as GAPs for Galpha proteins and form complexes with specific Gbeta subunits, adding to the combinatorial complexity of G protein-mediated signaling pathways (Snow, 1998b).

The G protein beta subunit Gbeta5 deviates significantly from the other four members of Gbeta-subunit family in amino acid sequence and subcellular localization. To detect the protein targets of Gbeta5 in vivo, a native Gbeta5 protein complex was isolated from the retinal cytosolic fraction and the protein tightly associated with Gbeta5 was identified as the regulator of G protein signaling (RGS) protein, RGS7. Complexes of Gbeta5 with RGS proteins can be formed in vitro from the recombinant proteins. The reconstituted Gbeta5-RGS dimers are similar to the native retinal complex in their behavior on gel-filtration and cation-exchange chromatographies and can be immunoprecipitated with either anti-Gbeta5 or anti-RGS7 antibodies. The specific Gbeta5-RGS7 interaction is determined by a distinct domain in RGS that has a striking homology to Ggamma subunits. Deletion of this domain prevents the RGS7-Gbeta5 binding, although the interaction with Galpha is retained. Substitution of the Ggamma-like domain of RGS7 with a portion of Ggamma1 changes its binding specificity from Gbeta5 to Gbeta1. The interaction of Gbeta5 with RGS7 blocks the binding of RGS7 to the Galpha subunit Galphao, indicating that Gbeta5 is a specific RGS inhibitor (Levay, 1999).

Heterotrimeric G protein alpha subunits, RGS proteins, and GoLoco motif proteins have been implicated in the control of mitotic spindle dynamics in C. elegans and D. melanogaster. RGS14 is expressed by the mouse embryonic genome immediately prior to the first mitosis, where it colocalizes with the anastral mitotic apparatus of the mouse zygote. Loss of Rgs14 expression in the mouse zygote results in cytofragmentation and failure to progress to the 2-cell stage. RGS14 is found in all tissues and segregates to the nucleus in interphase and to the mitotic spindle and centrioles during mitosis. Alteration of RGS14 levels in exponentially proliferating cells leads to cell growth arrest. These results indicate that RGS14 is one of the earliest essential products of the mammalian embryonic genome yet described and has a general role in mitosis (Martin-McCaffrey, 2004).

A clone of the regulator of G-protein signaling, RGS9, was isolated from a rat striatum-minus-cerebellum-minus-hippocampus subtracted library generated by directional tag polymerase chain reaction subtraction. The full-length cDNA clone encodes a 444 amino acid protein containing a 118 amino acid RGS domain, which corresponds to an evolutionarily conserved domain that is present in all members of the RGS family of proteins. Outside of the homology domain, RGS9 shows more extended similarity to human RGS6 and RGS7, rat RGS12, and the C. elegans protein EGL-10. During embryonic and early postnatal stages of development, two RGS9 transcripts of approximately 1.4 Kb and 1.8 Kb are detected in whole brain. After postnatal day 10, accumulation of the larger transcript increases progressively (until adulthood) at the expense of the smaller transcript, which is undetectable in the adult. In adult rat brain, the 1.8-Kb RGS9 transcript is detected in the striatum but not in other brain regions or peripheral tissues. In situ hybridization in rat and mouse demonstrates that RGS9 mRNA is expressed predominantly in medium-sized, spiny neurons of the neostriatum and in neurons of the nucleus accumbens and olfactory tubercle. Relatively strong signals are also detected in some hypothalamic nuclei. Its selective expression suggests that RGS9 may play an important role in modulation of the complex signaling pathways of the basal ganglia (Thomas, 1998).

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 reveal region-specific expression patterns in rat brain. RGS9 shows 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. A novel splice variant of RGS9, RGS9-2, 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. 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 (Rahman, 1999).

Regulators of G protein signaling (RGS) proteins serve as potent GTPase-activating proteins for the heterotrimeric G proteins alphai/o and aq/11. This study describes the immunohistochemical distribution of RGS7 throughout the adult rat brain and its cellular colocalization with Galphaq/11, an important G protein-coupled receptor signal transducer for phospholipase Cbeta-mediated activity. In general, both RGS7 and Galphaq/11 display a heterogeneous and overlapping regional distribution. RGS7 immunoreactivity is observed in cortical layers I-VI, being most intense in the neuropil of layer I. In the hippocampal formation, RGS7 immunoreactivity is concentrated in the strata oriens, strata radiatum, mossy fibers, and polymorphic cells, with faint to nondetectable immunolabeling within the dentate gyrus granule cells and CA1-CA3 subfield pyramidal cells. Numerous diencephalic and brainstem nuclei also display dense RGS7 immunostaining. Dual immunofluorescence labeling studies with the two protein-specific antibodies indicate a cellular selectivity in the colocalization between RGS7 and Galphaq/11 within many discrete brain regions, such as the superficial cortical layer I, hilus area of the hippocampal formation, and cerebellar Golgi cells. To assess the ability of Galphaq/11-mediated signaling pathways to dynamically modulate RGS expression, primary cortical neuronal cultures were incubated with phorbol 12,13-dibutyrate, a selective protein kinase C activator. A time-dependent increase in levels of mRNA for RGS7, but not RGS4, is observed. These results provide novel information on the region- and cell-specific pattern of distribution of RGS7 with the transmembrane signal transducer, Galphaq/11. A possible RGS7-selective neuronal feedback adaptation on Galphaq/11-mediated pathway function is described which may play an important role in signaling specificity in the brain (Khawaja, 1999).

Long-term neuronal plasticity is known to be dependent on rapid de novo synthesis of mRNA and protein. Recent studies provide insight into the molecules involved in this response. mRNA encoding a member of the regulator of G-protein signaling (RGS) family, RGS2, is rapidly induced in neurons of the hippocampus, cortex, and striatum in response to stimuli that evoke plasticity. Although several members of the RGS family are expressed in brain with discrete neuronal localizations, RGS2 appears unique in that its expression is dynamically responsive to neuronal activity. In biochemical assays, RGS2 stimulates the GTPase activity of the alpha subunit of Gq and Gi1. The effect on Gi1 was observed only after reconstitution of the protein in phospholipid vesicles containing M2 muscarinic acetylcholine receptors. RGS2 also inhibits both Gq- and Gi-dependent responses in transfected cells. These studies suggest a novel mechanism linking neuronal activity and signal transduction (Ingi, 1998).

A novel splice variant of RGS 9 was isolated from a rat hypothalamus, human retina, and a human kidney (Wilm's) tumor. This variant, termed RGS 9L, differs from the retinal form (termed RGS 9S) in that it contains a 211- (rat) or 205- (human) amino acid proline-rich domain on the carboxyl terminus. The pattern of RGS 9 mRNA splicing is tissue specific, with striatum, hypothalamus and nucleus accumbens expressing RGS 9L, whereas retina and pineal express RGS 9S almost exclusively. This pattern of mRNA splicing seemed to be highly conserved between human and rodents, suggesting cell-specific differences in the function of these variants. Transient expression of RGS 9L augments basal and beta-adrenergic receptor-stimulated adenylyl cyclase activity while suppressing dopamine D2 receptor-mediated inhibition. Furthermore, RGS 9L expression greatly accelerates the decay of dopamine D2 receptor-induced GIRK current. These results indicate that RGS 9L inhibits heterotrimeric Gi function in vivo, probably by acting as a GTPase-activating protein. The human RGS 9 gene was localized to chromosome 17 q23-24 by radiation hybrid and fluorescent in situ hybridization analyses. The RGS 9 gene is within a previously defined locus for retinitis pigmentosa (RP 17), a disease that has been linked to genes in the rhodopsin/transducin/cGMP signaling pathway (Granneman, 1998).

The present study demonstrates that the regulator of G-protein-signaling protein type 4 (RGS4) is differentially regulated in the locus coeruleus (LC) and the paraventricular nucleus (PVN) of the hypothalamus by chronic stress and glucocorticoid treatments. Acute or chronic administration of corticosterone to adult rats decreases RGS4 mRNA levels in the PVN but increases these levels in the LC. Similarly, chronic unpredictable stress decreases RGS4 mRNA levels in the PVN but has a strong trend to increase these levels in the LC. Chronic stress also decreases RGS4 mRNA levels in the pituitary. The molecular mechanisms of RGS4 mRNA regulation were further investigated in vitro in the LC-like CATH.a cell line and the neuroendocrine AtT20 cell line using the synthetic corticosterone analog dexamethasone. Consistent with the findings in vivo, dexamethasone treatment causes a dose- and time-dependent decrease in RGS4 mRNA levels in AtT20 cells but a dose- and time-dependent increase in CATH.a cells. RGS4 mRNA regulation seen in these two cell lines seems to be attributable, at least in part, to opposite changes in mRNA stability. The differential regulation of RGS4 expression in the LC and in key relays of the hypothalamic-pituitary-adrenal axis could contribute to the brain's region-specific and long-term adaptations to stress (Ni, 1999).

Multiple events are associated with the regulation of signaling by the M2 muscarinic cholinergic receptors (mAChRs). Desensitization of the attenuation of adenylyl cyclase by the M2 mAChRs appears to involve agonist-dependent phosphorylation of M2 mAChRs by G-protein coupled receptor kinases (GRKs) that phosphorylate the receptors in a serine/threonine rich motif in the 3rd intracellular domain of the receptors. Mutation of residues 307-311 from TVSTS to AVAAA in this domain of the human M2 mAChR results in a loss of receptor/G-protein uncoupling and a loss of arrestin binding. Agonist-induced sequestration of receptors away from their normal membrane environment is also regulated by agonist-induced phosphorylation of the M2 mAChRs on the 3rd intracellular domain, but in HEK cells, the predominant pathway of internalization is not regulated by GRKs or arrestins. This pathway of internalization is not inhibited by a dominant negative dynamin, and does not appear to involve either clathrin coated pits or caveolae. The signaling of the M2 mAChR to G-protein regulated inwardly rectifying K channels (GIRKs) can be modified by RGS proteins. In HEK cells, expression of RGS proteins leads to a constitutive activation of the channels through a mechanism that depends on Gbetagamma. RGS proteins appear to increase the concentration of free Gbetagamma in addition to acting as GAPs. Thus multiple mechanisms acting at either the level of the M2 mAChRs or the G-proteins can contribute to the regulation of signaling via the M2 mAChRs (Hosey, 1999).