Regulators of G-protein signaling and their Gα substrates: promises and challenges in their use as drug discovery targets.

Because G-protein coupled receptors (GPCRs) continue to represent excellent targets for the discovery and development of small-molecule therapeutics, it is posited that additional protein components of the signal transduction pathways emanating from activated GPCRs themselves are attractive as drug discovery targets. This review considers the drug discovery potential of two such components: members of the "regulators of G-protein signaling" (RGS protein) superfamily, as well as their substrates, the heterotrimeric G-protein α subunits. Highlighted are recent advances, stemming from mouse knockout studies and the use of "RGS-insensitivity" and fast-hydrolysis mutations to Gα, in our understanding of how RGS proteins selectively act in (patho)physiologic conditions controlled by GPCR signaling and how they act on the nucleotide cycling of heterotrimeric G-proteins in shaping the kinetics and sensitivity of GPCR signaling. Progress is documented regarding recent activities along the path to devising screening assays and chemical probes for the RGS protein target, not only in pursuits of inhibitors of RGS domain-mediated acceleration of Gα GTP hydrolysis but also to embrace the potential of finding allosteric activators of this RGS protein action. The review concludes in considering the Gα subunit itself as a drug target, as brought to focus by recent reports of activating mutations to GNAQ and GNA11 in ocular (uveal) melanoma. We consider the likelihood of several strategies for antagonizing the function of these oncogene alleles and their gene products, including the use of RGS proteins with Gα(q) selectivity.

Unique hydrophobic extension of the RGS2 amphipathic helix domain imparts increased plasma membrane binding and function relative to other RGS R4/B subfamily members.

RGS2 and RGS5 are inhibitors of G-protein signaling belonging to the R4/B subfamily of RGS proteins. We here show that RGS2 is a much more potent attenuator of M1 muscarinic receptor signaling than RGS5. We hypothesize that this difference is mediated by variation in their ability to constitutively associate with the plasma membrane (PM). Compared with full-length RGS2, the RGS-box domains of RGS2 and RGS5 both show reduced PM association and activity. Prenylation of both RGS-box domains increases activity to RGS2 levels, demonstrating that lipid bilayer targeting increases RGS domain function. Amino-terminal domain swaps confirm that key determinants of localization and function are found within this important regulatory domain. An RGS2 amphipathic helix domain mutant deficient for phospholipid binding (L45D) shows reduced PM association and activity despite normal binding to the M1 muscarinic receptor third intracellular loop and activated Galpha(q). Replacement of a unique dileucine motif adjacent to the RGS2 helix with corresponding RGS5 residues disrupts both PM localization and function. These data suggest that RGS2 contains a hydrophobic extension of its helical domain that imparts high efficiency binding to the inner leaflet of the lipid bilayer. In support of this model, disruption of membrane phospholipid composition with N-ethylmaleimide reduces PM association of RGS2, without affecting localization of the M1 receptor or Galpha(q). Together, these data indicate that novel features within the RGS2 amphipathic alpha helix facilitate constitutive PM targeting and more efficient inhibition of M1 muscarinic receptor signaling than RGS5 and other members of the R4/B subfamily.

How regulators of G protein signaling achieve selective regulation.

The regulators of G protein signaling (RGS) are a family of cellular proteins that play an essential regulatory role in G protein-mediated signal transduction. There are multiple RGS subfamilies consisting of over 20 different RGS proteins. They are basically the guanosine triphosphatase (GTPase)-accelerating proteins that specifically interact with G protein alpha subunits. RGS proteins display remarkable selectivity and specificity in their regulation of receptors, ion channels, and other G protein-mediated physiological events. The molecular and cellular mechanisms underlying such selectivity are complex and cooperate at many different levels. Recent research data have provided strong evidence that the spatiotemporal-specific expression of RGS proteins and their target components, as well as the specific protein-protein recognition and interaction through their characteristic structural domains and functional motifs, are determinants for RGS selectivity and specificity. Other molecular mechanisms, such as alternative splicing and scaffold proteins, also significantly contribute to RGS selectivity. To pursue a thorough understanding of the mechanisms of RGS selective regulation will be of great significance for the advancement of our knowledge of molecular and cellular signal transduction.

Localization of the GoLoco motif carrier regulator of G-protein signalling 12 and 14 proteins in monkey and rat brain.

Regulator of G-protein signalling (RGS)12 and -14 proteins possess the RGS domain, Ras-binding domains and the GoLoco motif. Emerging evidence suggests that these proteins are involved in several cellular functions in addition to stimulation of GTPase activity of G-protein alpha subunits. However, our understanding of the role of the two proteins in brain function remains marginal. Here, we have studied the expression pattern of RGS12 and RGS14 proteins in brain at regional, cellular and subcellular levels. Both proteins were expressed throughout the brain regions, including cortex, hippocampus, striatum, thalamus and substantia nigra. The most intense immunostaining for RGS12 was seen in cortex and that of RGS14 was found in striatum. In cortex, RGS12 and RGS14 proteins were associated with pyramidal and nonpyramidal cell types. Apical dendrites of pyramidal cells were also labelled. RGS12 was found in both nuclear and cytoplasmic compartments. In contrast to RGS12 protein, RGS14 was localized in astrocytes in addition to neurons. Pyramidal cells in the CA1 area showed labelling for both RGS proteins. The presence of RGS12 was predominantly nuclear in the striatum of rat brain; however, the labelling of this protein was non-nuclear in adult monkey brain. To our surprise, in 1-month-old monkey brain the immunostaining pattern of the same protein was changed to nuclear. Non-nuclear staining for RGS12 was also evident in thalamus of adult monkey brain; however, in 1-month-old monkey brain, it was seen into two different populations, one with nuclear and the other with cytoplasmic staining. Both RGS12 and RGS14 were exclusively localized at postsynaptic sites of excitatory synapses. Our results demonstrate a highly dynamic expression pattern of RGS12 and RGS14 proteins in the central nervous system, and support the view that these proteins may participate not only in G-protein receptor signalling pathways but also in other cellular activities.

RGS17/RGSZ2 and the RZ/A family of regulators of G-protein signaling.

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.

Mammalian RGS proteins: multifunctional regulators of cellular signalling.

Regulators of G-protein signalling (RGS) proteins are a large and diverse family initially identified as GTPase activating proteins (GAPs) of heterotrimeric G-protein Galpha-subunits. At least some can also influence Galpha activity through either effector antagonism or by acting as guanine nucleotide dissociation inhibitors (GDIs). As our understanding of RGS protein structure and function has developed, so has the realisation that they play roles beyond G-protein regulation. Such diversity of function is enabled by the variety of RGS protein structure and their ability to interact with other cellular molecules including phospholipids, receptors, effectors and scaffolds. The activity, sub-cellular distribution and expression levels of RGS proteins are dynamically regulated, providing a layer of complexity that has yet to be fully elucidated.

[The structure, classification and function of RGS proteins].

Regulators of G protein signaling (RGS) are a family of highly diverse, multifunctional signaling proteins that bind directly to activated Galpha subunits and markedly stimulates the GTPase activity of Galpha subunits leading to their deactivation and termination of downstream signals. RGS family members share a conserved 130-amino acid core domain which is responsible for binding Galpha subunits and negatively regulate G protein signal. Many RGS proteins possess additional non RGS domains and motifs. The non RGS domains are involved to integrate different G protein signaling pathways and as a saffolding protein that links G protein to related signaling proteins.

R7BP: a surprising new link between G proteins, RGS proteins, and nuclear signaling in the brain.

The regulators of G protein signaling (RGS proteins) bind directly to G protein alpha (Galpha) subunits in brain and other tissues to determine the strength, duration, and fidelity of neurotransmitter receptor signaling. A recent study shows, quite unexpectedly, that one class of RGS proteins [the R7 subfamily bound to Gbeta(5) (R7-Gbeta(5))] shuttles between the plasma membrane and the nucleus with assistance from a novel shuttle protein, R7BP. R7BP binds directly to R7-Gbeta(5) and the protein complex is tethered to the plasma membrane by addition of a lipid, palmitate, on R7BP. Removal of palmitate results in the translocation of the R7BP-R7-Gbeta(5) complex to the nucleus, presumably for nontraditional signaling functions. These findings suggest an entirely novel mechanism for regulating neurotransmitter signaling. That is, R7BP transduces signals directly from receptors and G proteins at the plasma membrane to the nucleus, and this plasma membrane-nuclear shuttling is controlled by reversible palmitoylation of R7BP.

Morphine alters the selective association between mu-opioid receptors and specific RGS proteins in mouse periaqueductal gray matter.

In the CNS, several regulators of G-protein signalling (RGS) modulate the activity of mu-opioid receptors. In pull-down assays performed on membranes from mouse periaqueductal gray matter (PAG), mu-opioid receptors co-precipitated with delta-opioid receptors, Gi/o/z/q proteins, and the regulators of G-protein signalling RGS4, RGS9-2, RGS14, RGSZ1 and RGSZ2. No RGS2, RGS7, RGS10 and RGS11 proteins were associated with the mu receptors in these PAG membranes. In mice, an intracerebroventricular dose of 10 nmol morphine produced acute tolerance at mu receptors but did not disrupt the co-precipitation of mu-delta receptor complexes. However, this opioid reduced by more than 50% the co-precipitation of G alpha i/o/z subunits with mu receptors, and altered their association with some of the RGS proteins at 30 min, 3 h and 24 h after its administration. The association of RGS9-2 with mu receptors diminished by 30-40% 24 h after the administration of morphine, while that of RGSZ2 and of RGSZ1 increased. Morphine treatment recruited RGS4 to the PAG membranes, and 30 min and 3 h after the opioid challenge its association with mu receptors had increased. However, 24 h after morphine administration, the co-precipitation of RGS4 had decreased by about 30%. The opioid produced no change in the membrane levels of RGS9-2, RGS14, RGSZ1 and RGSZ2. Thus, in PAG synaptosomal membranes, a dynamic and selective link exists between, mu-opioid receptors, Gi/o/z proteins and certain RGS proteins.

R7BP, a novel neuronal protein interacting with RGS proteins of the R7 family.

The R7 subfamily of the regulators of G protein signaling (RGS) proteins is represented by four members broadly expressed in the mammalian nervous system. Here we report that in the brain all four R7 proteins form tight complexes with a previously unidentified protein, which we call the R7-binding protein or R7BP. We initially identified R7BP as a protein co-precipitating with the R7 protein, RGS9, from extracts obtained from the striatal region of the brain. We further showed that R7BP forms a tight complex with RGS9 in vitro and that this binding occurs via the N-terminal DEP domain of RGS9. R7BP is expressed throughout the entire central nervous system but not in any of the tested non-neuronal tissues. All four R7 RGS proteins co-precipitate with R7BP from brain extracts and recombinant R7 proteins bind recombinant R7BP with high efficiency. The closest homolog of R7BP is R9AP which was previously found to interact with RGS9 in photoreceptors. Both R7BP and R9AP are related to the syntaxin subfamily of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins involved in vesicular trafficking and exocytosis. In photoreceptors R9AP regulates several critical properties of RGS9 including its intracellular targeting, stability and catalytic activity. This suggests that R7BP interactions with R7 proteins in the brain may also bear major functional significance.

[RGS proteins (regulators of G protein signaling) and their roles in regulation of immune response]. / Bialka regulujace przekazywanie sygnalu przez bialka G (bialka RGS) i ich znaczenie w regulacji odpowiedzi immunologicznej.

RGS proteins (Regulators of G-protein Signaling) comprise a protein family responsible for regulating G proteins. By enhancing the GTPase activity of the a subunit, they speed up the reconstruction of the heterotrimeric structure of G protein, thus inhibiting its signal transduction. Sst2 protein in yeast Saccharomyces cervisiae, FlbA in fungus Aspergillus nidulans, and Egl-10 in the nematode Caenorhabditis elegans are the first native G regulators with GTPase activity (GAPs:--GTPase-activating proteins). The existence of over 30 RGS human proteins has been confirmed thus far, and they have been grouped and classified into six subfamilies. In immunocompetent cells, RGS proteins are entangled in a complicate net of different interrelating signal pathways. They are connected with B- and T-cell chemokine susceptibility, efficient T cell proliferation, and the regulation of B cell maturation. They also take an essential part in inflammation. High hopes are held for drugs, which handle would be RGS proteins and which would further provide the possibility of modifying the pharmacokinetics of drugs acting through G protein- coupled receptors. The aim of this review is to discuss the new RGS protein family and explain the potential involvement of RGS proteins in the modulation of the immune response

Selective expression of regulators of G-protein signaling (RGS) in the human central nervous system.

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.

Regulators of G-protein signalling: multifunctional proteins with impact on signalling in the cardiovascular system.

Regulator of G-protein signalling (RGS) proteins form a superfamily of at least 25 proteins, which are highly diverse in structure, expression patterns, and function. They share a 120 amino acid homology domain (RGS domain), which exhibits GTPase accelerating activity for alpha-subunits of heterotrimeric G-proteins, and thus, are negative regulators of G-protein-mediated signalling. Based on the organisation of the Rgs genes, structural similarities, and differences in functions, they can be divided into at least six subfamilies of RGS proteins and three more families of RGS-like proteins. Many of these proteins regulate signalling processes within cells, not only via interaction with G-protein alpha-subunits, but are G-protein-regulated effectors, Gbetagamma scavenger, or scaffolding proteins in signal transduction complexes as well. The expression of at least 16 different RGS proteins in the mammalian or human myocardium have been described. A subgroup of at least eight was detected in a single atrial myocyte. The exact functions of these proteins remain mostly elusive, but RGS proteins such as RGS4 are involved in the regulation of G(i)-protein betagamma-subunit-gated K(+) channels. An up-regulation of RGS4 expression has been consistently found in human heart failure and some animal models. Evidence is increasing that the enhanced RGS4 expression counter-regulates the G(q/11)-induced signalling caused by hypertrophic stimuli. In the vascular system, RGS5 seems to be an important signalling regulator. It is expressed in vascular endothelial cells, but not in cultured smooth muscle cells. Its down-regulation, both in a model of capillary morphogenesis and in an animal model of stroke, render it a candidate gene, which may be involved in the regulation of capillary growth, angiogenesis, and in the pathophysiology of stroke.