Regulator Of G Protein Signaling Function Research Articles

Class VI G protein-coupled receptors in Aspergillus oryzae regulate sclerotia formation through GTPase-activating activity

G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors in eukaryotes that sense and transduce extracellular signals into cells. In Aspergillus oryzae, 16 canonical GPCR genes are identified and classified into nine classes based on the sequence similarity and proposed functions. Class VI GPCRs (AoGprK-1, AoGprK-2, and AoGprR in A. oryzae), unlike other GPCRs, feature a unique hybrid structure containing both the seven transmembrane (7-TM) and regulator of G-protein signaling (RGS) domains, which is not found in animal GPCRs. We report here that the mutants with double or triple deletion of class VI GPCR genes produced significantly increased number of sclerotia compared to the control strain when grown on agar plates. Interestingly, complementation analysis demonstrated that the expression of the RGS domain without the 7-TM domain is sufficient to restore the phenotype. In line with this, among the three Gα subunits in A. oryzae, AoGpaA, AoGpaB, and AoGanA, forced expression of GTPase-deficient mutants of either AoGpaA or AoGpaB caused an increase in the number of sclerotia formed, suggesting that RGS domains of class VI GPCRs are the negative regulators of these two GTPases. Finally, we measured the expression of velvet complex genes and sclerotia formation-related genes and found that the expression of velB was significantly increased in the multiple gene deletion mutants. Taken together, these results demonstrate that class VI GPCRs negatively regulate sclerotia formation through their GTPase-activating activity in the RGS domains.Key points• Class VI GPCRs in A. oryzae regulate sclerotia formation in A. oryzae• RGS function of class VI GPCRs is responsible for regulation of sclerotia formation• Loss of class VI GPCRs resulted in increased expression of sclerotia-related genes

Development of a bimolecular luminescence complementation assay for RGS: G protein interactions in cells

Cell based assessment tools and screening platforms are the preferred paradigm for small molecule identification and validation due to selectively identifying molecules with cellular activity and validation of compound activity against target proteins in their native environment. With respect to Regulator of G Protein Signaling (RGS) proteins, current cell based methodologies are either low throughput or monitor downstream signaling consequences. The increasing number of reports indicating RGS function in various disease pathogeneses highlights the need for a robust RGS inhibitor discovery and characterization paradigm. Promega's NanoBit Protein Complementation Assay utilizes NanoLuc, an engineered luciferase with enhanced luminescence characteristics which allow for both robust and kinetic assessment of protein interaction formation and disruption. Here we characterized 15 separate RGS: G protein interactions using this system. The binding profile of RGS: Gα interactions correlates to prior published biochemical binding profiles of these proteins. Additionally, we demonstrated this system is suitable for high throughput screening efforts via calculation of Z-factors for three of the interactions and demonstrated that a known small molecule inhibitor of RGS4 disrupts the RGS4: Gαi1 protein-protein interaction. In conclusion, the NanoBit Protein Complementation Assay holds promise as a robust platform for discovery and characterization of RGS inhibitors.

RGS-Insensitive G Proteins as In Vivo Probes of RGS Function.

Guanine nucleotide-binding proteins of the inhibitory (Gi/o) class play critical physiological roles and the receptors that activate them are important therapeutic targets (e.g., mu opioid, serotonin 5HT1a, etc.). Gi/o proteins are negatively regulated by regulator of G protein signaling (RGS) proteins. The redundant actions of the 20 different RGS family members have made it difficult to establish their overall physiological role. A unique G protein mutation (G184S in Gαi/o) prevents RGS binding to the Gα subunit and blocks all RGS action at that particular Gα subunit. The robust phenotypes of mice expressing these RGS-insensitive (RGSi) mutant G proteins illustrate the profound action of RGS proteins in cardiovascular, metabolic, and central nervous system functions. Specifically, the enhanced Gαi2 signaling through the RGSi Gαi2(G184S) mutant knock-in mice shows protection against cardiac ischemia/reperfusion injury and potentiation of serotonin-mediated antidepressant actions. In contrast, the RGSi Gαo mutant knock-in produces enhanced mu-opioid receptor-mediated analgesia but also a seizure phenotype. These genetic models provide novel insights into potential therapeutic strategies related to RGS protein inhibitors and/or G protein subtype-biased agonists at particular GPCRs.

A finer tuning of G-protein signaling through regulated control of RGS proteins

Regulators of G-protein signaling (RGS) proteins are GTPase-activating proteins (GAP) for various Gα subunits of heterotrimeric G proteins. Through this mechanism, RGS proteins regulate the magnitude and duration of G-protein-coupled receptor signaling and are often referred to as fine tuners of G-protein signaling. Increasing evidence suggests that RGS proteins themselves are regulated through multiple mechanisms, which may provide an even finer tuning of G-protein signaling and crosstalk between G-protein-coupled receptors and other signaling pathways. This review summarizes the current data on the control of RGS function through regulated expression, intracellular localization, and covalent modification of RGS proteins, as related to cell function and the pathogenesis of diseases.

RGS proteins in heart: brakes on the vagus

It has been nearly a century since Otto Loewi discovered that acetylcholine (ACh) release from the vagus produces bradycardia and reduced cardiac contractility. It is now known that parasympathetic control of the heart is mediated by ACh stimulation of Gi/o-coupled muscarinic M2 receptors, which directly activate G protein-coupled inwardly rectifying potassium (GIRK) channels via Gβγ resulting in membrane hyperpolarization and inhibition of action potential (AP) firing. However, expression of M2R–GIRK signaling components in heterologous systems failed to recapitulate native channel gating kinetics. The missing link was identified with the discovery of regulator of G protein signaling (RGS) proteins, which act as GTPase-activating proteins to accelerate the intrinsic GTPase activity of Gα resulting in termination of Gα- and Gβγ-mediated signaling to downstream effectors. Studies in mice expressing an RGS-insensitive Gαi2 mutant (G184S) implicated endogenous RGS proteins as key regulators of parasympathetic signaling in heart. Recently, two RGS proteins have been identified as critical regulators of M2R signaling in heart. RGS6 exhibits a uniquely robust expression in heart, especially in sinoatrial (SAN) and atrioventricular nodal regions. Mice lacking RGS6 exhibit increased bradycardia and inhibition of SAN AP firing in response to CCh as well as a loss of rapid activation and deactivation kinetics and current desensitization for ACh-induced GIRK current (IKACh). Similar findings were observed in mice lacking RGS4. Thus, dysregulation in RGS protein expression or function may contribute to pathologies involving aberrant electrical activity in cardiac pacemaker cells. Moreover, RGS6 expression was found to be up-regulated in heart under certain pathological conditions, including doxorubicin treatment, which is known to cause life-threatening cardiotoxicity and atrial fibrillation in cancer patients. On the other hand, increased vagal tone may be cardioprotective in heart failure where acetylcholinesterase inhibitors and vagal stimulation have been proposed as potential therapeutics. Together, these studies identify RGS proteins, especially RGS6, as new therapeutic targets for diseases such as sick sinus syndrome or other maladies involving abnormal autonomic control of the heart.

Integrating energy calculations with functional assays to decipher the specificity of G protein–RGS protein interactions

The diverse RGS protein family is responsible for the precise timing of G-protein signaling. To understand how RGS protein structure encodes their common ability to inactivate G-proteins and their selective G-protein recognition, we integrated structure-based energy calculations with biochemical measurements of RGS protein activity. We revealed that, in addition to previously identified conserved residues, RGS proteins contain another group of variable modulatory residues, which reside at the periphery of the RGS-domain–G-protein interface and fine-tune G-protein recognition. Mutations of modulatory residues in high-activity RGS proteins impaired RGS function, whereas redesign of low-activity RGS proteins in critical modulatory positions yielded complete gain-of-function. Therefore, RGS proteins combine a conserved core interface with peripheral modulatory residues to selectively optimize G-protein recognition and inactivation. Finally, we show that our quantitative framework for analyzing protein-protein interactions can be extended to analyze interaction specificity across other large protein families.

Genetic deletion of regulator of G-protein signaling 4 (RGS4) rescues a subset of fragile X related phenotypes in the FMR1 knockout mouse

Fragile X syndrome (FXS), the most common cause of inherited mental retardation, is caused by the loss of the mRNA binding protein, FMRP. Persons with FXS also display epileptic seizures, social anxiety, hyperactivity, and autistic behaviors. The metabotropic glutamate receptor theory of FXS postulates that in the absence of FMRP, enhanced signaling though G-protein coupled group I metabotropic glutamate receptors in the brain contributes to many of the abnormalities observed in the disorder. However, recent evidence suggests that alterations in cellular signaling through additional G-protein coupled receptors may also be involved in the pathogenesis of FXS, thus providing impetus for examining downstream molecules. One group of signaling molecules situated downstream of the receptors is the regulator of G-protein signaling (RGS) proteins. Notably, RGS4 is highly expressed in brain and has been shown to negatively regulate signaling through Group I mGluRs and GABA B receptors. To examine the potential role for RGS4 in the pathogenesis of FXS, we generated FXS/RGS4 double knockout mice. Characterization of these mice revealed that a subset of FXS related phenotypes, including increased body weight, altered synaptic protein expression, and abnormal social behaviors, were rescued in the double knockout mice. Other phenotypes, such as hyperactivity and macroorchidism, were not affected by the loss of RGS4. These findings suggest that tissue and cell-type specific differences in GPCR signaling and RGS function may contribute to the spectrum of phenotypic differences observed in FXS.

Thinking outside of the "RGS box": new approaches to therapeutic targeting of regulators of G protein signaling.

Regulators of G protein signaling (RGS) proteins are emerging as potentially important drug targets. The mammalian RGS protein family has more than 20 members and they share a common ∼120-residue RGS homology domain or "RGS box." RGS proteins regulate signaling via G protein-coupled receptors by accelerating GTPase activity at active α subunits of G proteins of the G(q) and G(i/o) families. Most studies searching for modulators of RGS protein function have been focused on inhibiting the GTPase accelerating protein activity. However, many RGS proteins contain additional domains that serve other functions, such as interactions with proteins or subcellular targeting. Here, we discuss a rationale for therapeutic targeting of RGS proteins by regulation of expression or allosteric modulation to permit either increases or decreases in RGS function. Several RGS proteins have reduced expression or function in pathophysiological states, so strategies to increase RGS function would be useful. Because several RGS proteins are rapidly degraded by the N-end rule pathway, finding ways to stabilize them may prove to be an effective way to enhance RGS protein function.

RGS2 is a negative regulator of Gq/11‐mediated signaling and cell proliferation in adult rat ventricular fibroblasts

Cardiac fibroblasts (CF) contribute to cardiac remodeling by assuming an “activated” myofibroblast (MyoFb) phenotype. Many G protein‐coupled receptors are involved in CF activation. G proteins are controlled by Regulators of G protein Signaling (RGS). RGS expression and function in CF and MyoFb are unknown. We set out to identify which RGS are expressed in isolated adult rat ventricular CF (passage P0‐P2) and to begin to delineate their function. Morphologic changes and expression of MyoFb markers (e.g., α‐smooth muscle actin) indicated gradual conversion from CF (P0) to MyoFb (P2). We detected mRNA of 14 RGS by RT‐PCR that were differentially regulated during CF to MyoFb conversion. Only RGS2 mRNA was markedly up‐regulated in response to short‐term stimulation of Gq/11‐coupled Angiotensin II (Ang II) and Gs‐coupled β‐adrenergic receptors. Adenoviral RGS2 expression blunted Ang II‐induced phospholipase C β activation by 65±2% (P0), 66±2% (P1) and 78±3% (P2) (n=3, P<0.05) and CF proliferation (BrdU incorporation) by 37±5% (n=10, P<0.05). RGS2 had no effect on Gs‐coupled cAMP accumulation. Thus, while RGS2 is selectively up‐regulated in response to Gq/11 and Gs activation, it negatively regulates only Gq/11‐mediated signaling and proliferation in both CF and MyoFb. These data point to RGS2 as an important regulator of CF and MyoFb signaling and function.Supported by the NIH and American Heart Association.

Isolation and characterization of a novel human RGS mutant displaying gain-of-function activity

Regulator of G protein signaling (RGS) proteins play a crucial role in the adaptation of cells to stimulation by G protein-coupled receptors via heterotrimeric G proteins. Alterations in RGS function have been implicated in a wide range of disease states, leading to many researchers focusing on controlling the action of these regulatory proteins. Previous studies have centered on reducing or inhibiting the action of RGS proteins, utilizing inactive mutants or small molecular RGS inhibitors. Here we describe the isolation and characterization of a novel human RGS4 mutant which displays enhanced or gain-of-function (GOF) activity. RGS4 S30C demonstrates GOF activity both in an in vivo yeast-based signalling pathway and in vitro against the Gα o1 subunit contained in an α 2A-adrenoreceptor-Gα o1 C351I fusion protein. Mutational analysis of serine 30 identified a number of alternative substitutions that result in GOF activity. GOF activity was retained upon transposition of the serine 30-cysteine mutation to the equivalent serine residue in human RGS16. As with previously identified GOF mutants, RGS4 S30C/S30F/S30K demonstrate increased steady state protein levels, however these mutants also demonstrate enhanced GAP activity through an additional mechanism distinct from the increased protein content. The identification of human RGS mutants with GOF activity may provide novel therapeutic agents for the treatment of signaling-based diseases and the ability to transpose these mutations to other human RGS proteins extends their application to multiple pathways.

Phosphorylation of Ser166 in RGS5 by protein kinase C causes loss of RGS function

RGS5 is a member of regulators of G protein signaling (RGS) proteins that attenuate heterotrimeric G protein signaling by functioning as GTPase-activating proteins (GAPs). We investigated phosphorylation of RGS5 and the resulting change of its function. In 293T cells, transiently expressed RGS5 was phosphorylated by endogenous protein kinases in the basal state. The phosphorylation was enhanced by phorbol 12-myristate 13-acetate (PMA) and endothelin-1 (ET-1), and suppressed by protein kinase C (PKC) inhibitors, H7, calphostin C and staurosporine. These results suggest involvement of PKC in phosphorylation of RGS5. In in vitro experiments, PKC phosphorylated recombinant RGS5 protein at serine residues. RGS5 protein phosphorylated by PKC showed much lower binding capacity for and GAP activity toward Gα subunits than did the unphosphorylated RGS5. In cells expressing RGS5, the inhibitory effect of RGS5 on ET-1-induced Ca 2+ responses was enhanced by staurosporine. Mass spectrometric analysis of the phosphorylated RGS5 revealed that Ser166 was one of the predominant phosphorylation sites. Substitution of Ser166 by aspartic acid abolished the binding capacity to Gα subunits and the GAP activity, and markedly reduced the inhibitory effect on ET-1-induced Ca 2+ responses. These results indicate that phosphorylation at Ser166 of RGS5 by PKC causes loss of the function of RGS5 in G protein signaling. Since this serine residue is conserved in RGS domains of many RGS proteins, the phosphorylation at Ser166 by PKC might act as a molecular switch and have functional significance.

N-Terminal Residues Control Proteasomal Degradation of RGS2, RGS4, and RGS5 in Human Embryonic Kidney 293 Cells

Regulator of G protein signaling (RGS) proteins modulate G protein-coupled receptor (GPCR) signaling. The N termini of some RGS4-family proteins provide receptor specificity and also contain an N-end rule determinant that results in ubiquitylation and decreased protein expression. The relevance of these mechanisms to other RGS proteins is not fully understood. Thus we examined function, receptor specificity, and expression of R4 subfamily RGS proteins (RGS2, -3, -4, -5, and -8). Although the N terminus plays a key role in protein stability in human embryonic kidney (HEK) 293 cells, we were unable to demonstrate specificity of RGS2, -3, -4, -5, or -8 for muscarinic receptors (M(1), M(3), and M(5)). However, cellular RGS activity (8 = 3 > 2) was strongly correlated with expression; RGS4 and -5 had minimal expression and activity. Stabilizing mutations of RGS4 and -5 (C2S) enhanced expression and function with a greater influence on RGS4 than on RGS5. We were surprised to find that a predicted destabilizing mutation in RGS8 (A2C) did not markedly affect expression and had no effect on function. In contrast, a destabilizing mutation in RGS2 (RGS2-Q2L) recently identified as a rare N-terminal genetic variant in a Japanese hypertensive cohort (J Hypertens 23:1497-1505, 2005) showed significantly reduced expression and inhibition of angiotensin II (AT(1)) receptor-stimulated accumulation of inositol phosphates. We were surprised to find that RGS2-Q2R, also predicted to be destabilizing, showed nearly normal expression and function. Thus, proteasomal regulation of RGS expression in HEK293 cells strongly controls RGS function and a novel RGS2 mutation with decreased protein expression could be relevant to the pathophysiology of hypertension in humans.

G Protein-coupled Receptor Kinase and β-Arrestin-mediated Desensitization of the Angiotensin II Type 1A Receptor Elucidated by Diacylglycerol Dynamics

Receptor desensitization progressively limits responsiveness of cells to chronically applied stimuli. Desensitization in the continuous presence of agonist has been difficult to study with available assay methods. Here, we used a fluorescence resonance energy transfer-based live cell assay for the second messenger diacylglycerol to measure desensitization of a model seven-transmembrane receptor, the Gq-coupled angiotensin II type 1(A) receptor, expressed in human embryonic kidney 293 cells. In response to angiotensin II, we observed a transient diacylglycerol response reflecting activation and complete desensitization of the receptor within 2-5 min. By utilizing a variety of approaches including graded tetracycline-inducible receptor expression, mutated receptors, and overexpression or short interfering RNA-mediated silencing of putative components of the cellular desensitization machinery, we conclude that the rate and extent of receptor desensitization are critically determined by the following: receptor concentration in the plasma membrane; the presence of phosphorylation sites on the carboxyl terminus of the receptor; kinase activity of G protein-coupled receptor kinase 2, but not of G protein-coupled receptor kinases 3, 5, or 6; and stoichiometric expression of beta-arrestin. The findings introduce the use of the biosensor diacylglycerol reporter as a powerful means for studying Gq-coupled receptor desensitization and document that, at the levels of receptor overexpression commonly used in such studies, the properties of the desensitization process are markedly perturbed and do not reflect normal cellular physiology.

Regulatory mechanisms involved in modulating RGS function.

Regulator of G-Protein Signaling (RGS) refers to a conserved 120-125 amino acid motif that was first identified by its ability to negatively regulate G-Protein-Coupled Receptor (GPCR) signalling. Mechanistically, RGSs were found to regulate GPCR responses by binding to and stimulating the GTPase activity of the receptor-activated GTP-bound G alpha subunits. There are now over 25 mammalian RGSs containing proteins that are reported to carry out a variety of functions, many of which are unrelated to GPCR signalling. RGS proteins range in size from small proteins that contain little more than an RGS box to very large proteins that contain a variety of domains. The selectivity of function of the RGS proteins is attributable to the divergence of the RGS sequences as well as the presence of a variety of functional motifs, which allow them to interact with other proteins. Here we focus on the RGSs that are involved in modulating GPCR signalling by reviewing the diversity of the mechanisms involved in regulating these RGSs.

Endogenous RGS Proteins and Gα Subtypes Differentially Control Muscarinic and Adenosine-Mediated Chronotropic Effects

Cardiac automaticity is controlled by G protein-coupled receptors, such as adrenergic, muscarinic, and adenosine receptors. The strength and duration of G protein signaling is attenuated by regulator of G protein signaling (RGS) proteins acting as GTPase-activating proteins for Galpha subunits; however, little is known about the role of endogenous RGS proteins in cardiac function. We created point mutations in Galpha subunits that disrupt Galpha-RGS binding and introduced them into embryonic stem (ES) cells by homologous recombination. Spontaneously contacting cardiocytes derived from the ES cells were used to evaluate the role of endogenous RGS proteins in chronotropic regulation. The RGS-insensitive GalphaoG184S homozygous knock-in (GalphaoGS/GS) cells demonstrated enhanced adenosine A1 and muscarinic M2 receptor-mediated bradycardic responses. In contrast, Galphai2GS/GS cells showed enhanced responses to M2 but not A1 receptors. Similarly M2 but not A1 bradycardic responses were dramatically enhanced in Galphai2GS/GS mice. Blocking G protein-coupled inward rectifying K+ (GIRK) channels largely abolished the mutation-induced enhancement of the M2 receptor-mediated response but had a minimal effect on A1 responses. The Galphas-dependent stimulation of beating rate by the beta2 adrenergic receptor agonist procaterol was significantly attenuated in GalphaoGS/GS and nearly abolished in Galphai2GS/GS cells because of enhanced signaling via a pertussis toxin sensitive mechanism. Thus, endogenous RGS proteins potently reduce the actions of Galpha(i/o)-linked receptors on cardiac automaticity. Furthermore, M2 and A1 receptors differentially use Galphai2 and Galphao and associated downstream effectors. Thus, alterations in RGS function may play a role in pathophysiological processes and RGS proteins could represent novel cardiovascular therapeutic targets.

Selective Inhibition of α1A-Adrenergic Receptor Signaling by RGS2 Association with the Receptor Third Intracellular Loop

Regulators of G-protein signaling (RGS) proteins act directly on Galpha subunits to increase the rate of GTP hydrolysis and to terminate signaling. However, the mechanisms involved in determining their specificities of action in cells remain unclear. Recent evidence has raised the possibility that RGS proteins may interact directly with G-protein-coupled receptors to modulate their activity. By using biochemical, fluorescent imaging, and functional approaches, we found that RGS2 binds directly and selectively to the third intracellular loop of the alpha1A-adrenergic receptor (AR) in vitro, and is recruited by the unstimulated alpha1A-AR to the plasma membrane in cells to inhibit receptor and Gq/11 signaling. This interaction was specific, because RGS2 did not interact with the highly homologous alpha1B- or alpha1D-ARs, and the closely related RGS16 did not interact with any alpha1-ARs. The N terminus of RGS2 was required for association with alpha1A-ARs and inhibition of signaling, and amino acids Lys219, Ser220, and Arg238 within the alpha1A-AR i3 loop were found to be essential for this interaction. These findings demonstrate that certain RGS proteins can directly interact with preferred G-protein-coupled receptors to modulate their signaling with a high degree of specificity.

Bimodal Regulation of the Human H1 Histamine Receptor by G Protein-coupled Receptor Kinase 2

The H1 histamine receptor (H1HR) is a member of the G protein-coupled receptor superfamily and regulates numerous cellular functions through its activation of the G(q/11) subfamily of heterotrimeric G proteins. Although the H1HR has been shown to undergo desensitization in multiple cell types, the mechanisms underlying the regulation of H1HR signaling are poorly defined. To address this issue, we examined the effects of wild type and mutant G protein-coupled receptor kinases (GRKs) on the phosphorylation and signaling of human H1HR in HEK293 cells. Overexpression of GRK2 promoted H1HR phosphorylation in intact HEK293 cells and completely inhibited inositol phosphate production stimulated by H1HR, whereas GRK5 and GRK6 had lesser effects on H1HR phosphorylation and signaling. Interestingly, catalytically inactive GRK2 (GRK2-K220R) also significantly attenuated H1HR-mediated inositol phosphate production, as did an N-terminal fragment of GRK2 previously characterized as a regulator of G protein signaling (RGS) protein for Galpha(q/11). Disruption of this RGS function in holo-GRK2 by mutation (GRK2-D110A) partially reversed the quenching effect of GRK2, whereas deletion of both the kinase activity and RGS function (GRK2-D110A/K220R) effectively relieved the inhibition of inositol phosphate generation. To evaluate the role of endogenous GRKs on H1HR regulation, we used small interfering RNAs to selectively target GRK2 and GRK5, two of the primary GRKs expressed in HEK293 cells. A GRK2-specific small interfering RNA effectively reduced GRK2 expression and resulted in a significant increase in histamine-promoted calcium flux. In contrast, knockdown of GRK5 expression was without effect on H1HR signaling. These findings demonstrate that GRK2 is the principal kinase mediating H1 histamine receptor desensitization in HEK293 cells and suggest that rapid termination of H1HR signaling is mediated by both the kinase activity and RGS function of GRK2.

Role of Palmitoylation in RGS Protein Function

Palmitoylation, the reversible, post-translational addition of palmitate to cysteine residues, occurs on several regulators of G-protein signaling (RGS) proteins. Palmitoylation can occur near the amino terminus, as for RGS4 and RGS16, but can also occur on a cysteine residue in the alpha4 helix of the RGS box, which is conserved in most RGS proteins. For some of the RGS proteins, palmitoylation is required to turn off G-protein signaling by accelerating GTP hydrolysis on the Galpha subunit. This article discusses the role of palmitoylation in RGS function and protocols are given for metabolic and in vitro labeling of RGS proteins with [3H]palmitate and measurement of GTP hydrolysis in membranes.

In Situ Hybridization Analysis of RGS mRNA Regulation and Behavioral Phenotyping of RGS Mutant Mice

To elucidate the functional role of regulators of G-protein signaling (RGS) in vivo, it will be critical to (i) determine how RGS activity is altered in response to a variety of manipulations and (ii) observe how the system is changed when RGS protein function is altered genetically. To facilitate studies of dynamic regulation of RGS protein activity, this article describes detailed methods for radioisotopic in situ hybridization for semiquantitative analyses of RGS mRNA abundances. Toward characterizing the functional differences in mice with genetically altered RGS activities, this article describes a subset of behavioral tests suitable for assaying sensitivities to drugs of abuse. These protocols should provide valuable guidance for investigators to establish these methodologies independently in their own laboratories and, over time, increase our understanding of RGS function in vivo.

Endogenous RGS Proteins Regulate Presynaptic and Postsynaptic Function: Functional Expression of RGS-Insensitive Gα Subunits in Central Nervous System Neurons

Regulators of G-protein signaling (RGS)-insensitive (RGSi) G-protein alpha subunits can be used to indirectly determine the function of endogenous RGS proteins in native cells. This article describes the application of RGSi Galpha subunits to the study of endogenous RGS function in central nervous system (CNS) neurons. Presynaptic inhibition of neurotransmitter release was reconstituted in primary neurons using RGSi Galpha(i/o) subunits, whereas postsynaptic regulation of potassium channels was reconstituted using RGSi chimeras of Galpha(q) and Galpha(i). These studies have shown that endogenous RGS proteins are essential for the rapid termination of some G-protein-mediated signals in CNS neurons, whereas these proteins are much less important for the regulation of other signals. Together, these techniques have helped reveal the complexity of RGS regulation of CNS function.