RGS-Insensitive G-Protein Mutations to Study the Role of Endogenous RGS Proteins

doi:10.1016/S0076-6879(04)89014-1

Methods in Enzymology

Volume 389, 2004, Pages 229-243

https://doi.org/10.1016/S0076-6879(04)89014-1 Get rights and content

Abstract

Regulator of G-protein signaling (RGS) proteins are very active GTPase-accelerating proteins (GAPs) in vitro and are expected to reduce signaling by G-protein coupled receptors in vivo. A novel method is presented to assess the in vivo role of RGS proteins in the function of a G protein in which Gα subunits do not bind to RGS proteins or respond with enhanced GTPase activity. A point mutation in the switch I region of Gα subunits (G184S Gα_o and G183S Gα_i1) blocks the interaction with RGS proteins but leaves intact the ability of Gα to couple to βγ subunits, receptors, and downstream effectors. Expression of the RGS-insensitive mutant G184S Gα_o in C6 glioma cells with the μ-opioid receptor dramatically enhances adenylylcyclase inhibition and activation of extracellular regulated kinase. Introducing the same G184S Gα_o protein into embryonic stem (ES) cells by gene targeting allows us to assess the functional importance of the endogenous RGS proteins using in vitro differentiation models and in intact mice. Using ES cell-derived cardiocytes, spontaneous and isoproterenol-stimulated beating rates were not different between wild-type and G184S Gα_o mutant cells; however, the bradycardiac response to adenosine A₁ receptor agonists was enhanced significantly (seven-fold decrease EC₅₀) in Gα_oRGSi mutant cells compared to wild-type Gα_o, indicating a significant role of endogenous RGS proteins in cardiac automaticity regulation. The approach of using RGS-insensitive Gα subunit knockins will reveal the role of RGS protein-mediated GAP activity in signaling by a given G_i⧸o protein. This will reveal the full extent of RGS regulation and will not be confounded by redundancy in the function of multiple RGS proteins.

Introduction

Regulator of G-protein signaling (RGS) proteins modulate the strength and duration of G-protein-mediated signaling by acting as GTPase-accelerating proteins (GAP) though their conserved RGS domain. In addition to their role as negative regulators, RGS proteins also serve as versatile modulators and integrators of G-protein signaling via their diverse functional domains and structural motifs (Hollinger and Hepler, 2002). Considerable knowledge has been gained regarding the biochemical mechanisms of RGS protein stimulation of the GTPase activity of Gα (Ross and Wilkie, 2000). In addition, studies have begun to reveal the physiological roles of RGS proteins (reviewed by Hollinger 2002, Zhong 2001). Much previous work, however, uses overexpression of the individual RGS protein, which complicates conclusions regarding the functional importance of endogenous RGS proteins in vivo. A few RGS knockout mice (i.e., RGS2, RGS9, and RGS14) have been generated (Chen et al., 2000; Oliveira-dos-Santos et al., 2000; Siderovski, personal communication). RGS2 and RGS9 knockout phenotypes are consistent with a negative regulatory function on the vasoconstrictor (Heximer 2003, Tang 2003) and dopamine (Martemyanov 2003, Rahman 2003) signaling, respectively. However, RGS proteins may be functionally redundant, which may make single knockouts of limited value in assessing the overall role of RGS protein function in vivo. Thus, knowledge of the role of RGS proteins in vivo is still in its infancy.

Section snippets

Characterization of an RGS-Insensitive Mutation

The defining feature of RGS proteins is the ∼120 amino acid-conserved RGS domain, which binds preferentially to the transition state of a Gα subunit and accelerates its intrinsic GTPase activity (Berman 1996, Hepler 1997, Hunt 1996). Yeast also has an RGS-regulated G-protein signaling system that is involved in mating pheromone responses. Disruption of the SST2 (“supersensitivity to pheromone-2”) gene leads to enhanced pheromone signaling in terms of both ligand concentration and duration of

Studying the Function of Endogenous RGS Proteins by Transfecting the RGSi Gα_o Mutant into Cells

Assessing the role of transfected G proteins in the face of endogenous G proteins is difficult. This problem has been solved by the use of pertussis toxin-insensitive (PTXi) mutants of the G_i⧸o family G proteins (Milligan, 1988). PTX treatment allows examination of responses mediated through the exogenous, expressed G protein without interference by endogenous G_i⧸o proteins. Using cultured hippocampal neurons, Chen and Lambert (2000) employed doubly mutated RGSi⧸PTXi Gα proteins expressed by

Acknowledgements

Supported by NIH Grants GM39561 (RRN), T32HL007853 (XH), MDRTC Grant (NIH P60 DK20572), and an AHA Predoctoral fellowship (YF). We thank Min Liu, Elizabeth Hughes, and Ginny Zawistowski for their technical support.

References (36)

D.M. Berman et al.
The GTPase-activating protein RGS4 stabilizes the transition state for nucleotide hydrolysis
J. Biol. Chem.
(1996)
M.J. Clark et al.
Endogenous RGS protein action modulates mu-opioid signaling through Gα: Effects on adenylyl cyclase, extracellular signal-regulated kinases, and intracellular calcium pathways
J. Biol. Chem.
(2003)
P.R. DiBello et al.
Selective uncoupling of RGS action by a single point mutation in the G protein alpha-subunit
J. Biol. Chem.
(1998)
H. Gu et al.
Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting
Cell
(1993)
K.L. Lan et al.
A point mutation in Gα_o and Gα_i1 blocks interaction with regulators of G protein signaling
J. Biol. Chem.
(1998)
K.L. Laugwitz et al.
Mu and delta opioid receptors differentially couple to G protein subtypes in membranes of human neuroblastoma SH-SY5Y cells
Neuron.
(1993)
K.A. Martemyanov et al.
Specificity of G protein-RGS protein recognition is regulated by affinity adapters
Neuron.
(2003)
R.M. Mortensen et al.
Inactivation of G-protein genes: Double knockout in cell lines
Methods Enzymol.
(1994)
Z. Rahman et al.
RGS9 modulates dopamine signaling in the basal ganglia
Neuron.
(2003)
N.A. Sarvazyan et al.
Determinants of G_i1α and βγ binding
J. Biol. Chem.
(1998)

P.C. Sternweis et al.

Isolation of two proteins with high affinity for guanine nucleotides from membranes of bovine brain

J. Biol. Chem.

(1984)

J.J. Tesmer et al.

Structure of RGS4 bound to AlF4-activated G(i alpha1): Stabilization of the transition state for GTP hydrolysis

Cell

(1997)

C. Ye et al.

Galpha(i2), alpha(i3) and Galpha(o) are all required for normal muscarinic inhibition of the cardiac calcium channels in nodal⧸atrial-like cultured cardiocytes

J. Mol. Cell Cardiol.

(1999)

K.R. Boheler et al.

Differentiation of pluripotent embryonic stem cells into cardiomyocytes

Circ. Res.

(2002)

R.K. Chan et al.

Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones

Mol. Cell Biol.

(1982)

H. Chen et al.

Endogenous regulators of G protein signaling proteins regulate presynaptic inhibition at rat hippocampal synapses

Proc. Natl. Acad. Sci. USA

(2000)

H.G. Dohlman et al.

Inhibition of G-protein signaling by dominant gain-of-function mutations in Sst2p, a pheromone desensitization factor in Saccharomyces cerevisiae

Mol. Cell Biol.

(1995)

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Mice carrying an RGS-insensitive Gαi2 mutation display growth retardation early after birth. Although the growth hormone (GH)-axis is a key endocrine modulator of postnatal growth, its functional state in these mice has not been characterized. The present study was undertaken to address this issue. Results revealed that pituitary mRNA levels for GH, prolactin (PRL), somatostatin (SST), GH-releasing-hormone receptor (GHRH-R) and GH secretagogue receptor (GHS-R) were decreased in mutants compared to controls. These changes were reflected by a significant decrease in plasma levels of GH, IGF-1 and IGF-binding protein-3 (IGFBP-3). Mutants were also less responsive to GHRH and ghrelin (GhL) on GH stimulation of release from pituitary primary cell cultures. In contrast, they were more sensitive to the inhibitory effect of SST. These data provide the first evidence for an alteration of the functional state of the GH-axis in Gαi2G184S mice that likely contributes to their growth retardation.
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Citation Excerpt :
For Gαi2 this is Gly184. The mutation prevents interaction of the Gα protein with all RGS proteins while maintaining normal enzyme kinetics and interactions with receptor and downstream effectors (Fu et al., 2004; Clark et al., 2003) and so provides the opportunity to determine the effect of removing RGS control of a specific Gα protein. Homozygous mice expressing the RGSi Gαi2 protein (Gαi2 GS/GS) display a baseline antidepressant-like phenotype which is fully reversible by 5-HT1AR antagonist treatment (Talbot et al., 2010), suggesting an important role for Gαi2 and RGS proteins downstream of the 5-HT1AR.
A single base mutation in the Gα_i2 protein (G184S) renders this Gα subunit insensitive to the negative modulatory effects of Regulator of G-protein Signaling (RGS) proteins. Mice expressing this RGS insensitive (RGSi) variant of Gα_i2 (RGSi Gα_i2) display a spontaneous antidepressant-like phenotype that is reversible by treatment with the 5-HT1A receptor (5-HT1AR) antagonist WAY100635. Here we test the hypothesis that increased activity of 5-HT1ARs in the hippocampus of RGSi Gα_i2 knock-in mice is responsible for the expression of the observed antidepressant-like behavior. We administered the 5-HT1AR antagonist WAY100635 or the agonist 8-OH-DPAT via bilateral intra-hippocampal infusion cannulae and evaluated antidepressant-like behavior using the tail suspension test (TST). WAY100635 reversed the antidepressant-like phenotype of the RGSi Gα_i2 knock-in mice and 8-OH-DPAT produced an antidepressant-like response in wild type mice that was blocked by systemic WAY100635. Furthermore, intra-hippocampal infusion of the RGS19/4 inhibitor CCG-203769 produced an antidepressant-like effect in female mice. Ex-vivo slice recording confirmed the 5-HT1AR-mediated decrease in hippocampal CA1 pyramidal neuron excitability was enhanced in the RGSi Gα_i2 knock-in mice. There was no change in hippocampal 5-HT1AR expression as measured by ligand binding but there was a compensatory reduction in Gαi proteins. The findings demonstrate that RGS protein control of hippocampal 5-HT1AR signaling is necessary and sufficient to account for the antidepressant-like phenotype in the RGSi Gα_i2 knock-in mice and that RGS proteins highly expressed in the hippocampus should be investigated as targets for novel antidepressant therapies.
RGS10 shapes the hemostatic response to injury through its differential effects on intracellular signaling by platelet agonists
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Platelets express ≥2 members of the regulators of G protein signaling (RGS) family. Here, we have focused on the most abundant, RGS10, examining its impact on the hemostatic response in vivo and the mechanisms involved. We have previously shown that the hemostatic thrombi formed in response to penetrating injuries consist of a core of fully activated densely packed platelets overlaid by a shell of less-activated platelets responding to adenosine 5′-diphosphate (ADP) and thromboxane A₂ (TxA₂). Hemostatic thrombi formed in RGS10^−/− mice were larger than in controls, with the increase due to expansion of the shell but not the core. Clot retraction was slower, and average packing density was reduced. Deleting RGS10 had agonist-specific effects on signaling. There was a leftward shift in the dose/response curve for the thrombin receptor (PAR4) agonist peptide AYPGKF but no increase in the maximum response. This contrasted with ADP and TxA₂, both of which evoked considerably greater maximum responses in RGS10^−/− platelets with enhanced G_q- and G_i-mediated signaling. Shape change, which is G₁₃-mediated, was unaffected. Finally, we found that free RGS10 levels in platelets are actively regulated. In resting platelets, RGS10 was bound to 2 scaffold proteins: spinophilin and 14-3-3γ. Platelet activation caused an increase in free RGS10, as did the endothelium-derived platelet antagonist prostacyclin. Collectively, these observations show that RGS10 serves as an actively regulated node on the platelet signaling network, helping to produce smaller and more densely packed hemostatic thrombi with a greater proportion of fully activated platelets.
The impact of RGS and other G-protein regulatory proteins on Gα<inf>i</inf>-mediated signaling in immunity
2016, Biochemical Pharmacology
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A single mutation in Gαi proteins was found to render them insensitive to RGS proteins as it blocked the interaction between the two proteins. This mutation does not affect Gαi binding to receptors, Gβγ, or effectors; nor does it affect Gαi expression [146]. Mice with such a mutation in the Gnai2 locus have been made [147].
Leukocyte chemoattractant receptors are members of the G-protein coupled receptor (GPCR) family. Signaling downstream of these receptors directs the localization, positioning and homeostatic trafficking of leukocytes; as well as their recruitment to, and their retention at, inflammatory sites. Ligand induced changes in the molecular conformation of chemoattractant receptors results in the engagement of heterotrimeric G-proteins, which promotes α subunits to undergo GTP/GDP exchange. This results in the functional release of βγ subunits from the heterotrimers, thereby activating downstream effector molecules, which initiate leukocyte polarization, gradient sensing, and directional migration. Pertussis toxin ADP ribosylates Gα_i subunits and prevents chemoattractant receptors from triggering Gα_i nucleotide exchange. The use of pertussis toxin revealed the essential importance of Gα_i subunit nucleotide exchange for chemoattractant receptor signaling. More recent studies have identified a range of regulatory mechanisms that target these receptors and their associated heterotrimeric G-proteins, thereby helping to control the magnitude, kinetics, and duration of signaling. A failure in these regulatory pathways can lead to impaired receptor signaling and immunopathology. The analysis of mice with targeted deletions of Gα_i isoforms as well as some of these G-protein regulatory proteins is providing insights into their roles in chemoattractant receptor signaling.
Introduction: G Protein-coupled Receptors and RGS Proteins
2015, Progress in Molecular Biology and Translational Science
Here, we provide an overview of the role of regulator of G protein-signaling (RGS) proteins in signaling by G protein-coupled receptors (GPCRs), the latter of which represent the largest class of cell surface receptors in humans responsible for transducing diverse extracellular signals into the intracellular environment. Given that GPCRs regulate virtually every known physiological process, it is unsurprising that their dysregulation plays a causative role in many human diseases and they are targets of 40–50% of currently marketed pharmaceuticals. Activated GPCRs function as GTPase exchange factors for Gα subunits of heterotrimeric G proteins, promoting the formation of Gα-GTP and dissociated Gβγ subunits that regulate diverse effectors including enzymes, ion channels, and protein kinases. Termination of signaling is mediated by the intrinsic GTPase activity of Gα subunits leading to reformation of the inactive Gαβγ heterotrimer. RGS proteins determine the magnitude and duration of cellular responses initiated by many GPCRs by functioning as GTPase-accelerating proteins (GAPs) for specific Gα subunits. Twenty canonical mammalian RGS proteins, divided into four subfamilies, act as functional GAPs while almost 20 additional proteins contain nonfunctional RGS homology domains that often mediate interaction with GPCRs or Gα subunits. RGS protein biochemistry has been well elucidated in vitro, but the physiological functions of each RGS family member remain largely unexplored. This book summarizes recent advances employing modified model organisms that reveal RGS protein functions in vivo, providing evidence that RGS protein modulation of G protein signaling and GPCRs can be as important as initiation of signaling by GPCRs.
5-HT1A receptor-mediated phosphorylation of extracellular signal-regulated kinases (ERK1/2) is modulated by regulator of G protein signaling protein 19
2014, Cellular Signalling
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Thus, RGS proteins act as GTPase accelerating proteins (GAPs) and afford a negative modulation of G-protein coupled receptor (GPCR) signaling [1–3]. Mutation of a glycine to serine in the switch I region of Gαi/o and Gαq proteins prevents the binding of Gα subunits to RH domain of all RGS proteins and so inhibits this negative regulation, leading to increased downstream signaling [4–8]. We have shown that a mouse with knock-in of a mutant Gαi2 that does not bind to RH domains (Gαi2GSGS) exhibits an anti-depressant- and anti-anxiety-like phenotype [9].
The 5-HT1A receptor is a G protein coupled receptor (GPCR) that activates G proteins of the Gαi/o family. 5-HT1A receptors expressed in the raphe, hippocampus and prefrontal cortex are implicated in the control of mood and are targets for anti-depressant drugs. Regulators of G protein signaling (RGS) proteins are members of a large family that play important roles in signal transduction downstream of G protein coupled receptors (GPCRs). The main role of RGS proteins is to act as GTPase accelerating proteins (GAPs) to dampen or negatively regulate GPCR-mediated signaling. We have shown that a mouse expressing Gαi2 that is insensitive to all RGS protein GAP activity has an anti-depressant-like phenotype due to increased signaling of postsynaptic 5-HT1A receptors, thus implicating the 5-HT1A receptor–Gαi2 complex as an important target. Here we confirm that RGS proteins act as GAPs to regulate signaling to adenylate cyclase and the mitogen-activated protein kinase (MAPK) pathway downstream of the 5-HT1A receptor, using RGS-insensitive Gαi2 protein expressed in C6 cells. We go on to use short hairpin RNA (shRNA) to show that RGS19 is responsible for the GAP activity in C6 cells and also that RGS19 acts as a GAP for 5-HT1A receptor signaling in human neuroblastoma SH-SY5Y cells and primary hippocampal neurons. In addition, in both cell types the synergy between 5-HT1A receptor and the fibroblast growth factor receptor 1 in stimulating the MAPK pathway is enhanced following shRNA reduction of RGS19 expression. Thus RGS19 may be a viable new target for anti-depressant medications.

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RGS-Insensitive G-Protein Mutations to Study the Role of Endogenous RGS Proteins

Abstract

Introduction

Section snippets

Characterization of an RGS-Insensitive Mutation

Studying the Function of Endogenous RGS Proteins by Transfecting the RGSi Gαo Mutant into Cells

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell

J. Biol. Chem.

Neuron.

Neuron.

Methods Enzymol.

Neuron.

J. Biol. Chem.

J. Biol. Chem.

Cell

J. Mol. Cell Cardiol.

Differentiation of pluripotent embryonic stem cells into cardiomyocytes

Circ. Res.

Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones

Mol. Cell Biol.

Endogenous regulators of G protein signaling proteins regulate presynaptic inhibition at rat hippocampal synapses

Proc. Natl. Acad. Sci. USA

Inhibition of G-protein signaling by dominant gain-of-function mutations in Sst2p, a pheromone desensitization factor in Saccharomyces cerevisiae

Mol. Cell Biol.

Studying the Function of Endogenous RGS Proteins by Transfecting the RGSi Gα_o Mutant into Cells