Structural basis for the 14-3-3 protein-dependent inhibition of the regulator of G protein signaling 3 (RGS3) function

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins for the α-subunit of heterotrimeric G proteins. The function of certain RGS proteins is negatively regulated by 14-3-3 proteins, a family of highly conserved regulatory molecules expressed in all eukaryotes. In this study, we provide a structural mechanism for 14-3-3-dependent inhibition of RGS3-Gα interaction. We have used small angle x-ray scattering, hydrogen/deuterium exchange kinetics, and Förster resonance energy transfer measurements to determine the low-resolution solution structure of the 14-3-3ζ·RGS3 complex. The structure shows the RGS domain of RGS3 bound to the 14-3-3ζ dimer in an as-yet-unrecognized manner interacting with less conserved regions on the outer surface of the 14-3-3 dimer outside its central channel. Our results suggest that the 14-3-3 protein binding affects the structure of the Gα interaction portion of RGS3 as well as sterically blocks the interaction between the RGS domain and the Gα subunit of heterotrimeric G proteins.     

Spinophilin regulates Ca2+ signalling by binding the N-terminal domain of RGS2 and the third intracellular loop of G-protein-coupled receptors

Signalling by G proteins is controlled by the regulator of G-protein signalling (RGS) proteins that accelerate the GTPase activity of Galpha subunits and act in a G-protein-coupled receptor (GPCR)-specific manner. The conserved RGS domain accelerates the G subunit GTPase activity, whereas the variable amino-terminal domain participates in GPCR recognition. How receptor recognition is achieved is not known. Here, we show that the scaffold protein spinophilin (SPL), which binds the third intracellular loop (3iL) of several GPCRs, binds the N-terminal domain of RGS2. SPL also binds RGS1, RGS4, RGS16 and GAIP. When expressed in Xenopus laevis oocytes, SPL markedly increased inhibition of alpha-adrenergic receptor (alphaAR) Ca2+ signalling by RGS2. Notably, the constitutively active mutant alphaAR(A293E) (the mutation being in the 3iL) did not bind SPL and was relatively resistant to inhibition by RGS2. Use of betaAR-alphaAR chimaeras identified the 288REKKAA293 sequence as essential for the binding of SPL and inhibition of Ca2+ signalling by RGS2. Furthermore, alphaAR-evoked Ca2+ signalling is less sensitive to inhibition by SPL in rgs2-/- cells and less sensitive to inhibition by RGS2 in spl-/- cells. These findings provide a general mechanism by which RGS proteins recognize GPCRs to confer signalling specificity.     

A Physiologically Required G Protein-coupled Receptor (GPCR)-Regulator of G Protein Signaling (RGS) Interaction That Compartmentalizes RGS Activity

G protein-coupled receptors (GPCRs) can interact with regulator of G protein signaling (RGS) proteins. However, the effects of such interactions on signal transduction and their physiological relevance have been largely undetermined. Ligand-bound GPCRs initiate by promoting exchange of GDP for GTP on the Gα subunit of heterotrimeric G proteins. Signaling is terminated by hydrolysis of GTP to GDP through intrinsic GTPase activity of the Gα subunit, a reaction catalyzed by RGS proteins. Using yeast as a tool to study GPCR signaling in isolation, we define an interaction between the cognate GPCR (Mam2) and RGS (Rgs1), mapping the interaction domains. This reaction tethers Rgs1 at the plasma membrane and is essential for physiological signaling response. In vivo quantitative data inform the development of a kinetic model of the GTPase cycle, which extends previous attempts by including GPCR-RGS interactions. In vivo and in silico data confirm that GPCR-RGS interactions can impose an additional layer of regulation through mediating RGS subcellular localization to compartmentalize RGS activity within a cell, thus highlighting their importance as potential targets to modulate GPCR signaling pathways.     

Macromolecular Composition Dictates Receptor and G Protein Selectivity of Regulator of G Protein Signaling (RGS) 7 and 9-2 Protein Complexes in Living Cells

Regulator of G protein signaling (RGS) proteins play essential roles in the regulation of signaling via G protein-coupled receptors (GPCRs). With hundreds of GPCRs and dozens of G proteins, it is important to understand how RGS regulates selective GPCR-G protein signaling. In neurons of the striatum, two RGS proteins, RGS7 and RGS9-2, regulate signaling by μ-opioid receptor (MOR) and dopamine D2 receptor (D2R) and are implicated in drug addiction, movement disorders, and nociception. Both proteins form trimeric complexes with the atypical G protein β subunit Gβ5 and a membrane anchor, R7BP. In this study, we examined GTPase-accelerating protein (GAP) activity as well as Gα and GPCR selectivity of RGS7 and RGS9-2 complexes in live cells using a bioluminescence resonance energy transfer-based assay that monitors dissociation of G protein subunits. We showed that RGS9-2/Gβ5 regulated both Gi and Go with a bias toward Go, but RGS7/Gβ5 could serve as a GAP only for Go. Interestingly, R7BP enhanced GAP activity of RGS7 and RGS9-2 toward Go and Gi and enabled RGS7 to regulate Gi signaling. Neither RGS7 nor RGS9-2 had any activity toward Gz, Gs, or Gq in the absence or presence of R7BP. We also observed no effect of GPCRs (MOR and D2R) on the G protein bias of R7 RGS proteins. However, the GAP activity of RGS9-2 showed a strong receptor preference for D2R over MOR. Finally, RGS7 displayed an four times greater GAP activity relative to RGS9-2. These findings illustrate the principles involved in establishing G protein and GPCR selectivity of striatal RGS proteins.     

RGS proteins have a signalling complex: interactions between RGS proteins and GPCRs, effectors, and auxiliary proteins

The intracellular regulator of G protein signalling (RGS) proteins were first identified as GTPase activating proteins (GAPs) for heterotrimeric G proteins, however, it was later found that they can also regulate G protein-effector interactions in other ways that are still not well understood. There is increasing evidence that some of the effects of RGS proteins occur due to their ability to interact with multiprotein signalling complexes. In this review, we will discuss recent evidence that supports the idea that RGS proteins can bind to proteins other than Galpha, such as G protein coupled receptors (GPCRs, e.g. muscarinic, dopaminergic, adrenergic, angiotensin, interleukin and opioid receptors) and effectors (e.g. adenylyl cyclase, GIRK channels, PDEgamma, PLC-beta and Ca(2+) channels). Furthermore, we will investigate novel RGS binding partners (e.g. GIPC, spinophilin, 14-3-3) that underlie the formation of signalling scaffolds or govern RGS protein availability and/or activity.     

Differential effects of RGS proteins on G alpha(q) and G alpha(11) activity

Heterotrimeric G proteins play a pivotal role in GPCR signalling; they link receptors to intracellular effectors and their inactivation by RGS proteins is a key factor in resetting the pathway following stimulation. The precise GPCR:G protein:RGS combination determines the nature and duration of the response. Investigating the activity of particular combinations is difficult in cells which contain multiples of each component. We have therefore utilised a previously characterised yeast system to express mammalian proteins in isolation. Human G alpha(q) and G alpha(11) spontaneously activated the yeast pheromone-response pathway by a mechanism which required the formation of G alpha-GTP. This provided an assay for the specific activity of human RGS proteins. RGS1, RGS2, RGS3 and RGS4 inhibited the spontaneous activity of both G alpha(q) and G alpha(11) but, in contrast, RGS5 and RGS16 were much less effective against G alpha(11) than G alpha(q). Interestingly, RGS2 and RGS3 were able to inhibit signalling from the constitutively active G alpha(q)QL/G alpha(11)QL mutants, confirming the GAP-independent activity of these RGS proteins. To determine if the RGS-G alpha specificity was maintained under conditions of GPCR stimulation, minor modifications to the C-terminus of G alpha(q)/G alpha(11) enabled coupling to an endogenous receptor. RGS2 and RGS3 were effective inhibitors of both G alpha subunits even at high levels of receptor stimulation, emphasising their GAP-independent activity. At low levels of stimulation RGS5 and RGS16 retained their differential G alpha activity, further highlighting that RGS proteins can discriminate between two very closely related G alpha subunits.     

14-3-3 proteins and the response to abiotic and biotic stress

14-3-3 proteins function as regulators of a wide range of target proteins in all eukaryotes by effecting direct protein-protein interactions. Primarily, interactions between 14-3-3 proteins and their targets are mediated by phosphorylation at specific sites on the target protein. Hence, interactions with 14-3-3s are subject to environmental control through signalling pathways which impact on 14-3-3 binding sites. Because 14-3-3 proteins regulate the activities of many proteins involved in signal transduction, there are multiple levels at which 14-3-3 proteins may play roles in stress responses in higher plants. In this article, we review evidence which implicates 14-3-3 proteins in responses to environmental, metabolic and nutritional stresses, as well as in defence responses to wounding and pathogen attack. This evidence includes stress-inducible changes in 14-3-3 gene expression, interactions between 14-3-3 proteins and signalling proteins and interactions between 14-3-3 proteins and proteins with defensive functions.     

The N-terminal non-RGS domain of human regulator of G-protein signalling 1 contributes to its ability to inhibit pheromone receptor signalling in yeast

Regulators of G-protein signalling (RGS) are a family of proteins that interact with G-proteins to regulate negatively G-protein coupled receptor (GPCR) signalling. In addition to a conserved core domain that is necessary and sufficient for their GTPase activating protein (GAP) like activity, RGSs possess N- and C-terminal motifs that confer distinct functional differences. In order to identify the role of the non-RGS region of human RGS1, we have characterized a series of fusions between RGS1 and GFP in a yeast mutant lacking the RGS containing SST2 gene. Using both halo assays as well as a GPCR responsive FUS1-LacZ reporter gene, we demonstrate that a RGS1-GFP fusion inhibits GPCR signalling in yeast while GFP fusions containing either the N-terminus non RGS sequence of RGS1(1-68) or the sequence containing the RGS box of RGS1(68-197) produce proteins that retain RGS1 activity. These results suggest that both the N-terminal and the RGS box of RGS1 function to inhibit signalling. Analysis of a series of mutants spanning the entire N-terminal non-RGS region of RGS1 produced by conservative segment exchange (CSE) mutagenesis showed little loss of function in yeast. This suggests that the overall structure of the N-terminal region of RGS1 rather than specific motifs or residues is required for its function.     

Regulator of G Protein Signaling 2 (RGS2) and RGS4 Form Distinct G Protein-Dependent Complexes with Protease Activated-Receptor 1 (PAR1) in Live Cells

Protease-activated receptor 1 (PAR1) is a G-protein coupled receptor (GPCR) that is activated by natural proteases to regulate many physiological actions. We previously reported that PAR1 couples to G_i, G_q and G₁₂ to activate linked signaling pathways. Regulators of G protein signaling (RGS) proteins serve as GTPase activating proteins to inhibit GPCR/G protein signaling. Some RGS proteins interact directly with certain GPCRs to modulate their signals, though cellular mechanisms dictating selective RGS/GPCR coupling are poorly understood. Here, using bioluminescence resonance energy transfer (BRET), we tested whether RGS2 and RGS4 bind to PAR1 in live COS-7 cells to regulate PAR1/Gα-mediated signaling. We report that PAR1 selectively interacts with either RGS2 or RGS4 in a G protein-dependent manner. Very little BRET activity is observed between PAR1-Venus (PAR1-Ven) and either RGS2-Luciferase (RGS2-Luc) or RGS4-Luc in the absence of Gα. However, in the presence of specific Gα subunits, BRET activity was markedly enhanced between PAR1-RGS2 by Gα_q/11, and PAR1-RGS4 by Gα_o, but not by other Gα subunits. Gα_q/11-YFP/RGS2-Luc BRET activity is promoted by PAR1 and is markedly enhanced by agonist (TFLLR) stimulation. However, PAR1-Ven/RGS-Luc BRET activity was blocked by a PAR1 mutant (R205A) that eliminates PAR1-G_q/11 coupling. The purified intracellular third loop of PAR1 binds directly to purified His-RGS2 or His-RGS4. In cells, RGS2 and RGS4 inhibited PAR1/Gα-mediated calcium and MAPK/ERK signaling, respectively, but not RhoA signaling. Our findings indicate that RGS2 and RGS4 interact directly with PAR1 in Gα-dependent manner to modulate PAR1/Gα-mediated signaling, and highlight a cellular mechanism for selective GPCR/G protein/RGS coupling.     

14-3-3 interacts with regulator of G protein signaling proteins and modulates their activity

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins (GAPs) that stimulate the inactivation of heterotrimeric G proteins. We have recently shown that RGS proteins may be regulated on a post-translational level (Benzing, T., Brandes, R., Sellin, L., Schermer, B., Lecker, S., Walz, G., and Kim, E. (1999) Nat. Med. 5, 913-918). However, mechanisms controlling the GAP activity of RGS proteins are poorly understood. Here we show that 14-3-3 proteins associate with RGS7 and RGS3. Binding of 14-3-3 is mediated by a conserved phosphoserine located in the Galpha-interacting portion of the RGS domain; interaction with 14-3-3 inhibits the GAP activity of RGS7, depends upon phosphorylation of a conserved residue within the RGS domain, and results in inhibition of GAP function. Collectively, these data indicate that phosphorylation-dependent binding of 14-3-3 may act as molecular switch that controls the GAP activity keeping a substantial fraction of RGS proteins in a dormant state.     

Integration of G Protein α (Gα) Signaling by the Regulator of G Protein Signaling 14 (RGS14)

RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gα_i/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gα_i1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gα_i1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gα_o-AlF₄⁻. Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gα_i1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gα_o-AlF₄⁻ and an AlF₄⁻-insensitive mutant (G42R) of Gα_i1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gα_i1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gα_o-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity.     

Molecular organization and dynamics of the melatonin MT1 receptor/RGS20/Gi protein complex reveal asymmetry of receptor dimers for RGS and Gi coupling

Functional asymmetry of G‐protein‐coupled receptor (GPCR) dimers has been reported for an increasing number of cases, but the molecular architecture of signalling units associated to these dimers remains unclear. Here, we characterized the molecular complex of the melatonin MT₁ receptor, which directly and constitutively couples to G_i proteins and the regulator of G‐protein signalling (RGS) 20. The molecular organization of the ternary MT₁/G_i/RGS20 complex was monitored in its basal and activated state by bioluminescence resonance energy transfer between probes inserted at multiple sites of the complex. On the basis of the reported crystal structures of G_i and the RGS domain, we propose a model wherein one G_i and one RGS20 protein bind to separate protomers of MT₁ dimers in a pre‐associated complex that rearranges upon agonist activation. This model was further validated with MT₁/MT₂ heterodimers. Collectively, our data extend the concept of asymmetry within GPCR dimers, reinforce the notion of receptor specificity for RGS proteins and highlight the advantage of GPCRs organized as dimers in which each protomer fulfils its specific task by binding to different GPCR‐interacting proteins.     

Differential contribution of GTPase activation and effector antagonism to the inhibitory effect of RGS proteins on Gq-mediated signaling in vivo

RGS proteins act as negative regulators of G protein signaling by serving as GTPase-activating proteins (GAP) for alpha subunits of heterotrimeric G proteins (Galpha), thereby accelerating G protein inactivation. RGS proteins can also block Galpha-mediated signal production by competing with downstream effectors for Galpha binding. Little is known about the relative contribution of GAP and effector antagonism to the inhibitory effect of RGS proteins on G protein-mediated signaling. By comparing the inhibitory effect of RGS2, RGS3, RGS5, and RGS16 on Galpha(q)-mediated phospholipase Cbeta (PLCbeta) activation under conditions where GTPase activation is possible versus nonexistent, we demonstrate that members of the R4 RGS subfamily differ significantly in their dependence on GTPase acceleration. COS-7 cells were transiently transfected with either muscarinic M3 receptors, which couple to endogenous Gq protein and mediate a stimulatory effect of carbachol on PLCbeta, or constitutively active Galphaq*, which is inert to GTP hydrolysis and activates PLCbeta independent of receptor activation. In M3-expressing cells, all of the RGS proteins significantly blunted the efficacy and potency of carbachol. In contrast, Galphaq* -induced PLCbeta activation was inhibited by RGS2 and RGS3 but not RGS5 and RGS16. The observed differential effects were not due to changes in M3, Galphaq/Galphaq*, PLCbeta, or RGS expression, as shown by receptor binding assays and Western blots. We conclude that closely related R4 RGS family members differ in their mechanism of action. RGS5 and RGS16 appear to depend on G protein inactivation, whereas GAP-independent mechanisms (such as effector antagonism) are sufficient to mediate the inhibitory effect of RGS2 and RGS3.     

Opioid-induced down-regulation of RGS4: role of ubiquitination and implications for receptor cross-talk

Regulator of G protein signaling protein 4 (RGS4) acts as a GTPase accelerating protein to modulate μ- and δ- opioid receptor (MOR and DOR, respectively) signaling. In turn, exposure to MOR agonists leads to changes in RGS4 at the mRNA and/or protein level. Here we have used human neuroblastoma SH-SY5Y cells that endogenously express MOR, DOR, and RGS4 to study opioid-mediated down-regulation of RGS4. Overnight treatment of SH-SY5Y cells with the MOR agonist DAMGO or the DOR agonist DPDPE decreased RGS4 protein by ～60% accompanied by a profound loss of opioid receptors but with no change in RGS4 mRNA. The decrease in RGS4 protein was prevented by the pretreatment with pertussis toxin or the opioid antagonist naloxone. The agonist-induced down-regulation of RGS4 proteins was completely blocked by treatment with the proteasome inhibitors MG132 or lactacystin or high concentrations of leupeptin, indicating involvement of ubiquitin-proteasome and lysosomal degradation. Polyubiquitinated RGS4 protein was observed in the presence of MG132 or the specific proteasome inhibitor lactacystin and promoted by opioid agonist. The loss of opioid receptors was not prevented by MG132, demonstrating a different degradation pathway. RGS4 is a GTPase accelerating protein for both Gα(i/o) and Gα(q) proteins. After overnight treatment with DAMGO to reduce RGS4 protein, signaling at the Gα(i/o)-coupled DOR and the Gα(q)-coupled M(3) muscarinic receptor (M(3)R) was increased but not signaling of the α(2) adrenergic receptor or bradykinin BK(2) receptor, suggesting the development of cross-talk between the DOR and M(3)R involving RGS4.     

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.     

The Ras‐binding domain region of RGS14 regulates its functional interactions with heterotrimeric G proteins

RGS14 is a 60 kDa protein that contains a regulator of G protein signaling (RGS) domain near its N‐terminus, a central region containing a pair of tandem Ras‐binding domains (RBD), and a GPSM (G protein signaling modulator) domain (a.k.a. Gi/o‐Loco binding [GoLoco] motif) near its C‐terminus. The RGS domain of RGS14 exhibits GTPase accelerating protein (GAP) activity toward Gαi/o proteins, while its GPSM domain acts as a guanine nucleotide dissociation inhibitor (GDI) on Gαi1 and Gαi3. In the current study, we investigate the contribution of different domains of RGS14 to its biochemical functions. Here we show that the full‐length protein has a greater GTPase activating activity but a weaker inhibition of nucleotide dissociation relative to its isolated RGS and GPSM regions, respectively. Our data suggest that these differences may be attributable to an inter‐domain interaction within RGS14 that promotes the activity of the RGS domain, but simultaneously inhibits the activity of the GPSM domain. The RBD region seems to play an essential role in this regulatory activity. Moreover, this region of RGS14 is also able to bind to members of the B/R4 subfamily of RGS proteins and enhance their effects on GPCR‐activated Gi/o proteins. Overall, our results suggest a mechanism wherein the RBD region associates with the RGS domain region, producing an intramolecular interaction within RGS14 that enhances the GTPase activating function of its RGS domain while disfavoring the negative effect of its GPSM domain on nucleotide dissociation. J. Cell. Biochem. 114: 1414–1423, 2013. © 2012 Wiley Periodicals, Inc.     

Anchoring of both PKA and 14-3-3 inhibits the Rho-GEF activity of the AKAP-Lbc signaling complex

A-kinase anchoring proteins (AKAPs) target the cAMP-regulated protein kinase (PKA) to its physiological substrates. We recently identified a novel anchoring protein, called AKAP-Lbc, which functions as a PKA-targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We demonstrated that AKAP-Lbc Rho-GEF activity is stimulated by the alpha subunit of the heterotrimeric G protein G12. Here, we identified 14-3-3 as a novel regulatory protein interacting with AKAP-Lbc. Elevation of the cellular concentration of cAMP activates the PKA holoenzyme anchored to AKAP-Lbc, which phosphorylates the anchoring protein on the serine 1565. This phosphorylation event induces the recruitment of 14-3-3, which inhibits the Rho-GEF activity of AKAP-Lbc. AKAP-Lbc mutants that fail to interact with PKA or with 14-3-3 show a higher basal Rho-GEF activity as compared to the wild-type protein. This suggests that, under basal conditions, 14-3-3 maintains AKAP-Lbc in an inactive state. Therefore, while it is known that AKAP-Lbc activity can be stimulated by Galpha12, in this study we demonstrated that it is inhibited by the anchoring of both PKA and 14-3-3.     

Phosphatidic acid binding inhibits RGS1 activity to affect specific signaling pathways in Arabidopsis

Modulation of the active versus inactive forms of the Gα protein is critical for the signaling processes mediated by the heterotrimeric G‐protein complex. We have recently established that in Arabidopsis, the regulator of G‐protein signaling (RGS1) protein and a lipid‐hydrolyzing enzyme, phospholipase Dα1 (PLDα1), both act as GTPase‐activity accelerating proteins (GAPs) for the Gα protein to attenuate its activity. RGS1 and PLDα1 interact with each other, and RGS1 inhibits the activity of PLDα1 during regulation of a subset of responses. In this study, we present evidence that this regulation is bidirectional. Phosphatidic acid (PA), a second messenger typically derived from the lipid‐hydrolyzing activity of PLDα1, is a molecular target of RGS1. PA binds and inhibits the GAP activity of RGS1. A conserved lysine residue in RGS1 (Lys²⁵⁹) is directly involved in RGS1–PA binding. Introduction of this RGS1 protein variant in the rgs1 mutant background makes plants hypersensitive to a subset of abscisic acid‐mediated responses. Our data point to the existence of negative feedback loops between these two regulatory proteins that precisely modulate the level of active Gα, consequently generating a highly controlled signal–response output.