The RGS14 GoLoco domain discriminates among Galphai isoforms

Regulators of G protein signaling (RGS) modulate G protein activity by functioning as GTPase-activating proteins (GAPs) for alpha-subunits of heterotrimeric G proteins. RGS14 regulates G protein nucleotide exchange and hydrolysis by acting as a GAP through its RGS domain and as a guanine nucleotide dissociation inhibitor (GDI) through its GoLoco motif. RGS14 exerts GDI activity on Galphai1, but not Galphao. Selective interactions are mediated by contacts between the alphaA and alphaB helices of the Galphai1 helical domain and the GoLoco C terminus (Kimple, R. J., Kimple, M. E., Betts, L., Sondek, J., and Siderovski, D. P. (2002) Nature 416, 878-881). Three isoforms of Galphai exist in mammalian cells. In this study, we tested whether all three isoforms were subject to RGS14 GDI activity. We found that RGS14 inhibits guanine nucleotide exchange on Galphai1 and Galphai3 could, but not Galphai2. Galphai2 be rendered sensitive to RGS14 GDI activity by replacement of residues within the alpha-helical domain. In addition to the contact residues in the alphaA and alphaB helices previously identified, we found that the alphaA/alphaB and alphaB/alphaC loops are important determinants of Galphai selectivity. The striking selectivity observed for RGS14 GDI activity in vitro points to Galphai1 and Galphai3 as the likely targets of RGS14-GoLoco regulation in vivo.     

Structural basis for the selectivity of the RGS protein, GAIP, for Galphai family members. Identification of a single amino acid determinant for selective interaction of Galphai subunits with GAIP.

GAIP is a regulator of G protein signaling (RGS) that accelerates the rate of GTP hydrolysis by some G protein alpha subunits. In the present studies, we have examined the structural basis for the ability of GAIP to discriminate among members of the Galphai family. Galphai1, Galphai3, and Galphao interacted strongly with GAIP, whereas Galphai2 interacted weakly and Galphas did not interact at all. A chimeric G protein composed of a Galphai2 N terminus and a Galphai1 C terminus interacted as strongly with GAIP as native Galphai1, whereas a chimeric N-terminal Galphai1 with a Galphai2 C terminus did not interact. These results suggest that the determinants responsible for GAIP selectivity between these two Galphais reside within the C-terminal GTPase domain of the G protein. To further localize residues contributing to G protein-GAIP selectivity, a panel of 15 site-directed Galphai1 and Galphai2 mutants were assayed. Of the Galphai1 mutants tested, only that containing a mutation at aspartate 229 located at the N terminus of Switch 3 did not interact with GAIP. Furthermore, the only Galphai2 variant that interacted strongly with GAIP contained a replacement of the corresponding Galphai2 Switch 3 residue (Ala230) with aspartate. To determine whether GAIP showed functional preferences for Galpha subunits that correlate with the binding data, the ability of GAIP to enhance the GTPase activity of purified alpha subunits was tested. GAIP catalyzed a 3-5-fold increase in the rate of GTP hydrolysis by Galphai1 and Galphai2(A230D) but no increase in the rate of Galphai2 and less than a 2-fold increase in the rate of Galphai1(D229A) under the same conditions. Thus, GAIP was able to discriminate between Galphai1 and Galphai2 in both binding and functional assays, and in both cases residue 229/230 played a critical role in selective recognition.     

G beta 5.RGS7 inhibits G alpha q-mediated signaling via a direct protein-protein interaction

A subfamily of regulators of G protein signaling (RGS) proteins consisting of RGS6, -7, -9, and -11 is characterized by the presence of a unique Ggamma-like domain through which they form obligatory dimers with the G protein subunit Gbeta5 in vivo. In Caenorhabditis elegans, orthologs of Gbeta5.RGS dimers are implicated in regulating both Galphai and Galphaq signaling, and in cell-based assays these dimers regulate Galphai/o- and Galphaq/11-mediated pathways. However, initial studies with purified Gbeta5.RGS6 or Gbeta5.RGS7 showed that they only serve as GTPase activating proteins for Galphao. Pull-down assays and co-immunoprecipitation with these dimers failed to detect their binding to either Galphao or Galphaq, indicating that the interaction might require additional factors present in vivo. Here, we asked if the RGS7.Gbeta5 complex binds to Galphaq using fluorescence resonance energy transfer (FRET) in transiently transfected mammalian cells. RGS7, Gbeta5, and Galpha subunits were tagged with yellow variants of green fluorescent protein. First we confirmed the functional activity of the fusion proteins by co-immunoprecipitation and also their effect on signaling. Second, we again demonstrate the interaction between RGS7 and Gbeta5 using FRET. Finally, using both FRET spectroscopy on cell suspensions and microscopy of individual cells, we showed FRET between the yellow fluorescence protein-tagged RGS7.Gbeta5 complex and cyan fluorescence protein-tagged Galphaq, indicating a direct interaction between these molecules.     

The alpha-helical domain of Galphat determines specific interaction with regulator of G protein signaling 9

RGS proteins (regulators of G protein signaling) are potent accelerators of the intrinsic GTPase activity of G protein alpha subunits (GAPs), thus controlling the response kinetics of a variety of cell signaling processes. Most RGS domains that have been studied have relatively little GTPase activating specificity especially for G proteins within the Gi subfamily. Retinal RGS9 is unique in its ability to act synergistically with a downstream effector cGMP phosphodiesterase to stimulate the GTPase activity of the alpha subunit of transducin, Galphat. Here we report another unique property of RGS9: high specificity for Galphat. The core (RGS) domain of RGS9 (RGS9) stimulates Galphat GTPase activity by 10-fold and Galphai1 GTPase activity by only 2-fold at a concentration of 10 microM. Using chimeric Galphat/Galphai1 subunits we demonstrated that the alpha-helical domain of Galphat imparts this specificity. The functional effects of RGS9 were well correlated with its affinity for activated Galpha subunits as measured by a change in fluorescence of a mutant Galphat (Chi6b) selectively labeled at Cys-210. Kd values for RGS9 complexes with Galphat and Galphai1 calculated from the direct binding and competition experiments were 185 nM and 2 microM, respectively. The gamma subunit of phosphodiesterase increases the GAP activity of RGS9. We demonstrate that this is because of the ability of Pgamma to increase the affinity of RGS9 for Galphat. A distinct, nonoverlapping pattern of RGS and Pgamma interaction with Galphat suggests a unique mechanism of effector-mediated GAP function of the RGS9.     

Characterization of a novel C. elegans RGS protein with a C2 domain: evidence for direct association between C2 domain and Galphaq subunit

RGS (regulator of G protein signaling) proteins are GTPase-activating proteins (GAPs) for heterotrimeric G protein alpha subunits and negatively regulate G protein-mediated signal transduction. In this study, we determined the cDNA sequence of a novel Caenorhabditis elegans (C. elegans) RGS protein. The predicted protein, termed C2-RGS, consists of 782 amino acids, and contains a C2 domain and an RGS domain. C2 domains are typically known to be Ca(2+) and phospholipid binding sites, found in many proteins involved in membrane traffic or signal transduction, and most of their biological roles are not identified. To study the function of C2-RGS protein, a series of six truncated versions of C2-RGS were constructed. When the full-length protein of C2-RGS was expressed transiently in AT1a-293T cells, ET-1-induced Ca(2+) responses were strongly suppressed. When each of the mutants with either RGS domain or C2 domain was expressed, the Ca(2+) responses were suppressed moderately. Furthermore, we found that C2 domain of PLC-beta1 also had a similar moderate inhibitory effect. RGS domain of C2-RGS bound to mammalian and C. elegans Galphai/o and Galphaq subunits only in the presence of GDP/AlF(4)(-), and had GAP activity to Galphai3. On the other hand, C2 domains of C2-RGS and PLC-beta1 also bound strongly to Galphaq subunit, in the presence of GDP, GDP/AlF(4)(-), and GTPgammaS, suggesting the stable persistent association between these C2 domains and Galphaq subunit at any stage during GTPase cycle. These results indicate that both the RGS domain and the C2 domain are responsible for the inhibitory effect of the full-length C2-RGS protein on Galphaq-mediated signaling, and suggest that C2 domains of C2-RGS and PLC-beta1 may act as a scaffold module to organize Galphaq and the respective whole protein molecule in a stable signaling complex, both in the absence and presence of stimulus.     

RGS2 binds directly and selectively to the M1 muscarinic acetylcholine receptor third intracellular loop to modulate Gq/11alpha signaling

RGS proteins serve as GTPase-activating proteins and/or effector antagonists to modulate Galpha signaling events. In live cells, members of the B/R4 subfamily of RGS proteins selectively modulate G protein signaling depending on the associated receptor (GPCR). Here we examine whether GPCRs selectively recruit RGS proteins to modulate linked G protein signaling. We report the novel finding that RGS2 binds directly to the third intracellular (i3) loop of the G(q/11)-coupled M1 muscarinic cholinergic receptor (M1 mAChR; M1i3). This interaction is selective because closely related RGS16 does not bind M1i3, and neither RGS2 nor RGS16 binds to the G(i/o)-coupled M2i3 loop. When expressed in cells, RGS2 and M1 mAChR co-localize to the plasma membrane whereas RGS16 does not. The N-terminal region of RGS2 is both necessary and sufficient for binding to M1i3, and RGS2 forms a stable heterotrimeric complex with both activated G(q)alpha and M1i3. RGS2 potently inhibits M1 mAChR-mediated phosphoinositide hydrolysis in cell membranes by acting as an effector antagonist. Deletion of the N terminus abolishes this effector antagonist activity of RGS2 but not its GTPase-activating protein activity toward G(11)alpha in membranes. These findings predict a model where the i3 loops of GPCRs selectively recruit specific RGS protein(s) via their N termini to regulate the linked G protein. Consistent with this model, we find that the i3 loops of the mAChR subtypes (M1-M5) exhibit differential profiles for binding distinct B/R4 RGS family members, indicating that this novel mechanism for GPCR modulation of RGS signaling may generally extend to other receptors and RGS proteins.     

Palmitoylation regulates regulators of G-protein signaling (RGS) 16 function. I. Mutation of amino-terminal cysteine residues on RGS16 prevents its targeting to lipid rafts and palmitoylation of an internal cysteine residue

Regulators of G-protein signaling (RGS) proteins down-regulate signaling by heterotrimeric G-proteins by accelerating GTP hydrolysis on the G alpha subunits. Palmitoylation, the reversible addition of palmitate to cysteine residues, occurs on several RGS proteins and is critical for their activity. For RGS16, mutation of Cys-2 and Cys-12 blocks its incorporation of [3H]palmitate and ability to turn-off Gi and Gq signaling and significantly inhibited its GTPase activating protein activity toward aG alpha subunit fused to the 5-hydroxytryptamine receptor 1A, but did not reduce its plasma membrane localization based on cell fractionation studies and immunoelectron microscopy. Palmitoylation can target proteins, including many signaling proteins, to membrane microdomains, called lipid rafts. A subpopulation of endogenous RGS16 in rat liver membranes and overexpressed RGS16 in COS cells, but not the nonpalmitoylated cysteine mutant of RGS16, localized to lipid rafts. However, disruption of lipid rafts by treatment with methyl-beta-cyclodextrin did not decrease the GTPase activating protein activity of RGS16. The lipid raft fractions were enriched in protein acyltransferase activity, and RGS16 incorporated [3H]palmitate into a peptide fragment containing Cys-98, a highly conserved cysteine within the RGS box. These results suggest that the amino-terminal palmitoylation of an RGS protein promotes its lipid raft targeting that allows palmitoylation of a poorly accessible cysteine residue that we show in the accompanying article (Osterhout, J. L., Waheed, A. A., Hiol, A., Ward, R. J., Davey, P. C., Nini, L., Wang, J., Milligan, G., Jones, T. L. Z., and Druey, K. M. (2003) J. Biol. Chem. 278, 19309-19316) was critical for RGS16 and RGS4 GAP activity.     

Ca2+/Calmodulin reverses phosphatidylinositol 3,4, 5-trisphosphate-dependent inhibition of regulators of G protein-signaling GTPase-activating protein activity

Regulators of G protein signaling (RGS proteins) are GTPase-activating proteins (GAPs) for G(i) and/or G(q) class G protein alpha subunits. RGS GAP activity is inhibited by phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) but not by other lipid phosphoinositides or diacylglycerol. Both the negatively charged head group and long chain fatty acids (C16) are required for binding and inhibition of GAP activity. Amino acid substitutions in helix 5 within the RGS domain of RGS4 reduce binding affinity and inhibition by PIP(3) but do not affect inhibition of GAP activity by palmitoylation. Conversely, the GAP activity of a palmitoylation-resistant mutant RGS4 is inhibited by PIP(3). Calmodulin binds all RGS proteins we tested in a Ca(2+)-dependent manner but does not directly affect GAP activity. Indeed, Ca(2+)/calmodulin binds a complex of RGS4 and a transition state analog of Galpha(i1)-GDP-AlF(4)(-). Ca(2+)/calmodulin reverses PIP(3)-mediated but not palmitoylation-mediated inhibition of GAP activity. Ca(2+)/calmodulin competition with PIP(3) may provide an intracellular mechanism for feedback regulation of Ca(2+) signaling evoked by G protein-coupled agonists.     

Differential effects of regulator of G protein signaling (RGS) proteins on serotonin 5-HT1A, 5-HT2A, and dopamine D2 receptor-mediated signaling and adenylyl cyclase activity

Regulator of G protein signaling (RGS) proteins function as GTPase accelerating proteins (GAP) for Galpha subunits, attenuating G-protein-coupled receptor signal transduction. The present study tested the ability of members of different subfamilies of RGS proteins to modulate both G-protein-dependent and -independent signaling in mammalian cells. RGS4, RGS10, and RGSZ1 significantly attenuated Galphai-mediated signaling by 5-HT1A, but not by dopamine D2, receptor-expressing cells. Additionally, RGS4 and RGS10 significantly inhibited forskolin-stimulated cAMP production in both cell lines. In contrast, RGS2, RGS7, and RGSZ1 had no effect on forskolin-stimulated cAMP production in these cells. RGS2 and RGS7 significantly decreased Galphaq-mediated signaling by 5-HT2A receptors, confirming that the RGS4 and RGS10 effects on forskolin-stimulated cAMP production were specific, and not simply due to overexpression. Interestingly, similar expression levels of RGS4 protein resulted in greater inhibition of G-protein-independent cAMP production compared to G-protein-dependent GAP activity. Our results suggest specificity and selectivity of RGS proteins on G-protein-dependent and -independent signaling in mammalian cells.     

Palmitoylation regulates regulator of G-protein signaling (RGS) 16 function. II. Palmitoylation of a cysteine residue in the RGS box is critical for RGS16 GTPase accelerating activity and regulation of Gi-coupled signalling

Palmitoylation is a reversible post-translational modification used by cells to regulate protein activity. The regulator of G-protein signaling (RGS) proteins RGS4 and RGS16 share conserved cysteine (Cys) residues that undergo palmitoylation. In the accompanying article (Hiol, A., Davey, P. C., Osterhout, J. L., Waheed, A. A., Fischer, E. R., Chen, C. K., Milligan, G., Druey, K. M., and Jones, T. L. Z. (2003) J. Biol. Chem. 278, 19301-19308), we determined that mutation of NH2-terminal cysteine residues in RGS16 (Cys-2 and Cys-12) reduced GTPase accelerating (GAP) activity toward a 5-hydroxytryptamine (5-HT1A)/G alpha o1 receptor fusion protein in cell membranes. NH2-terminal acylation also permitted palmitoylation of a cysteine residue in the RGS box of RGS16 (Cys-98). Here we investigated the role of internal palmitoylation in RGS16 localization and GAP activity. Mutation of RGS16 Cys-98 or RGS4 Cys-95 to alanine reduced GAP activity on the 5-HT1A/G alpha o1 fusion protein and regulation of adenylyl cyclase inhibition. The C98A mutation had no effect on RGS16 localization or GAP activity toward purified G-protein alpha subunits. Enzymatic palmitoylation of RGS16 resulted in internal palmitoylation on residue Cys-98. Palmitoylated RGS16 or RGS4 WT but not C98A or C95A preincubated with membranes expressing 5-HT1a/G alpha o1 displayed increased GAP activity over time. These results suggest that palmitoylation of a Cys residue in the RGS box is critical for RGS16 and RGS4 GAP activity and their ability to regulate Gi-coupled signaling in mammalian cells.     

RGS14 is a bifunctional regulator of Gαi/o activity that exists in multiple populations in brain

Members of the regulators of G protein signaling (RGS) family modulate Galpha-directed signals as a result of the GTPase-activating protein (GAP) activity of their conserved RGS domain. In addition to its RGS domain, RGS14 contains a Rap binding domain (RBD) and a GoLoco motif. To define the cellular and biochemical properties of RGS14 we utilized two different affinity purified antisera that specifically recognize recombinant and native RGS14. In brain, we observed two RGS14-like immunoreactive bands of distinct size (60 kDa and 55 kDa). Both forms are present in brain cytosol and in two, biochemically distinct, membrane subpopulations: one detergent-extractable and the other detergent-insensitive. Recombinant RGS14 binds specifically to activated Galphai/o, but not Galphaq/11, Galpha12/13, or Galphas in brain membranes. In reconstitution studies, we found that RGS14 is a non-selective GAP for Galphai1 and Galphao and that full-length RGS14 is an approximately 10-fold more potent stimulator of Galpha GTPase activity than the RGS domain alone. In contrast, neither full-length RGS14 nor the isolated RBD domain is a GAP for Rap1. RGS14 is also a highly selective guanine nucleotide dissociation inhibitor (GDI) for Galphai but not Galphao, and this activity is restricted to the C-terminus containing the GoLoco domain. These findings highlight previously unknown biochemical properties of RGS14 in brain, and provide one of the first examples of an RGS protein that is a bifunctional regulator of Galpha actions.     

Identification of a novel alternative splicing variant of RGS5 mRNA in human ocular tissues

Regulator of G protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) for Galpha subunits and negatively regulate G protein-coupled receptor signaling. Using RGS5 gene-specific RT-PCR, we have identified a novel alternative splicing variant of RGS5 mRNA in human ocular tissues. The alternative splicing of RGS5 mRNA occurred at position +44 (GenBank NM_003617), spliced out 174 bp (+44 to +218 bp) of the coding region, and encoded an RGS5s protein with a 108 amino acid N-terminal deletion. This study is the first to document alternative splicing of an RGS5 gene. We therefore studied RGS5 and RGS5s mRNA distribution in human tissues. In the eye, RGS5s was found to be highly expressed in the ciliary body and trabecular meshwork. It was also expressed in the kidney, brain, spleen, skeletal muscle and small intestine, but was not detectable in the liver, lung, heart. RGS5s was not found in monkey and rat ocular tissues, indicating species specificity for the eye. Comparing the recombinant RGS5 and RGS5s expression in HEK293/EBNA cells, RGS5s was present almost exclusively in the cytosolic fraction, whereas RGS5 was present in both membrane and cytosolic fractions. The data suggest that the N-terminal of RGS5 may be important for protein translocation to the cell membrane. Both RGS5 and RGS5s antagonized the rapid phosphorylation of p44/42 MAP kinase induced by Galphai coupled cannibinoid receptor-1 activation. RGS5, but not RGS5s, inhibited the Ca2+ signaling initiated by activation of Galphaq coupled angiotensin II receptors (AT1) and prostaglandin FP receptors. Cotransfection of RGS5s with RGS5 resulted in the blockade of RGS5 actions with respect to inhibition of the signal transduction initiated by activation of both AT1 and FP receptor, suggesting that RGS5s may contain functional domains that compete with RGS5 in the regulation of the Galphaq coupled AT1 and FP receptors. The unique expression pattern, cellular localization and functions of RGS5s suggest that RGS5s may play a critical role in the regulation of intracellular signaling pathways.     

G protein-coupled receptors and resistance to inhibitors of cholinesterase-8A (Ric-8A) both regulate the regulator of g protein signaling 14 RGS14·Gαi1 complex in live cells

Regulator of G protein Signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates both conventional and unconventional G protein signaling pathways. Like other RGS (regulator of G protein signaling) proteins, RGS14 acts as a GTPase accelerating protein to terminate conventional Gα(i/o) signaling. However, unlike other RGS proteins, RGS14 also contains a G protein regulatory/GoLoco motif that specifically binds Gα(i1/3)-GDP in cells and in vitro. The non-receptor guanine nucleotide exchange factor Ric-8A can bind and act on the RGS14·Gα(i1)-GDP complex to play a role in unconventional G protein signaling independent of G protein-coupled receptors (GPCRs). Here we demonstrate that RGS14 forms a Gα(i/o)-dependent complex with a G(i)-linked GPCR and that this complex is regulated by receptor agonist and Ric-8A (resistance to inhibitors of cholinesterase-8A). Using live cell bioluminescence resonance energy transfer, we show that RGS14 functionally associates with the α(2A)-adrenergic receptor (α(2A)-AR) in a Gα(i/o)-dependent manner. This interaction is markedly disrupted after receptor stimulation by the specific agonist UK14304, suggesting complex dissociation or rearrangement. Agonist-mediated dissociation of the RGS14·α(2A)-AR complex occurs in the presence of Gα(i/o) but not Gα(s) or Gα(q). Unexpectedly, RGS14 does not dissociate from Gα(i1) in the presence of stimulated α(2A)-AR, suggesting preservation of RGS14·Gα(i1) complexes after receptor activation. However, Ric-8A facilitates dissociation of both the RGS14·Gα(i1) complex and the Gα(i1)-dependent RGS14·α(2A)-AR complex after receptor activation. Together, these findings indicate that RGS14 can form complexes with GPCRs in cells that are dependent on Gα(i/o) and that these RGS14·Gα(i1)·GPCR complexes may be substrates for other signaling partners such as Ric-8A.     

Regulation of Protease-activated Receptor 1 Signaling by the Adaptor Protein Complex 2 and R4 Subfamily of Regulator of G Protein Signaling Proteins

The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of “regulator of G protein signaling” (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 ⁴²⁰AKKAA⁴²⁴ mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gα_q association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.     

RGS7 and RGS8 differentially accelerate G protein-mediated modulation of K+ currents

The recently discovered family of RGS (regulators of G protein signaling) proteins acts as GTPase activating proteins which bind to alpha subunits of heterotrimeric G proteins. We previously showed that a brain-specific RGS, RGS8 speeds up the activation and deactivation kinetics of the G protein-coupled inward rectifier K+ channel (GIRK) upon receptor stimulation (Saitoh, O., Kubo, Y., Miyatani, Y., Asano, T., and Nakata, H. (1997) Nature 390, 525-529). Here we report the isolation of a full-length rat cDNA of another brain-specific RGS, RGS7. In situ hybridization study revealed that RGS7 mRNA is predominantly expressed in Golgi cells within granule cell layer of cerebellar cortex. We observed that RGS7 recombinant protein binds preferentially to Galphao, Galphai3, and Galphaz. When co-expressed with GIRK1/2 in Xenopus oocytes, RGS7 and RGS8 differentially accelerate G protein-mediated modulation of GIRK. RGS7 clearly accelerated activation of GIRK current similarly with RGS8 but the acceleration effect of deactivation was significantly weaker than that of RGS8. These acceleration properties of RGS proteins may play important roles in the rapid regulation of neuronal excitability and the cellular responses to short-lived stimulations.     

FBXO44-Mediated Degradation of RGS2 Protein Uniquely Depends on a Cullin 4B/DDB1 Complex

The ubiquitin-proteasome system for protein degradation plays a major role in regulating cell function and many signaling proteins are tightly controlled by this mechanism. Among these, Regulator of G Protein Signaling 2 (RGS2) is a target for rapid proteasomal degradation, however, the specific enzymes involved are not known. Using a genomic siRNA screening approach, we identified a novel E3 ligase complex containing cullin 4B (CUL4B), DNA damage binding protein 1 (DDB1) and F-box protein 44 (FBXO44) that mediates RGS2 protein degradation. While the more typical F-box partners CUL1 and Skp1 can bind FBXO44, that E3 ligase complex does not bind RGS2 and is not involved in RGS2 degradation. These observations define an unexpected DDB1/CUL4B-containing FBXO44 E3 ligase complex. Pharmacological targeting of this mechanism provides a novel therapeutic approach to hypertension, anxiety, and other diseases associated with RGS2 dysregulation.     

Modules in the photoreceptor RGS9-1.Gbeta 5L GTPase-accelerating protein complex control effector coupling, GTPase acceleration, protein folding, and stability

RGS (regulators of G protein signaling) proteins regulate G protein signaling by accelerating GTP hydrolysis, but little is known about regulation of GTPase-accelerating protein (GAP) activities or roles of domains and subunits outside the catalytic cores. RGS9-1 is the GAP required for rapid recovery of light responses in vertebrate photoreceptors and the only mammalian RGS protein with a defined physiological function. It belongs to an RGS subfamily whose members have multiple domains, including G(gamma)-like domains that bind G(beta)(5) proteins. Members of this subfamily play important roles in neuronal signaling. Within the GAP complex organized around the RGS domain of RGS9-1, we have identified a functional role for the G(gamma)-like-G(beta)(5L) complex in regulation of GAP activity by an effector subunit, cGMP phosphodiesterase gamma and in protein folding and stability of RGS9-1. The C-terminal domain of RGS9-1 also plays a major role in conferring effector stimulation. The sequence of the RGS domain determines whether the sign of the effector effect will be positive or negative. These roles were observed in vitro using full-length proteins or fragments for RGS9-1, RGS7, G(beta)(5S), and G(beta)(5L). The dependence of RGS9-1 on G(beta)(5) co-expression for folding, stability, and function has been confirmed in vivo using transgenic Xenopus laevis. These results reveal how multiple domains and regulatory polypeptides work together to fine tune G(talpha) inactivation.     

5-HT1A receptor-mediated phosphorylation of extracellular signal-regulated kinases (ERK1/2) is modulated by regulator of G protein signaling protein 19

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.     

Regulator of G-protein signaling 3 (RGS3) inhibits Gbeta1gamma 2-induced inositol phosphate production, mitogen-activated protein kinase activation, and Akt activation

Regulator of G-protein signaling 3 (RGS3) enhances the intrinsic rate at which Galpha(i) and Galpha(q) hydrolyze GTP to GDP, thereby limiting the duration in which GTP-Galpha(i) and GTP-Galpha(q) can activate effectors. Since GDP-Galpha subunits rapidly combine with free Gbetagamma subunits to reform inactive heterotrimeric G-proteins, RGS3 and other RGS proteins may also reduce the amount of Gbetagamma subunits available for effector interactions. Although RGS6, RGS7, and RGS11 bind Gbeta(5) in the absence of a Ggamma subunit, RGS proteins are not known to directly influence Gbetagamma signaling. Here we show that RGS3 binds Gbeta(1)gamma(2) subunits and limits their ability to trigger the production of inositol phosphates and the activation of Akt and mitogen-activated protein kinase. Co-expression of RGS3 with Gbeta(1)gamma(2) inhibits Gbeta(1)gamma(2)-induced inositol phosphate production and Akt activation in COS-7 cells and mitogen-activated protein kinase activation in HEK 293 cells. The inhibition of Gbeta(1)gamma(2) signaling does not require an intact RGS domain but depends upon two regions in RGS3 located between acids 313 and 390 and between 391 and 458. Several other RGS proteins do not affect Gbeta(1)gamma(2) signaling in these assays. Consistent with the in vivo results, RGS3 inhibits Gbetagamma-mediated activation of phospholipase Cbeta in vitro. Thus, RGS3 may limit Gbetagamma signaling not only by virtue of its GTPase-activating protein activity for Galpha subunits, but also by directly interfering with the activation of effectors.