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.     

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.     

β-arrestin2 plays permissive roles in the inhibitory activities of RGS9-2 on G protein-coupled receptors by maintaining RGS9-2 in the open conformation

Together with G protein-coupled receptor (GPCR) kinases (GRKs) and β-arrestins, RGS proteins are the major family of molecules that control the signaling of GPCRs. The expression pattern of one of these RGS family members, RGS9-2, coincides with that of the dopamine D(3) receptor (D(3)R) in the brain, and in vivo studies have shown that RGS9-2 regulates the signaling of D2-like receptors. In this study, β-arrestin2 was found to be required for scaffolding of the intricate interactions among the dishevelled-EGL10-pleckstrin (DEP) domain of RGS9-2, Gβ5, R7-binding protein (R7BP), and D(3)R. The DEP domain of RGS9-2, under the permission of β-arrestin2, inhibited the signaling of D(3)R in collaboration with Gβ5. β-Arrestin2 competed with R7BP and Gβ5 so that RGS9-2 is placed in the cytosolic region in an open conformation which is able to inhibit the signaling of GPCRs. The affinity of the receptor protein for β-arrestin2 was a critical factor that determined the selectivity of RGS9-2 for the receptor it regulates. These results show that β-arrestins function not only as mediators of receptor-G protein uncoupling and initiators of receptor endocytosis but also as scaffolding proteins that control and coordinate the inhibitory effects of RGS proteins on the signaling of certain GPCRs.     

GPR158/179 regulate G protein signaling by controlling localization and activity of the RGS7 complexes

The extent and temporal characteristics of G protein-coupled receptor (GPCR) signaling are shaped by the regulator of G protein signaling (RGS) proteins, which promote G protein deactivation. With hundreds of GPCRs and dozens of RGS proteins, compartmentalization plays a key role in establishing signaling specificity. However, the molecular details and mechanisms of this process are poorly understood. In this paper, we report that the R7 group of RGS regulators is controlled by interaction with two previously uncharacterized orphan GPCRs: GPR158 and GPR179. We show that GPR158/179 recruited RGS complexes to the plasma membrane and augmented their ability to regulate GPCR signaling. The loss of GPR179 in a mouse model of night blindness prevented targeting of RGS to the postsynaptic compartment of bipolar neurons in the retina, illuminating the role of GPR179 in night vision. We propose that the interaction of RGS proteins with orphan GPCRs promotes signaling selectivity in G protein pathways.     

Spinophilin/neurabin reciprocally regulate signaling intensity by G protein-coupled receptors

Spinophilin (SPL) and neurabin (NRB) are structurally similar scaffolding proteins with several protein binding modules, including actin and PP1 binding motifs and PDZ and coiled-coil domains. SPL also binds regulators of G protein signaling (RGS) proteins and the third intracellular loop (3iL) of G protein-coupled receptors (GPCRs) to reduce the intensity of Ca(2+) signaling by GPCRs. The role of NRB in Ca(2+) signaling is not known. In the present work, we used biochemical and functional assays in model systems and in SPL(-/-) and NRB(-/-) mice to show that SPL and NRB reciprocally regulate Ca(2+) signaling by GPCRs. Thus, SPL and NRB bind all members of the R4 subfamily of RGS proteins tested (RGS1, RGS2, RGS4, RGS16) and GAIP. By contract, SPL, but not NRB, binds the 3iL of the GPCRs alpha(1B)-adrenergic (alpha(1B)AR), dopamine, CCKA, CCKB and the muscarinic M3 receptors. Coexpression of SPL or NRB with the alpha(1B)AR in Xenopus oocytes revealed that SPL reduces, whereas NRB increases, the intensity of Ca(2+) signaling by alpha(1B)AR. Accordingly, deletion of SPL in mice enhanced binding of RGS2 to NRB and Ca(2+) signaling by alphaAR, whereas deletion of NRB enhanced binding of RGS2 to SPL and reduced Ca(2+) signaling by alphaAR. This was due to reciprocal modulation by SPL and NRB of the potency of RGS2 to inhibit Ca(2+) signaling by alphaAR. These findings suggest a novel mechanism of regulation of GPCR-mediated Ca(2+) signaling in which SPL/NRB forms a functional pair of opposing regulators that modulates Ca(2+) signaling intensity by GPCRs by determining the extent of inhibition by the R4 family of RGS proteins.     

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.     

GIPC recruits GAIP (RGS19) to attenuate dopamine D2 receptor signaling

Pleiotropic G proteins are essential for the action of hormones and neurotransmitters and are activated by stimulation of G protein-coupled receptors (GPCR), which initiates heterotrimer dissociation of the G protein, exchange of GDP for GTP on its Galpha subunit and activation of effector proteins. Regulator of G protein signaling (RGS) proteins regulate this cascade and can be recruited to the membrane upon GPCR activation. Direct functional interaction between RGS and GPCR has been hypothesized. We show that recruitment of GAIP (RGS19) by the dopamine D2 receptor (D2R), a GPCR, required the scaffold protein GIPC (GAIP-interacting protein, C terminus) and that all three were coexpressed in neurons and neuroendocrine cells. Dynamic translocation of GAIP to the plasma membrane and coassembly in a protein complex in which GIPC was a required component was dictated by D2R activation and physical interactions. In addition, two different D2R-mediated responses were regulated by the GTPase activity of GAIP at the level of the G protein coupling in a GIPC-dependent manner. Since GIPC exclusively interacted with GAIP and selectively with subsets of GPCR, this mechanism may serve to sort GPCR signaling in cells that usually express a large repertoire of GPCRs, G proteins, and RGS.     

G protein-coupled receptor kinase function is essential for chemosensation in C. elegans

G protein-coupled receptors (GPCRs) mediate diverse signaling processes, including olfaction. G protein-coupled receptor kinases (GRKs) are important regulators of G protein signal transduction that specifically phosphorylate activated GPCRs to terminate signaling. Despite previously described roles for GRKs in GPCR signal downregulation, animals lacking C. elegans G protein-coupled receptor kinase-2 (Ce-grk-2) function are not hypersensitive to odorants. Instead, decreased Ce-grk-2 function in adult sensory neurons profoundly disrupts chemosensation, based on both behavioral analysis and Ca(2+) imaging. Although mammalian arrestin proteins cooperate with GRKs in receptor desensitization, loss of C. elegans arrestin-1 (arr-1) does not disrupt chemosensation. Either overexpression of the C. elegans Galpha subunit odr-3 or loss of eat-16, which encodes a regulator of G protein signaling (RGS) protein, restores chemosensation in Ce-grk-2 mutants. These results demonstrate that loss of GRK function can lead to reduced GPCR signal transduction and suggest an important role for RGS proteins in the regulation of chemosensation.     

Cellular mechanisms that determine selective RGS protein regulation of G protein-coupled receptor signaling

Regulators of G protein signaling (RGS proteins) bind directly to activated Galpha subunits to inhibit their signaling. However, recent findings show that RGS proteins selectively regulate signaling by certain G protein-coupled receptors (GPCRs) in cells, irrespective of the coupled G protein. New studies support an emerging model that suggests RGS proteins utilize both direct and indirect mechanisms to form stable functional pairs with preferred GPCRs to selectively modulate the signaling functions of those receptors and linked G proteins. Here, we discuss these findings and their implications for established models of GPCR signaling.     

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.     

Toll-like receptor signaling alters the expression of regulator of G protein signaling proteins in dendritic cells: implications for G protein-coupled receptor signaling

Conserved structural motifs on pathogens trigger pattern recognition receptors present on APCs such as dendritic cells (DCs). An important class of such receptors is the Toll-like receptors (TLRs). TLR signaling triggers a cascade of events in DCs that includes modified chemokine and cytokine production, altered chemokine receptor expression, and changes in signaling through G protein-coupled receptors (GPCRs). One mechanism by which TLR signaling could modify GPCR signaling is by altering the expression of regulator of G protein signaling (RGS) proteins. In this study, we show that human monocyte-derived DCs constitutively express significant amounts of RGS2, RGS10, RGS14, RGS18, and RGS19, and much lower levels of RGS3 and RGS13. Engagement of TLR3 or TLR4 on monocyte-derived DCs induces RGS16 and RGS20, markedly increases RGS1 expression, and potently down-regulates RGS18 and RGS14 without modifying other RGS proteins. A similar pattern of Rgs protein expression occurred in immature bone marrow-derived mouse DCs stimulated to mature via TLR4 signaling. The changes in RGS18 and RGS1 expression are likely important for DC function, because both proteins inhibit G alpha(i)- and G alpha(q)-mediated signaling and can reduce CXC chemokine ligand (CXCL)12-, CC chemokine ligand (CCL)19-, or CCL21-induced cell migration. Providing additional evidence, bone marrow-derived DCs from Rgs1(-/-) mice have a heightened migratory response to both CXCL12 and CCL19 when compared with similar DCs prepared from wild-type mice. These results indicate that the level and functional status of RGS proteins in DCs significantly impact their response to GPCR ligands such as chemokines.     

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.     

FMRFamide-like peptides encoded on the flp-18 precursor gene activate two isoforms of the orphan Caenorhabditis elegans G-protein-coupled receptor Y58G8A.4 heterologously expressed in mammalian cells

Two alternatively spliced variants of an orphan Caenorhabditis elegans G-protein-coupled receptors (GPCRs; Y58G8A.4a and Y58G8A.4b) were cloned and functionally expressed in Chinese hamster ovary (CHO) cells. The Y58G8A.4a and Y58G8A.4b proteins (397 and 433 amino acid residues, respectively) differ both in amino acid sequence and length of the C-terminal tail of the receptor. A calcium mobilization assay was used as a read-out for receptor function. Both receptors were activated, with nanomolar potencies, by putative peptides encoded by the flp-18 precursor gene, leading to their designation as FLP-18R1a (Y58G8A.4a) and FLP-18R1b (Y58G8A.4b). Three Ascaris suum neuropeptides AF3, AF4, and AF20 all sharing the same FLP-18 C-terminal signature, -PGVLRF-NH(2), were also potent agonists. In contrast to other previously reported C. elegans GPCRs expressed in mammalian cells, both FLP-18R1 variants were fully functional at 37 degrees C. However, a 37 to 28 degrees C temperature shift improved their activity, an effect that was more pronounced for FLP-18R1a. Despite differences in the C-terminus, the region implicated in distinct G-protein recognition for many other GPCRs, the same signaling pathways were observed for both Y58G8A.4 isoforms expressed in CHO cells. Gq protein coupling seems to be the main but not the exclusive signaling pathway, because pretreatment of cells with U-73122, a phospholipase inhibitor, attenuated but did not completely abolish the Ca(2+) signal. A weak Gs-mediated receptor activation was also detected as reflected in an agonist-triggered concentration-dependent cAMP increase. The matching of the FLP-18 peptides with their receptor(s) allows for the evaluation of the pharmacology of this system in the worm in vivo.     

RGS expression rate-limits recovery of rod photoresponses

Signaling through G protein-coupled receptors (GPCRs) underlies many cellular processes, yet it is not known which molecules determine the duration of signaling in intact cells. Two candidates are G protein-coupled receptor kinases (GRKs) and Regulators of G protein signaling (RGSs), deactivation enzymes for GPCRs and G proteins, respectively. Here we investigate whether GRK or RGS governs the overall rate of recovery of the light response in mammalian rod photoreceptors, a model system for studying GPCR signaling. We show that overexpression of rhodopsin kinase (GRK1) increases phosphorylation of the GPCR rhodopsin but has no effect on photoresponse recovery. In contrast, overexpression of the photoreceptor RGS complex (RGS9-1.Gbeta5L.R9AP) dramatically accelerates response recovery. Our results show that G protein deactivation is normally at least 2.5 times slower than rhodopsin deactivation, resolving a long-standing controversy concerning the mechanism underlying the recovery of rod visual transduction.     

Disease-causing mutation in PKR2 receptor reveals a critical role of positive charges in the second intracellular loop for G-protein coupling and receptor trafficking

Prokineticins are a pair of signal factors involved in many physiological processes by binding to two closely related G-protein-coupled receptors, PKR1 and PKR2. Recently, mutations in prokineticin 2 (PK2) and PKR2 are found to be associated with Kallmann syndrome and/or idiopathic hypogonadotropic hypogonadism, disorders characterized by delayed puberty and infertility. However, little is known how PKRs interact and activate G-proteins to elicit signal transduction. In the present study, we took advantage of one disease-associated mutation (R164Q) located in the second intracellular (IL2) loop of PKR2, to investigate the role of IL2 loop in the cell signaling, G-protein binding and receptor trafficking. R164Q mutant PKR2 showed normal cell surface expression and ligand binding capacity. However, the PKR2 signaling was abolished by R164Q mutation. We demonstrated that R164Q mutation disrupted the interaction of IL2 loop to the Gα(q), Gα(i), and Gα(16)-proteins. A positive-charged amino acid at this position is required for proper function, and the signaling efficacy and potency depend on the net amount of positive charges. We also demonstrated that the interactive partner of Arg-164 may localize in the C-terminal five residues of Gα(q)-protein. A series of mutation analysis indicated that the basic amino acids at the C terminus of IL2 loop may function cooperatively in GPCRs. Furthermore, R164Q mutation also results in minimal ligand-induced endocytosis of PKR2. As many GPCRs share structural homology in the C terminus of IL2 loop, our findings may have general application in understanding structure and function of GPCRs.     

Selective inhibition of alpha1A-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.     

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.     

Two cytoplasmic loops of the glucagon receptor are required to elevate cAMP or intracellular calcium.

The glucagon receptor is a member of a distinct class of G protein-coupled receptors (GPCRs) sharing little amino acid sequence homology with the larger rhodopsin-like GPCR family. To identify the components of the glucagon receptor necessary for G-protein coupling, we replaced sequentially all or part of each intracellular loop (i1, i2, and i3) and the C-terminal tail of the glucagon receptor with the 11 amino acids comprising the first intracellular loop of the D4 dopamine receptor. When expressed in transiently transfected COS-1 cells, the mutant receptors fell into two different groups with respect to hormone-mediated signaling. The first group included the loop i1 mutants, which bound glucagon and signaled normally. The second group comprised the loop i2 and i3 chimeras, which caused no detectable adenylyl cyclase activation in COS-1 cells. However, when expressed in HEK 293T cells, the loop i2 or i3 chimeras caused very small glucagon-mediated increases in cAMP levels and intracellular calcium concentrations, with EC50 values nearly 100-fold higher than those measured for wild-type receptor. Replacement of both loops i2 and i3 simultaneously was required to completely abolish G protein signaling as measured by both cAMP accumulation and calcium flux assays. These results show that the i2 and i3 loops play a role in glucagon receptor signaling, consistent with recent models for the mechanism of activation of G proteins by rhodopsin-like GPCRs.     

RGS17/RGSZ2, a novel regulator of Gi/o, Gz, and Gq signaling

To identify novel regulators of Galpha(o), the most abundant G-protein in brain, we used yeast two-hybrid screening with constitutively active Galpha(o) as bait and identified a new regulator of G-protein signaling (RGS) protein, RGS17 (RGSZ2), as a novel human member of the RZ (or A) subfamily of RGS proteins. RGS17 contains an amino-terminal cysteine-rich motif and a carboxyl-terminal RGS domain with highest homology to hRGSZ1- and hRGS-Galpha-interacting protein. RGS17 RNA was strongly expressed as multiple species in cerebellum and other brain regions. The interactions between hRGS17 and active forms of Galpha(i1-3), Galpha(o), Galpha(z), or Galpha(q) but not Galpha(s) were detected by yeast two-hybrid assay, in vitro pull-down assay, and co-immunoprecipitation studies. Recombinant RGS17 acted as a GTPase-activating protein (GAP) on free Galpha(i2) and Galpha(o) under pre-steady-state conditions, and on M2-muscarinic receptor-activated Galpha(i1), Galpha(i2), Galpha(i3), Galpha(z), and Galpha(o) in steady-state GTPase assays in vitro. Unlike RGSZ1, which is highly selective for G(z), RGS17 exhibited limited selectivity for G(o) among G(i)/G(o) proteins. All RZ family members reduced dopamine-D2/Galpha(i)-mediated inhibition of cAMP formation and abolished thyrotropin-releasing hormone receptor/Galpha(q)-mediated calcium mobilization. RGS17 is a new RZ member that preferentially inhibits receptor signaling via G(i/o), G(z), and G(q) over G(s) to enhance cAMP-dependent signaling and inhibit calcium signaling. Differences observed between in vitro GAP assays and whole-cell signaling suggest additional determinants of the G-protein specificity of RGS GAP effects that could include receptors and effectors.     

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.