ARF6 activation by Galpha q signaling: Galpha q forms molecular complexes with ARNO and ARF6

G protein-coupled receptors (GPCRs) are widely expressed hepta-helical receptors with tightly regulated pleiotropic effects. ADP-Ribosylation Factor 6 (ARF6) plays an important role in GPCR trafficking and is the subject of intense research. However, the mechanisms underlying activation and regulation of ARF6 by GPCRs are poorly characterized. Here we report that Galpha(q) signaling leads to the activation of ARF6. Stimulation of the TPbeta receptor triggered ARF6 activation which was completely inhibited by the RGS domain of GRK2 known to specifically bind and sequester Galpha(q). Co-immunoprecipitation studies revealed that ARNO (a guanine nucleotide exchange factor for ARF6) and ARF6 formed complexes preferentially with activated Galpha(q) compared to non-activated Galpha(q). Formation of the Galpha(q) complexes with ARNO and ARF6 was detected early and was optimal after 30 min of receptor stimulation corresponding with the profile of ARF6 activation. Interestingly, binding experiments using purified proteins showed that Galpha(q) interacted directly with ARNO. Galpha(q)-dependent TPbeta receptor-mediated activation of ARF6 resulted in phosphoinositol-4,5-bisphosphate production which was potently inhibited by dominant negative mutants of ARNO and ARF6. Furthermore, our data show that the expression of ARNO and ARF6 promoted, whereas dominant negative mutants of these proteins inhibited the internalization of the TPbeta receptor. This further elucidates our previous data on the PLCbeta- and PKC-independent mechanism involved in Galpha(q)-mediated internalization of the TPbeta receptor. Taken altogether, our results support a novel model where activated Galpha(q) forms molecular complexes with ARNO and ARF6, possibly through a direct interaction with ARNO, leading to ARF6 activation.     

Socius, a novel binding partner of Galpha12/13, promotes the Galpha12-induced RhoA activation

Heterotrimeric G proteins act as a molecular switch that conveys signals from G protein-coupled receptors in the cell membrane to intracellular downstream effectors. The Galpha subunits of the G(12) family of heterotrimeric G proteins, defined by Galpha(12) and Galpha(13), have many cellular functions through their specific downstream effectors. On the other hand, regulatory systems of the activity of Galpha(12) and Galpha(13) have not been fully clear. Here, we show that Socius, a previously identified Rho family small GTPase Rnd1 interacting protein, binds directly to Galpha(12) and Galpha(13) through its NH(2)-terminal region. Socius increased the amounts of GTP-bound active form of Galpha(12) in 293T cells. Furthermore, Socius promotes the Galpha(12)-induced RhoA activation in 293T cells. These results demonstrate that Socius is a novel activator of the Galpha(12) family.     

Unresponsiveness of platelets lacking both Galpha(q) and Galpha(13). Implications for collagen-induced platelet activation

The diffusible platelet stimuli ADP and thromboxane A(2) activate multiple G protein-mediated signaling pathways and function as important secondary mediators of platelet activation as they are released from activated platelets. Because they can also increase their own formation and release, their effects are amplified; eventually, all major G protein-mediated signaling pathways are activated. The multiple positive feedback mechanisms operating during platelet activation have obscured the exact analysis of the roles individual G protein-mediated signaling pathways play during the platelet activation process. In this report, we show that platelets lacking G(q) and G(13) are completely unresponsive to diffusible stimuli such as ADP, thromboxane A(2), or thrombin, even when applied at very high concentrations in combination, whereas all stimuli are able to induce platelet aggregation, shape change, and RhoA activation in platelets lacking only one Galpha subunit. This shows that G(q) or G(13) is required to induce some platelet activation, whereas the activation of G(i)-mediated signaling alone is not sufficient to induceactivation of mouse platelets. In addition, platelets lacking Galpha(q) and Galpha(13) adhered normally to collagen under high shearbut did not aggregate any more in response to collagen, indicating that collagen-induced platelet activation but not platelet adhesion requires intact G protein-mediated signaling pathways.     

Galpha(13) mediates activation of a depolarizing chloride current that accompanies RhoA activation in both neuronal and nonneuronal cells

Loss of membrane potential (membrane depolarization) is one of the earliest and most striking responses of quiescent cells to stimulation with serum or G protein-coupled receptor (GPCR) agonists such as lysophosphatidic acid and thrombin. Membrane depolarization is due to the activation of a chloride conductance. While this response has received relatively little attention in the past, it is clear that the acute loss of membrane potential may have important physiological consequences. However, the dissection of the underlying G protein pathway and the establishment of cause-effect relationships have remained elusive to date. Here we report that, in neuronal cells, the depolarizing chloride current invariably accompanies GPCR-induced activation of RhoA and subsequent neurite retraction, and neither of these events requires phosphoinositide hydrolysis or Ca2+ mobilization. Through antibody microinjections and a genetic approach, we demonstrate that activation of the chloride conductance is mediated by Galpha(13) in a RhoA-independent manner in both neuronal cells and fibroblasts. We further show that, in neuronal cells, this newly described Galpha(13) pathway may profoundly modulate membrane excitability during RhoA-regulated neurite remodeling.     

Activation of RhoA by association of Galpha(13) with Dbl

RhoA is a small G protein that is implicated in the regulation of the actin cytoskeleton, gene expression, and cell cycle progression. It is activated by many agonists whose receptors are linked to heterotrimeric G proteins, but the mechanisms are incompletely understood. In this study, we show that the constitutively active alpha-subunit of the heterotrimeric G protein G(13) associated with the Rho family guanine nucleotide exchange factor Dbl in NIH 3T3 cells and that this resulted in activation of RhoA. This activation was not seen with wild-type Galpha(13) or if Dbl and active Galpha(13) were expressed separately and mixed. In contrast, coexpression of constitutively active Galpha(q) with Dbl did not lead to their association and caused a weak activation of RhoA that was no greater than that observed with wild-type Galpha(q). These findings illustrate that activated Galpha(13) and Dbl can associate in vivo and that this leads to Rho activation.     

An inwardly rectifying whole cell current induced by Gq-coupled receptors

Ca(2+) influx across the plasma membrane after stimulation of G protein-coupled receptors is important for many physiological functions. Here we studied the regulation of an inwardly rectifying whole cell current and its putative role in Ca(2+) entry in Xenopus oocytes. Expression of P2Y(1) or M1 receptors in Xenopus oocytes elicited a characteristic inwardly rectifying current without receptor stimulation. This current displayed distinct activation and inactivation kinetics and was highly Ca(2+)-dependent. After stimulation of endogenous G(q)-coupled receptors in water-injected cells similar currents were observed. We therefore speculated that the current could be activated via Ca(2+) store depletion induced by constitutive stimulation of the IP(3) cascade in cells overexpressing G(q)-coupled receptors. Receptor-independent Ca(2+) store depletion also induced the current. In conclusion, this current is activated after store depletion suggesting a role in Ca(2+) entry after stimulation of G(q)-coupled receptors. Finally, our data do not support the proposed ionotropic properties of the P2Y(1) receptor.     

Activation of G-protein Galpha subunits by receptors through Galpha-Gbeta and Galpha-Ggamma interactions

Activation of the Galpha subunit of heterotrimeric GTP-binding proteins by transmembrane receptors requires the propagation of structural signals from the receptor-binding site to the nucleotide-binding site at the opposite side of the protein. In a previous model, it was suggested that the Gbeta-Ggamma dimer is tilted away from Galpha by a lever-arm motion of the Galpha N-terminal helix. Here, we propose that the motion occurs in the opposite direction, close-packing the Galpha-Gbeta interface and creating a novel interface between the helical domain of Galpha and the N terminus of Ggamma, which determines the specificity of activation.     

Gbetagamma signaling and Ca2+ mobilization co-operate synergistically in a Sos and Rac-dependent manner in the activation of JNK by Gq-coupled receptors

The mechanism by which G(q)-coupled receptors stimulate the c-Jun N-terminal kinase (JNK) activity has not been fully delineated. Here, we showed that stimulation of endogenous G(q)-coupled receptors in human hepatocarcinoma HepG2 cells resulted in an Src family kinase- and Ca(2+)-dependent JNK activation. Cos-7 cells transfected with HA-tagged JNK and various G(q)-coupled receptors also exhibited similar characteristics and provided further evidence for the involvement of Gbetagamma, an upstream intermediate for Src family kinases. The Ca(2+) and Gbetagamma signals operate in a high degree of independence. Transient expression of Gbetagamma subunits and elevation of cytoplasmic Ca(2+) level by thapsigargin activated JNK in a synergistic fashion. JNK activities triggered by G(q)-coupled receptors, Gbetagamma and thapsigargin were all suppressed by dominant negative (DN) mutants of Son of sevenless (Sos) and Rac. We propose that the co-operative effect between Gbetagamma-mediated signaling and the increased intracellular Ca(2+) level represents a robust mechanism for the stimulation of JNK by G(q)-coupled receptors.     

Action of Pasteurella multocida toxin on Galpha(q) is persistent and independent of interaction with G-protein-coupled receptors

Pasteurella multocida toxin (PMT) activates Galpha(q) and facilitates stimulation of inositol phosphate accumulation induced by agonists via G(q)-coupled membrane receptors. Here, we studied the effects of PMT on agonist-induced GTPgammaS binding to G(q) in cell membranes and a role of G-protein-coupled receptors in the action of PMT. Pre-treatment of Swiss 3T3 cells with PMT increased bombesin or vasopressin-induced GTPgammaS-binding in cell membranes by about 50 to 150%. Increase in agonist-stimulated GTPgammaS-binding caused by PMT pretreatment was specific for Galpha(q) and not observed with Galpha(11). PMT-induced effects on GTPgammaS-binding were persistent after removing the toxin or in the presence of anti-PMT antibody. Stimulation of agonist-induced GTPgammaS-binding by PMT was independent of phosphorylation of the C-terminal tyrosine356 of Galpha(q). Activation of phospholipase C by PMT occurred via Galpha(q) which was fused to the alpha(1b)-adrenoceptor and also with a C-terminally deleted Galpha(q), which is not able to interact with G protein-coupled membrane receptors. The data indicate that activation of Galpha(q) by PMT is persistent and independent of a functional interaction of G(q) with G-protein-coupled receptors.     

Enhanced activation of Akt and extracellular-regulated kinase pathways by simultaneous occupancy of Gq-coupled 5-HT2A receptors and Gs-coupled 5-HT7A receptors in PC12 cells

The most commonly prescribed antidepressants, the serotonin (5-HT) selective reuptake inhibitors, increase 5-HT without targeting specific receptors. Yet, little is known about the interaction of multiple receptor subtypes expressed by individual neurons. Specifically, the effect of increases in cAMP induced by Gs-coupled 5-HT receptor subtypes on the signaling pathways modulated by other receptor subtypes has not been studied. We have, therefore, examined the activation of the extracellular-regulated kinase (ERK) and Akt pathways by Gs-coupled 5-HT7A receptors and Gq-coupled 5-HT2A receptors, which are co-expressed in discrete brain regions. Agonists for both receptors were found to activate ERK and Akt in transfected PC12 cells. 5-HT2A receptor-mediated activation of the two pathways was found to be Ca2+-dependent. In contrast, 5-HT7A receptor-mediated activation of Akt required increases in both [cAMP] and intracellular [Ca2+], while activation of ERK was inhibited by Ca2+. The activation of ERK and Akt stimulated by simultaneous treatment of cells with 5-HT2A and 5-HT7A receptor agonists was found to be at least additive. Cell-permeable cAMP analogs mimicked 5-HT7A receptor agonists in enhancing 5-HT2A receptor-mediated activation of ERK and Akt. A role was identified for the cAMP-guanine exchange factor, Epac, in this augmentation of ERK, but not Akt, activation. Our finding of enhanced activation of neuroprotective Akt and ERK pathways by simultaneous occupancy of 5-HT2A and 5-HT7A receptors may also be relevant to the interaction of other neuronally expressed Gq- and Gs-coupled receptors.     

Modulation of distinct isoforms of L-type calcium channels by Gq-coupled receptors in Xenopus oocytes

L-type voltage dependent Ca²⁺ channels (L-VDCCs; Ca_v1.2) are crucial in cardiovascular physiology. In heart and smooth muscle, hormones and transmitters operating via G_q enhance L-VDCC currents via essential protein kinase C (PKC) involvement. Heterologous reconstitution studies in Xenopus oocytes suggested that PKC and G_q-coupled receptors increased L-VDCC currents only in cardiac long N-terminus (NT) isoforms of α_1C, whereas known smooth muscle short-NT isoforms were inhibited by PKC and G_q activators. We report a novel regulation of the long-NT α_1C isoform by Gβγ. Gβγ inhibited whereas a Gβγ scavenger protein augmented the G_q- but not phorbol ester-mediated enhancement of channel activity, suggesting that Gβγ acts upstream from PKC. In vitro binding experiments reveal binding of both Gβγ and PKC to α_1C-NT. However, PKC modulation was not altered by mutations of multiple potential phosphorylation sites in the NT, and was attenuated by a mutation of C-terminally located serine S1928. The insertion of exon 9a in intracellular loop 1 rendered the short-NT α_1C sensitive to PKC stimulation and to Gβγ scavenging. Our results suggest a complex antagonistic interplay between G_q-activated PKC and Gβγ in regulation of L-VDCC, in which multiple cytosolic segments of α_1C are involved.     

N-terminal polybasic motifs are required for plasma membrane localization of Galpha(s) and Galpha(q)

Heterotrimeric G proteins typically localize at the cytoplasmic face of the plasma membrane where they interact with heptahelical receptors. For G protein alpha subunits, multiple membrane targeting signals, including myristoylation, palmitoylation, and interaction with betagamma subunits, facilitate membrane localization. Here we show that an additional membrane targeting signal, an N-terminal polybasic region, plays a key role in plasma membrane localization of non-myristoylated alpha subunits. Mutations of N-terminal basic residues in alpha(s) and alpha(q) caused defects in plasma membrane localization, as assessed through immunofluorescence microscopy and biochemical fractionations. In alpha(s), mutation of four basic residues to glutamine was sufficient to cause a defect, whereas in alpha(q) a defect in membrane localization was not observed unless nine basic residues were mutated to glutamine or if three basic residues were mutated to glutamic acid. betagamma co-expression only partially rescued the membrane localization defects; thus, the polybasic region is also important in the context of the heterotrimer. Introduction of a site for myristoylation into the polybasic mutants of alpha(s) and alpha(q) recovered strong plasma membrane localization, indicating that myristoylation and polybasic motifs may have complementary roles as membrane targeting signals. Loss of plasma membrane localization coincided with defects in palmitoylation. The polybasic mutants of alpha(s) and alpha(q) were still capable of assuming activated conformations and stimulating second messenger production, as demonstrated through GST-RGS4 interaction assays, cAMP assays, and inositol phosphate assays. Electrostatic interactions with membrane lipids have been found to be important in plasma membrane targeting of many proteins, and these results provide evidence that basic residues play a role in localization of G protein alpha subunits.     

Interaction with Gbetagamma is required for membrane targeting and palmitoylation of Galpha(s) and Galpha(q)

Peripheral membrane proteins utilize a variety of mechanisms to attach tightly, and often reversibly, to cellular membranes. The covalent lipid modifications, myristoylation and palmitoylation, are critical for plasma membrane localization of heterotrimeric G protein alpha subunits. For alpha(s) and alpha(q), two subunits that are palmitoylated but not myristoylated, we examined the importance of interacting with the G protein betagamma dimer for their proper plasma membrane localization and palmitoylation. Conserved alpha subunit N-terminal amino acids predicted to mediate binding to betagamma were mutated to create a series of betagamma binding region mutants expressed in HEK293 cells. These alpha(s) and alpha(q) mutants were found in soluble rather than particulate fractions, and they no longer localized to plasma membranes as demonstrated by immunofluorescence microscopy. The mutations also inhibited incorporation of radiolabeled palmitate into the proteins and abrogated their signaling ability. Additional alpha(q) mutants, which contain these mutations but are modified by both myristate and palmitate, retained their localization to plasma membranes and ability to undergo palmitoylation. These findings identify binding to betagamma as a critical membrane attachment signal for alpha(s) and alpha(q) and as a prerequisite for their palmitoylation, while myristoylation can restore membrane localization and palmitoylation of betagamma binding-deficient alpha(q) subunits.     

Regulator of G-protein signalling 3 redirects prototypical Gi-coupled receptors from Rac1 to RhoA activation

The small GTPases, Rac1 and RhoA, are pivotal regulators of several essential, but distinct cellular processes. Numerous G-protein-coupled receptors signal to these GTPases, but with different specificities. Specifically, Gi-coupled receptors (GiPCRs) are generally believed to activate Rac1, but not RhoA, a process involving Gbetagamma-dimers and phosphatidylinositol 3-kinase (PI3K). Here we show that, depending on the expression level of the 519 amino acid isoform of regulator of G-protein signalling 3 (RGS3L), prototypical GiPCRs, like M2 muscarinic, A1 adenosine, and alpha2-adrenergic receptors, activate either Rac1 or RhoA in human embryonic kidney cells and neonatal rat cardiomyocyte-derived H10 cells. The switch from Rac1 to RhoA activation in H10 cells was controlled by fibroblast growth factor-2 (FGF-2), lowering the expression of RGS3L. Activation of both, Rac1 and RhoA, seen at low and high expression levels of RGS3L, respectively, was sensitive to pertussis toxin and the PI3K inhibitor LY294002 and mediated by Gbetagamma-dimers. We conclude that RGS3L functions as a molecular switch, redirecting GiPCRs via Gbetagamma-dimers and PI3K from Rac1 to RhoA activation. Considering the essential roles of Rac1 and RhoA in many signalling pathways, this additional function of RGS3L indicates a specific role of this protein in cellular signalling networks.     

Regulation of GTP-binding protein alpha q (Galpha q) signaling by the ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50)

Although ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a PDZ domain-containing protein known to bind to various channels, receptors, cytoskeletal elements, and cytoplasmic proteins, there is still very little evidence for a role of EBP50 in the regulation of receptor signal transduction. In this report, we show that EBP50 inhibits the phospholipase C (PLC)-beta-mediated inositol phosphate production of a Galpha(q)-coupled receptor as well as PLC-beta activation by the constitutively active Galpha(q)-R183C mutant. Coimmunoprecipitation experiments revealed that EBP50 interacts with Galpha(q) and to a greater extent with Galpha(q)-R183C. Agonist stimulation of the thromboxane A(2) receptor (TP receptor) resulted in an increased interaction between EBP50 and Galpha(q), suggesting that EBP50 preferentially interacts with activated Galpha(q). We also demonstrate that EBP50 inhibits Galpha(q) signaling by preventing the interaction between Galpha(q) and the TP receptor and between activated Galpha(q) and PLC-beta1. Investigation of the EBP50 regions involved in Galpha(q) binding indicated that its two PDZ domains are responsible for this interaction. This study constitutes the first demonstration of an interaction between a G protein alpha subunit and another protein through a PDZ domain, with broad implications in the regulation of diverse physiological systems.     

Inhibition of cell differentiation by Galpha q in the renal epithelial cell line LLC-PK1

LLC-PK₁, an epithelial cellline derived from the kidney proximal tubule, was used to study theability of the G protein -subunit, G_q, to regulate celldifferentiation. A constitutively active mutant protein,_qQ209L, was expressed using theLacSwitch-inducible mammalian expression system. Induction of_qQ209L expression with isopropyl--D-thiogalactopyranoside(IPTG) enhanced phospholipase C activity maximally by 6- to 7.5-fold.Increasing concentrations of IPTG progressively inhibited the activityof two differentiation markers,Na⁺-dependent hexose transport andalkaline phosphatase activity. Induction of_qQ209L expression also caused achange from an epithelial to a spindle-shaped morphology. The effectsof _qQ209L expression on celldifferentiation were similar to those observed with12-O-tetradecanoylphorbol 13-acetate(TPA) treatment. However, protein kinase C (PKC) levels weredownregulated in TPA-treated cells but not in_qQ209L-expressing cells,suggesting that the regulation of PKC byG_q may be different fromregulation by TPA. Interestingly, the PKC inhibitor GF-109203X did notinhibit the effect of IPTG on the development ofNa⁺-dependent hexose transport in_qQ209L-expressing cells. These data implicate PKC and PKC in the pathway used byG_q to block the development ofNa⁺-dependent hexose transport inIPTG-treated cells.

     

A transgenic mouse model of heart failure using inducible Galpha q

Receptors coupled to Galpha q play a key role in the development of heart failure. Studies using genetically modified mice suggest that Galpha q mediates a hypertrophic response in cardiac myocytes. Galpha q signaling in these models is modified during early growth and development, whereas most heart failure in humans occurs after cardiac damage sustained during adulthood. To determine the phenotype of animals that express increased Galpha q signaling only as adults, we generated transgenic mice that express a silent Galpha q protein (Galpha qQ209L-hbER) in cardiac myocytes that can be activated by tamoxifen. Following drug treatment to activate Galpha q Q209L-hbER, these mice rapidly develop a dilated cardiomyopathy and heart failure. This phenotype does not appear to involve myocyte hypertrophy but is associated with dephosphorylation of phospholamban (PLB), decreased sarcoplasmic reticulum Ca2+-ATPase activity, and a decrease in L-type Ca2+ current density. Changes in Ca2+ handling and decreased cardiac contractility are apparent 1 week after Galpha qQ209L-hbER activation. In contrast, transgenic mice that express an inducible Galpha q mutant that cannot activate phospholipase Cbeta (PLCbeta) do not develop heart failure or changes in PLB phosphorylation, but do show decreased L-type Ca2+ current density. These results demonstrate that activation of Galpha q in cardiac myocytes of adult mice causes a dilated cardiomyopathy that requires the activation of PLCbeta. However, increased PLCbeta signaling is not required for all of the Galpha q-induced cardiac abnormalities.     

Regulation of hematopoietic-specific G-protein Galpha15 and Galpha16 by protein kinase C

Heterotrimeric G proteins mediate cell growth and differentiation by coupling cell surface receptors to intracellular effector enzymes. The G-protein alpha subunit, Galpha(16), and its murine homologue Galpha(15), are expressed specifically in hematopoietic cells and their expression is highly regulated during differentiation of normal and leukemic cells. In this study, we examined the phosphorylation of Galpha(15)/Galpha(16) and its role in receptor and effector coupling. We observed a PMA-stimulated intact cell phosphorylation of Galpha(15) in COS7 cells transfected with Galpha(15) and protein kinase Calpha (PKCalpha), and phosphorylation of endogenous Galpha(16) in HL60 cells. We also showed that peptides derived from the two G-proteins were phosphorylated in vitro using purified brain PKC. Furthermore, we identified the putative phosphorylation site and showed that mutation or deletion of this PKC phosphorylation site inhibited phospholipase C (PLC) activation. The behavior of double mutants with the constitutively active G-protein mutation (QL-mutant) and mutation in the putative phosphorylation site suggests that the phosphorylation site of Galpha(15/16) is essential for receptor-coupled activation of PLC, but not for direct interaction of the G-protein with PLC-beta.