Neutrophil elastase acts as a biased agonist for proteinase-activated receptor-2 (PAR2)

Human neutrophil proteinases (elastase, proteinase-3, and cathepsin-G) are released at sites of acute inflammation. We hypothesized that these inflammation-associated proteinases can affect cell signaling by targeting proteinase-activated receptor-2 (PAR(2)). The PAR family of G protein-coupled receptors is triggered by a unique mechanism involving the proteolytic unmasking of an N-terminal self-activating tethered ligand (TL). Proteinases can either activate PAR signaling by unmasking the TL sequence or disarm the receptor for subsequent enzyme activation by cleaving downstream from the TL sequence. We found that none of neutrophil elastase, cathepsin-G, and proteinase-3 can activate G(q)-coupled PAR(2) calcium signaling; but all of these proteinases can disarm PAR(2), releasing the N-terminal TL sequence, thereby preventing G(q)-coupled PAR(2) signaling by trypsin. Interestingly, elastase (but neither cathepsin-G nor proteinase-3) causes a TL-independent PAR(2)-mediated activation of MAPK that, unlike the canonical trypsin activation, does not involve either receptor internalization or recruitment of β-arrestin. Cleavage of synthetic peptides derived from the extracellular N terminus of PAR(2), downstream of the TL sequence, demonstrated distinct proteolytic sites for all three neutrophil-derived enzymes. We conclude that in inflammation, neutrophil proteinases can modulate PAR(2) signaling by preventing/disarming the G(q)/calcium signal pathway and, via elastase, can selectively activate the p44/42 MAPK pathway. Our data illustrate a new mode of PAR regulation that involves biased PAR(2) signaling by neutrophil elastase and a disarming/silencing effect of cathepsin-G and proteinase-3.     

Independent regulation of Rap1 and mitogen-activated protein kinase by the alpha chain of Go

Receptors coupled to G(i/o) proteins stimulate the mitogen-activated protein kinase (MAPK) cascade. The intracellular pathways linking the alpha chains of these G proteins to MAPK activation are not completely understood. One of the signaling molecules which has been suggested to act downstream of Galpha(i/o) is the small G protein Rap1. We investigated the role of Rap1 in MAPK stimulation by Galpha(o) in Chinese hamster ovary (CHO) cells. Our previous results have shown that in this cell system activated Galpha(o) strongly potentiates the MAPK response to the epidermal growth factor (EGF) receptor. Rap1 regulation was examined in cells transfected with Rap1 and wild-type Galpha(o) or the activated mutant Galpha(o)-Q205L. Immunocytochemical analysis detected both Rap1 and the Galpha(o) subunit at the plasma membrane as well as on perinuclear cytoplasmic vesicles. Expression of wild-type Galpha(o) had no significant effect on the levels of activated Rap1. In contrast, Galpha(o)-Q205L virtually abolished the activation of Rap1 induced by EGF. Further experiments showed that MAPK stimulation by EGF was greatly inhibited by expression of activated Rap1, suggesting that Rap1 inhibition could mediate the effect of Galpha(o) on the MAPK cascade. However, Galpha(o)-Q205L efficiently inhibited the activation of Rap1 induced by fibroblast growth factor (FGF). We have previously found that the ability of FGF to activate MAPK is not modified by Galpha(o). In addition, expression of the GAP protein RAP1GAPII blocked Rap1 activation without affecting EGF- or FGF-dependent MAPK stimulation. These findings provide evidence for independent regulation of Rap1 and MAPK by the G(o )alpha chain.     

Two distinct aspects of coupling between Gα(i) protein and G protein-activated K+ channel (GIRK) revealed by fluorescently labeled Gα(i3) protein subunits

G protein-activated K(+) channels (Kir3 or GIRK) are activated by direct interaction with Gβγ. Gα is essential for specific signaling and regulates basal activity of GIRK (I(basal)) and kinetics of the response elicited by activation by G protein-coupled receptors (I(evoked)). These regulations are believed to occur within a GIRK-Gα-Gβγ signaling complex. Fluorescent energy resonance transfer (FRET) studies showed strong GIRK-Gβγ interactions but yielded controversial results regarding the GIRK-Gα(i/o) interaction. We investigated the mechanisms of regulation of GIRK by Gα(i/o) using wild-type Gα(i3) (Gα(i3)WT) and Gα(i3) labeled at three different positions with fluorescent proteins, CFP or YFP (xFP). Gα(i3)xFP proteins bound the cytosolic domain of GIRK1 and interacted with Gβγ in a guanine nucleotide-dependent manner. However, only an N-terminally labeled, myristoylated Gα(i3)xFP (Gα(i3)NT) closely mimicked all aspects of Gα(i3)WT regulation except for a weaker regulation of I(basal). Gα(i3) labeled with YFP within the Gα helical domain preserved regulation of I(basal) but failed to restore fast I(evoked). Titrated expression of Gα(i3)NT and Gα(i3)WT confirmed that regulation of I(basal) and of the kinetics of I(evoked) of GIRK1/2 are independent functions of Gα(i). FRET and direct biochemical measurements indicated much stronger interaction between GIRK1 and Gβγ than between GIRK1 and Gα(i3). Thus, Gα(i/o)βγ heterotrimer may be attached to GIRK primarily via Gβγ within the signaling complex. Our findings support the notion that Gα(i/o) actively regulates GIRK. Although regulation of I(basal) is a function of Gα(i)(GDP), our new findings indicate that regulation of kinetics of I(evoked) is mediated by Gα(i)(GTP).     

The G alpha(o/i)-coupled cannabinoid receptor-mediated neurite outgrowth involves Rap regulation of Src and Stat3

The study of the signaling pathways regulating neurite outgrowth in culture is important because of their potential role in neuronal differentiation in vivo. We have previously shown that the G alpha(o/i)-coupled CB1 cannabinoid receptor (CB1R) activates Rap1 to induce neurite outgrowth. G alpha(o/i) also activates the Src-Stat3 pathway. Here, we studied the relationship between the G alpha(o/i)-Rap1 and Src-Stat3 pathways and the role of these signaling pathways in CB1R-mediated neurite outgrowth in Neuro-2A cells. The CB1 agonist HU-210 induced pertussis toxin-sensitive Src and Stat3 phosphorylation. Dominant negative (DN) mutants of Src and Stat3 blocked CB1R-induced neurite outgrowth. Constitutively active Rap 1B and Ral-activated Src and CB1R-induced Src phosphorylation was inhibited by Rap1-DN and Ral-DN, indicating that both Rap1 and Ral mediate downstream signaling from G alpha(o/i) for Src activation. Rap1-activated Ral and Ral-DN blocked Rap-induced Src phosphorylation. G alpha(o)-induced Stat3 activation was blocked by Ral-DN, whereas v-Src-induced Stat3 activation was not inhibited by Ral-DN, indicating that the CB1R, through G alpha(o), mediates the sequential activation of Rap1 to Ral to Src to Stat3 in Neuro-2A cells. Downstream of Src, the CB1R also activated Rac1 and JNK, which enhanced CBR1-mediated Stat3 activation. Rac-DN blocked CB1R-induced activation of JNK. Pharmacological inhibition of JNK blocked Src and CB1R activation of Stat3, indicating that Rac and JNK are also involved in CB1R-mediated neurite outgrowth. Overall, this study demonstrated that G alpha(o/i)-coupled CB1R triggers neurite outgrowth in Neuro-2A through the activation of a signaling network containing two pathways that bifurcate at Src and converge at Stat3.     

Optogenetic control of motor coordination by Gi/o protein-coupled vertebrate rhodopsin in cerebellar Purkinje cells

G protein-coupled receptors are involved in the modulation of complex neuronal networks in the brain. To investigate the impact of a cell-specific G(i/o) protein-mediated signaling pathway on brain function, we created a new optogenetic mouse model in which the G(i/o) protein-coupled receptor vertebrate rhodopsin can be cell-specifically expressed with the aid of Cre recombinase. Here we use this mouse model to study the functional impact of G(i/o) modulation in cerebellar Purkinje cells (PCs). We show that in vivo light activation of vertebrate rhodopsin specifically expressed in PCs reduces simple spike firing that is comparable with the reduction in firing observed for the activation of cerebellar G(i/o)-coupled GABA(B) receptors. Notably, the light exposure of the cerebellar vermis in freely moving mice changes the motor behavior. Thus, our studies directly demonstrate that spike modulation via G(i/o)-mediated signaling in cerebellar PCs affects motor coordination and show a new promising approach for studying the physiological function of G protein-coupled receptor-mediated signaling in a cell type-specific manner.     

Regulation of α(2B)-adrenergic receptor-mediated extracellular signal-regulated kinase 1/2 (ERK1/2) activation by ADP-ribosylation factor 1

A number of signaling molecules are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway by G protein-coupled receptors. In this study, we have demonstrated that α(2B)-adrenergic receptor (α(2B)-AR) interacts with ADP-ribosylation factor 1 (ARF1), a small GTPase involved in vesicle-mediated trafficking, in an agonist activation-dependent manner and that the interaction is mediated through a unique double Trp motif in the third intracellular loop of the receptor. Interestingly, mutation of the double Trp motif and siRNA-mediated depletion of ARF1 attenuate α(2B)-AR-mediated activation of extracellular signal-regulated kinases 1/2 (ERK1/2) without altering receptor intracellular trafficking, whereas expression of the constitutively active mutant ARF1Q71L and ARNO, a GDP-GTP exchange factor of ARF1, markedly enhances the activation of Raf1, MEK1, and ERK1/2. These data strongly demonstrate that the small GTPase ARF1 modulates ERK1/2 activation by α(2B)-AR and provide the first evidence indicating a novel function for ARF1 in regulating the MAPK signaling pathway.     

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.     

Roles of phosphorylation-dependent and -independent mechanisms in the regulation of histamine H2 receptor by G protein-coupled receptor kinase 2

It is widely assumed that G protein-coupled receptor kinase 2 (GRK2)-mediated specific inhibition of G protein-coupled receptors (GPCRs) response involves GRK-mediated receptor phosphorylation followed by β-arrestin binding and subsequent uncoupling from the heterotrimeric G protein. It has recently become evident that GRK2-mediated GPCRs regulation also involves phosphorylation-independent mechanisms. In the present study we investigated whether the histamine H2 receptor (H2R), a Gα(s)-coupled GPCR known to be desensitized by GRK2, needs to be phosphorylated for its desensitization and/or internalization and resensitization. For this purpose we evaluated the effect of the phosphorylating-deficient GRK2K220R mutant on H2R signaling in U937, COS7, and HEK293T cells. We found that although this mutant functioned as dominant negative concerning receptor internalization and resensitization, it desensitized H2R signaling in the same degree as the GRK2 wild type. To identify the domains responsible for the kinase-independent receptor desensitization, we co-transfected the receptor with constructions encoding the GRK2 RGS-homology domain (RH) and the RH or the kinase domain fused to the pleckstrin-homology domain. Results demonstrated that the RH domain of GRK2 was sufficient to desensitize the H2R. Moreover, disruption of RGS functions by the use of GRK2D110A/K220R double mutant, although coimmunoprecipitating with the H2R, reversed GRK2K220R-mediated H2R desensitization. Overall, these results indicate that GRK2 induces desensitization of H2R through a phosphorylation-independent and RGS-dependent mechanism and extends the GRK2 RH domain-mediated regulation of GPCRs beyond Gα(q)-coupled receptors. On the other hand, GRK2 kinase activity proved to be necessary for receptor internalization and the resulting resensitization.     

Distinct roles for Galpha(i)2 and Gbetagamma in signaling to DNA synthesis and Galpha(i)3 in cellular transformation by dopamine D2S receptor activation in BALB/c 3T3 cells

Control of cell proliferation depends on intracellular mediators that determine the cellular response to external cues. In neuroendocrine cells, the dopamine D2 receptor short form (D2S receptor) inhibits cell proliferation, whereas in mesenchymal cells the same receptor enhances cell proliferation. Nontransformed BALB/c 3T3 fibroblast cells were stably transfected with the D2S receptor cDNA to study the G proteins that direct D2S signaling to stimulate cell proliferation. Pertussis toxin inactivates G(i) and G(o) proteins and blocks signaling of the D2S receptor in these cells. D2S receptor signaling was reconstituted by individually transfecting pertussis toxin-resistant Galpha(i/o) subunit mutants and measuring D2-induced responses in pertussis toxin-treated cells. This approach identified Galpha(i)2 and Galpha(i)3 as mediators of the D2S receptor-mediated inhibition of forskolin-stimulated adenylyl cyclase activity; Galpha(i)2-mediated D2S-induced stimulation of p42 and p44 mitogen-activated kinase (MAPK) and DNA synthesis, whereas Galpha(i)3 was required for formation of transformed foci. Transfection of toxin-resistant Galpha(i)1 cDNA induced abnormal cell growth independent of D2S receptor activation, while Galpha(o) inhibited dopamine-induced transformation. The role of Gbetagamma subunits was assessed by ectopic expression of the carboxyl-terminal domain of G protein receptor kinase to selectively antagonize Gbetagamma activity. Mobilization of Gbetagamma subunits was required for D2S-induced calcium mobilization, MAPK activation, and DNA synthesis. These findings reveal a remarkable and distinct G protein specificity for D2S receptor-mediated signaling to initiate DNA synthesis (Galpha(i)2 and Gbetagamma) and oncogenic transformation (Galpha(i)3), and they indicate that acute activation of MAPK correlates with enhanced DNA synthesis but not with transformation.     

Sprouty2-mediated inhibition of fibroblast growth factor signaling is modulated by the protein kinase DYRK1A

Raf-MEK-extracellular signal-regulated kinase (Erk) signaling initiated by growth factor-engaged receptor tyrosine kinases (RTKs) is modulated by an intricate network of positive and negative feedback loops which determine the specificity and spatiotemporal characteristics of the intracellular signal. Well-known antagonists of RTK signaling are the Sprouty proteins. The activity of Sprouty proteins is modulated by phosphorylation. However, little is known about the kinases responsible for these posttranslational modifications. We identify DYRK1A as one of the protein kinases of Sprouty2. We show that DYRK1A interacts with and regulates the phosphorylation status of Sprouty2. Moreover, we identify Thr75 on Sprouty2 as a DYRK1A phosphorylation site in vitro and in vivo. This site is functional, since its mutation enhanced the repressive function of Sprouty2 on fibroblast growth factor (FGF)-induced Erk signaling. Further supporting the idea of a functional interaction, DYRK1A and Sprouty2 are present in protein complexes in mouse brain, where their expression overlaps in several structures. Moreover, both proteins copurify with the synaptic plasma membrane fraction of a crude synaptosomal preparation and colocalize in growth cones, pointing to a role in nerve terminals. Our results suggest, therefore, that DYRK1A positively regulates FGF-mitogen-activated protein kinase signaling by phosphorylation-dependent impairment of the inhibitory activity of Sprouty2.     

Targeted deletion of LPA5 identifies novel roles for lysophosphatidic acid signaling in development of neuropathic pain

Lysophosphatidic acid (LPA) is a bioactive lipid that serves as an extracellular signaling molecule acting through cognate G protein-coupled receptors designated LPA(1-6) that mediate a wide range of both normal and pathological effects. Previously, LPA(1), a G(αi)-coupled receptor (which also couples to other G(α) proteins) to reduce cAMP, was shown to be essential for the initiation of neuropathic pain in the partial sciatic nerve ligation (PSNL) mouse model. Subsequent gene expression studies identified LPA(5), a G(α12/13)- and G(q)-coupled receptor that increases cAMP, in a subset of dorsal root ganglion neurons and also within neurons of the spinal cord dorsal horn in a pattern complementing, yet distinct from LPA(1), suggesting its possible involvement in neuropathic pain. We therefore generated an Lpar5 null mutant by targeted deletion followed by PSNL challenge. Homozygous null mutants did not show obvious base-line phenotypic defects. However, following PSNL, LPA(5)-deficient mice were protected from developing neuropathic pain. They also showed reduced phosphorylated cAMP response element-binding protein expression within neurons of the dorsal horn despite continued up-regulation of the characteristic pain-related markers Caα(2)δ(1) and glial fibrillary acidic protein, results that were distinct from those previously observed for LPA(1) deletion. These data expand the influences of LPA signaling in neuropathic pain through a second LPA receptor subtype, LPA(5), involving a mechanistically distinct downstream signaling pathway compared with LPA(1).     

Real-time analysis of the inside-out regulation of lymphocyte function-associated antigen-1 revealed similarities to and differences from very late antigen-4

Ten years ago, we introduced a fluorescent probe that shed light on the inside-out regulation of one of the major leukocyte integrins, very late antigen-4 (VLA-4, CD49d/CD29). Here we describe the regulation of another leukocyte integrin, lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18) using a novel small fluorescent probe in real time on live cells. We found that multiple signaling mechanisms regulate LFA-1 conformation in a manner analogous to VLA-4. LFA-1 can be rapidly activated by Gα(i)-coupled G protein-coupled receptors (GPCRs) and deactivated by Gα(s)-coupled GPCRs. The effects of Gα(s)-coupled GPCR agonists can be reversed in real time by receptor-specific antagonists. The specificity of the fluorescent probe binding has been assessed in a competition assay using the natural LFA-1 ligand ICAM-1 and the LFA-1-specific α I allosteric antagonist BIRT0377. Similar to VLA-4 integrin, modulation of the ligand dissociation rate can be observed for different LFA-1 affinity states. However, we also found a striking difference in the binding of the small fluorescent ligand. In the absence of inside-out activation ligand, binding to LFA-1 is extremely slow, at least 10 times slower than expected for diffusion-limited binding. This implies that an additional structural mechanism prevents ligand binding to inactive LFA-1. We propose that such a mechanism explains the inability of LFA-1 to support cell rolling, where the absence of its rapid engagement by a counterstructure in the inactive state leads to a requirement for a selectin-mediated rolling step.     

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.     

Heterotrimeric Gi/Go proteins modulate endothelial TLR signaling independent of the MyD88-dependent pathway

The innate immune recognition of bacterial lipopolysaccharide (LPS) is mediated by Toll-like receptor 4 (TLR4) and results in activation of proinflammatory signaling including NF-κB and MAPK pathways. Heterotrimeric G proteins have been previously implicated in LPS signaling in macrophages and monocytes. In the present study, we show that pertussis toxin sensitive heterotrimeric G proteins (Gα(i/o)) are involved in the activation of MAPK and Akt downstream of TLR2, TLR3, and TLR4 in endothelial cells. Gα(i/o) are also required for full activation of interferon signaling downstream of TLR3 and TLR4 but are not required for the activation of NF-κB. We find that Gα(i/o)-mediated activation of the MAPK is independent of the canonical MyD88, interleukin-1 receptor-associated kinase, and tumor necrosis factor receptor-associated factor 6 signaling cascade in LPS-stimulated cells. Taken together, the data presented here suggest that heterotrimeric G proteins are widely involved in TLR pathways along a signaling cascade that is distinct from MyD88-TRAF6.     

Cannabinoid receptor-induced neurite outgrowth is mediated by Rap1 activation through G(alpha)o/i-triggered proteasomal degradation of Rap1GAPII

The G(alpha)o/i-coupled CB1 cannabionoid receptor induces neurite outgrowth in Neuro-2A cells. The mechanisms of signaling through G(alpha)o/i to induce neurite outgrowth were studied. The expression of G(alpha)o/i reduces the stability of its direct interactor protein, Rap1GAPII, by targeting it for ubiquitination and proteasomal degradation. This results in the activation of Rap1. G(alpha)o/i-induced activation of endogenous Rap1 in Neuro-2A cells is blocked by the proteasomal inhibitor lactacystin. G(alpha)o/i stimulates neurite outgrowth that is blocked by the expression of dominant negative Rap1. Expression of Rap1GAPII also blocks the G(alpha)o/i-induced neurite outgrowth and treatment with proteasomal inhibitors potentiates this inhibition. The endogenous G(alpha)o/i-coupled cannabinoid (CB1) receptor in Neuro-2A cells stimulates the degradation of Rap1GAPII; activation of Rap1 and treatment with pertussis toxin or lactacystin blocks these effects. The CB1 receptor-stimulated neurite outgrowth is blocked by treatment with pertussis toxin, small interfering RNA for Rap, lactacystin, and expression of Rap1GAPII. Thus, the G(alpha)o/i-coupled cannabinoid receptor, by regulating the proteasomal degradation of Rap1GAPII, activates Rap1 to induce neurite outgrowth.     

Synergistic Ca2+ responses by G{alpha}i- and G{alpha}q-coupled G-protein-coupled receptors require a single PLC{beta} isoform that is sensitive to both G{beta}{gamma} and G{alpha}q

Cross-talk between Gα(i)- and Gα(q)-linked G-protein-coupled receptors yields synergistic Ca(2+) responses in a variety of cell types. Prior studies have shown that synergistic Ca(2+) responses from macrophage G-protein-coupled receptors are primarily dependent on phospholipase Cβ3 (PLCβ3), with a possible contribution of PLCβ2, whereas signaling through PLCβ4 interferes with synergy. We here show that synergy can be induced by the combination of Gβγ and Gα(q) activation of a single PLCβ isoform. Synergy was absent in macrophages lacking both PLCβ2 and PLCβ3, but it was fully reconstituted following transduction with PLCβ3 alone. Mechanisms of PLCβ-mediated synergy were further explored in NIH-3T3 cells, which express little if any PLCβ2. RNAi-mediated knockdown of endogenous PLCβs demonstrated that synergy in these cells was dependent on PLCβ3, but PLCβ1 and PLCβ4 did not contribute, and overexpression of either isoform inhibited Ca(2+) synergy. When synergy was blocked by RNAi of endogenous PLCβ3, it could be reconstituted by expression of either human PLCβ3 or mouse PLCβ2. In contrast, it could not be reconstituted by human PLCβ3 with a mutation of the Y box, which disrupted activation by Gβγ, and it was only partially restored by human PLCβ3 with a mutation of the C terminus, which partly disrupted activation by Gα(q). Thus, both Gβγ and Gα(q) contribute to activation of PLCβ3 in cells for Ca(2+) synergy. We conclude that Ca(2+) synergy between Gα(i)-coupled and Gα(q)-coupled receptors requires the direct action of both Gβγ and Gα(q) on PLCβ and is mediated primarily by PLCβ3, although PLCβ2 is also competent.     

Activation of B-Raf and regulation of the mitogen-activated protein kinase pathway by the G(o) alpha chain

Many receptors coupled to the pertussis toxin-sensitive G(i/o) proteins stimulate the mitogen-activated protein kinase (MAPK) pathway. The role of the alpha chains of these G proteins in MAPK activation is poorly understood. We investigated the ability of Galpha(o) to regulate MAPK activity by transient expression of the activated mutant Galpha(o)-Q205L in Chinese hamster ovary cells. Galpha(o)-Q205L was not sufficient to activate MAPK but greatly enhanced the response to the epidermal growth factor (EGF) receptor. This effect was not associated with changes in the state of tyrosine phosphorylation of the EGF receptor. Galpha(o)-Q205L also potentiated MAPK stimulation by activated Ras. In Chinese hamster ovary cells, EGF receptors activate B-Raf but not Raf-1 or A-Raf. We found that expression of activated Galpha(o) stimulated B-Raf activity independently of the activation of the EGF receptor or Ras. Inactivation of protein kinase C and inhibition of phosphatidylinositol-3 kinase abolished both B-Raf activation and EGF receptor-dependent MAPK stimulation by Galpha(o). Moreover, Galpha(o)-Q205L failed to affect MAPK activation by fibroblast growth factor receptors, which stimulate Raf-1 and A-Raf but not B-Raf activity. These results suggest that Galpha(o) can regulate the MAPK pathway by activating B-Raf through a mechanism that requires a concomitant signal from tyrosine kinase receptors or Ras to efficiently stimulate MAPK activity. Further experiments showed that receptor-mediated activation of Galpha(o) caused a B-Raf response similar to that observed after expression of the mutant subunit. The finding that Galpha(o) induces Ras-independent and protein kinase C- and phosphatidylinositol-3 kinase-dependent activation of B-Raf and conditionally stimulates MAPK activity provides direct evidence for intracellular signals connecting this G protein subunit to the MAPK pathway.