Sphingosine 1-phosphate-induced signal transduction in cat esophagus smooth muscle cells

We investigated the mechanism of contraction induced by S1P in esophageal smooth muscle cells. Western blot analysis demonstrated that S1P(1), S1P(2), S1P(3), and S1P(5) receptors existed in the cat esophagus. Only penetration of EDG-5 (S1P(2)) antibody into permeabilized cells inhibited S1P-induced contraction. Pertussis toxin (PTX) also inhibited contraction, suggesting that it was mediated by S1P(2) receptors coupled to a PTX-sensitive G(i) protein. Specific antibodies to G(i2), G(q) and G(beta) inhibited contraction, implying that the S1P-induced contraction depends on PTX-insensitive G(q) and G(beta) dimers as well as the PTX-sensitive G(i2). Contraction was not affected by the phospholipase A2 inhibitor DEDA, or the PLD inhibitor rho-chloromer-curibenzoate, but it was abolished by the PLC inhibitor U73122. Incubation of permeabilized cells with PLCb3 antibody also inhibited contraction. Contraction involved the activation of a PKC pathway since it was affected by GF109203X and chelerythrine. Since PKCepsilon antibody inhibited contraction, PKCe may be required. Preincubation of the muscle cells with the MEK inhibitor PD98059 blocked S1P-induced contraction, but the p38 MAP kinase inhibitor SB202190 did not. In addition, co-treatment of cells with GF 109203X and PD98059 did not have a synergistic effect, suggesting that these two kinases are involved in the same signaling pathway. Our data suggest that S1P-induced contraction in esophageal smooth muscle cells is mediated by S1P(2) receptors coupled to PTX-sensitive G(i2) proteins, and PTX-insensitive G(q) and G(beta) proteins, and that the resulting activation of the PLCb3 and PKCepsilon pathway leads to activation of a p44/p42 MAPK pathway.     

Receptor-mediated inhibition of G protein-coupled inwardly rectifying potassium channels involves G(alpha)q family subunits, phospholipase C, and a readily diffusible messenger

G protein-coupled inwardly rectifying K+ (GIRK) channels can be activated or inhibited by distinct classes of receptor (G(alpha)i/o- and G(alpha)q-coupled), providing dynamic regulation of cellular excitability. Receptor-mediated activation involves direct effects of G(beta)gamma subunits on GIRK channels, but mechanisms involved in GIRK channel inhibition have not been fully elucidated. An HEK293 cell line that stably expresses GIRK1/4 channels was used to test G protein mechanisms that mediate GIRK channel inhibition. In cells transiently or stably cotransfected with 5-HT1A (G(alpha)i/o-coupled) and TRH-R1 (G(alpha)q-coupled) receptors, 5-HT (5-hydroxytryptamine; serotonin) enhanced GIRK channel currents, whereas thyrotropin-releasing hormone (TRH) inhibited both basal and 5-HT-activated GIRK channel currents. Inhibition of GIRK channel currents by TRH primarily involved signaling by G(alpha)q family subunits, rather than G(beta)gamma dimers: GIRK channel current inhibition was diminished by Pasteurella multocida toxin, mimicked by constitutively active members of the G(alpha)q family, and reduced by minigene constructs that disrupt G(alpha)q signaling, but was completely preserved in cells expressing constructs that interfere with signaling by G(beta)gamma subunits. Inhibition of GIRK channel currents by TRH and constitutively active G(alpha)q was reduced by, an inhibitor of phospholipase C (PLC). Moreover, TRH- R1-mediated GIRK channel inhibition was diminished by minigene constructs that reduce membrane levels of the PLC substrate phosphatidylinositol bisphosphate, further implicating PLC. However, we found no evidence for involvement of protein kinase C, inositol trisphosphate, or intracellular calcium. Although these downstream signaling intermediaries did not contribute to receptor-mediated GIRK channel inhibition, bath application of TRH decreased GIRK channel activity in cell-attached patches. Together, these data indicate that receptor-mediated inhibition of GIRK channels involves PLC activation by G(alpha) subunits of the G(alpha)q family and suggest that inhibition may be communicated at a distance to GIRK channels via unbinding and diffusion of phosphatidylinositol bisphosphate away from the channel.     

Down-regulation of GABA(A) receptor via promiscuity with the vasoactive peptide urotensin II receptor. Potential involvement in astrocyte plasticity

GABA(A) receptor (GABA(A)R) expression level is inversely correlated with the proliferation rate of astrocytes after stroke or during malignancy of astrocytoma, leading to the hypothesis that GABA(A)R expression/activation may work as a cell proliferation repressor. A number of vasoactive peptides exhibit the potential to modulate astrocyte proliferation, and the question whether these mechanisms may imply alteration in GABA(A)R-mediated functions and/or plasma membrane densities is open. The peptide urotensin II (UII) activates a G protein-coupled receptor named UT, and mediates potent vasoconstriction or vasodilation in mammalian vasculature. We have previously demonstrated that UII activates a PLC/PIPs/Ca(2+) transduction pathway, via both G(q) and G(i/o) proteins and stimulates astrocyte proliferation in culture. It was also shown that UT/G(q)/IP(3) coupling is regulated by the GABA(A)R in rat cultured astrocytes. Here we report that UT and GABA(A)R are co-expressed in cerebellar glial cells from rat brain slices, in human native astrocytes and in glioma cell line, and that UII inhibited the GABAergic activity in rat cultured astrocytes. In CHO cell line co-expressing human UT and combinations of GABA(A)R subunits, UII markedly depressed the GABA current (β(3)γ(2)>α(2)β(3)γ(2)>α(2)β(1)γ(2)). This effect, characterized by a fast short-term inhibition followed by drastic and irreversible run-down, is not relayed by G proteins. The run-down partially involves Ca(2+) and phosphorylation processes, requires dynamin, and results from GABA(A)R internalization. Thus, activation of the vasoactive G protein-coupled receptor UT triggers functional inhibition and endocytosis of GABA(A)R in CHO and human astrocytes, via its receptor C-terminus. This UII-induced disappearance of the repressor activity of GABA(A)R, may play a key role in the initiation of astrocyte proliferation.     

Cell cycle-dependent coupling of the vasopressin V1a receptor to different G proteins

Arginine vasopressin (AVP) regulates biological processes by binding to G protein-coupled receptors. In Swiss 3T3 fibroblasts, expressing the V(1a) subtype of vasopressin receptors, AVP mobilizes calcium from intracellular stores. In proliferating cells, the AVP-induced increase in intracellular calcium concentration ([Ca(2+)](i)) was mediated by G proteins of the G(q) family, which are insensitive to pertussis toxin (PTX) pretreatment of the cells. In quiescent cells, the AVP-induced increase in [Ca(2+)](i) was partially PTX-sensitive, suggesting an involvement of G(i) proteins. We confirmed this by photoaffinity labeling of G proteins in Swiss 3T3 cell membranes activated by AVP. In Swiss 3T3 cells arrested in the G(0)/G(1) phase of the cell cycle, the AVP-induced increase in [Ca(2+)](i) was also partially PTX-sensitive but was PTX-insensitive in cells arrested in other phases of the cell cycles. The blocking effect of PTX pretreatment in G(0)/G(1) cells was mimicked by microinjection of antisense oligonucleotides suppressing the expression of the Galpha(i3) subunits. These results were confirmed by microinjection of antibodies directed against the C terminus of G protein alpha-subunits. The data presented indicate that in Swiss 3T3 fibroblasts synchronized in the G(0)/G(1) phase of the cell cycle the V(1a) receptor couples to G(q/11) and G(i3) to activate the phospholipase C-beta, leading to release of intracellular calcium.     

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

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

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.     

Constitutively active Gq/11-coupled receptors enable signaling by co-expressed G(i/o)-coupled receptors

Co-expression of guanine nucleotide-binding regulatory (G) protein-coupled receptors (GPCRs), such as the G(i/o)-coupled human 5-hydroxytryptamine receptor 1B (5-HT(1B)R), with the G(q/11)-coupled human histamine 1 receptor (H1R) results in an overall increase in agonist-independent signaling, which can be augmented by 5-HT(1B)R agonists and inhibited by a selective inverse 5-HT(1B)R agonist. Interestingly, inverse H1R agonists inhibit constitutively H1R-mediated as well as 5-HT(1B)R agonist-induced signaling in cells co-expressing both receptors. This phenomenon is not solely characteristic of 5-HT(1B)R; it is also evident with muscarinic M2 and adenosine A1 receptors and is mimicked by mastoparan-7, an activator of G(i/o) proteins, or by over-expression of Gbetagamma subunits. Likewise, expression of the G(q/11)-coupled human cytomegalovirus (HCMV)-encoded chemokine receptor US28 unmasks a functional coupling of G(i/o)-coupled CCR1 receptors that is mediated via the constitutive activity of receptor US28. Consequently, constitutively active G(q/11)-coupled receptors, such as the H1R and HCMV-encoded chemokine receptor US28, constitute a regulatory switch for signal transduction by G(i/o)-coupled receptors, which may have profound implications in understanding the role of both constitutive GPCR activity and GPCR cross-talk in physiology as well as in the observed pathophysiology upon HCMV infection.     

Galpha(q/11) and gbetagamma proteins and membrane signaling of calcitriol and estradiol.

17beta-estradiol and 1,25-dihydroxyvitamin D(3)()(calcitriol) rapidly increase (< 5 sec) the concentration of intracellular calcium by mobilizing Ca(2+) from the endoplasmic reticulum and forming inositol 1,4,5-trisphosphate (InsP(3)) and diacylglycerol. Calcitriol increases InsP(3) formation via activation of phospholipase C (PLC)-beta1 linked to a pertussis toxin (PTX)-insensitive G-protein, and estradiol via activation of PLC-beta2 linked to a PTX-sensitive G-protein. Since PLC are effectors of different subunits of various G-proteins, we looked for and identified several G-subunits (Galpha(q/11), Galphas, Galphai, Gbeta and Ggamma) in female rat osteoblasts using Western immunoblotting. The action of calcitriol on InsP(3) formation and Ca(2+) mobilization in Fura-2-loaded confluent osteoblasts involved Galpha(q/11). The membrane effects of estradiol involved Gbetagamma; subunits, and principally Gbeta subunits, but not alpha-subunits. These results may provide additional evidence for membrane receptors of steroid hormones. Since PLC-beta1 is the target effector of Galpha(q/11), whereas PLC-beta2 is only activated by betagamma subunits, this specificity may help to generate membrane receptor-specific responses in vivo.     

G alpha(q/11) coupled to mammalian phospholipase C beta 3-like enzyme mediates the ginsenoside effect on Ca(2+)-activated Cl(-) current in the Xenopus oocyte.

Recently we demonstrated that ginsenosides, the active ingredients of Panax ginseng, enhanced Ca(2+)-activated Cl(-) current in the Xenopus oocyte through a signal transduction mechanism involving the activation of pertussis toxin-insensitive G protein and phospholipase C (PLC). However, it has not yet been determined precisely which G protein subunit(s) and which PLC isoform(s) participate in the ginsenoside signaling. To provide answers to these questions, we investigated the changes in ginsenoside effect on the Cl(-) current after intraoocyte injections of the cRNAs coding various G protein subunits, a regulator of G protein signaling (RGS2), and G beta gamma-binding proteins. In addition, we examined which of mammalian PLC beta 1-3 antibodies injected into the oocyte inhibited the action of ginsenosides on the Cl(-) current. Injection of G alpha(q) or G alpha(11) cRNA increased the basal Cl(-) current recorded 48 h after, and it further prevented ginsenosides from enhancing the Cl(-) current, whereas G alpha(i2) and G alpha(oA) cRNA injection had no significant effect. The changes following G alpha(q) cRNA injection were prevented when G beta(1)gamma(2) and G alpha(q) subunits were co-expressed by simultaneous injection of the cRNAs coding these subunits. Injection of cRNA coding G alpha(q)Q209L, a constitutively active mutant that does not bind to G beta gamma, produced effects similar to those of G alpha(q) cRNA injection. The effects of G alpha(q)Q209L cRNA injection, however, were not prevented by co-injection of G beta(1)gamma(2) cRNA. Injection of the cRNA coding RGS2, which interacts most selectively with G alpha(q/11) among various identified RGS isoforms and stimulates the hydrolysis of GTP to GDP in active GTP-bound G alpha subunit, resulted in a severe attenuation of ginsenoside effect on the Cl(-) current. Finally, antibodies against PLC beta 3, but not -beta 1 and -beta 2, markedly attenuated the ginsenoside effect examined at 3-h postinjection. These results suggest that G alpha(q/11) coupled to mammalian PLC beta 3-like enzyme mediates ginsenoside effect on Ca(2+)-activated Cl(-) current in the Xenopus oocyte.     

Eliminating phosphorylation sites of the parathyroid hormone receptor type 1 differentially affects stimulation of phospholipase C and receptor internalization

The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTH1R) belongs to family B of seven-transmembrane-spanning receptors and is activated by PTH and PTHrP. Upon PTH stimulation, the rat PTH1R becomes phosphorylated at seven serine residues. Elimination of all PTH1R phosphorylation sites results in prolonged cAMP accumulation and impaired internalization in stably transfected LLC-PK1 cells. The present study explores the role of individual PTH1R phosphorylation sites in PTH1R signaling through phospholipase C, agonist-dependent receptor internalization, and regulation by G protein-coupled receptor kinases. By means of transiently transfected COS-7 cells, we demonstrate that the phosphorylation-deficient (pd) PTH1R confers dramatically enhanced coupling to G(q/11) proteins upon PTH stimulation predominantly caused by elimination of Ser(491/492/493), Ser(501), or Ser(504). Reportedly, impaired internalization of the pd PTH1R, however, is not dependent on a specific phosphorylation site. In addition, we show that G protein-coupled receptor kinase 2 interferes with pd PTH1R signaling to G(q/11) proteins at least partially by direct binding to G(q/11) proteins.     

G protein beta 5 subunit interactions with alpha subunits and effectors

When the beta(5) (short form) and gamma(2) subunits of heterotrimeric G proteins were expressed with hexahistidine-tagged alpha(i) in insect cells, a heterotrimeric complex was formed that bound to a Ni-NTA-agarose affinity matrix. Binding to the Ni-NTA-agarose column was dependent on expression of hexahistidine-tagged alpha(i) and resulted in purification of beta(5)gamma(2) to near homogeneity. Subsequent anion-exchange chromatography of beta(5)gamma(2) resulted in resolution of beta(5) from gamma(2) and further purification of beta(5). The purified beta(5) eluted as a monomer from a size-exclusion column and was resistant to trypsin digestion suggesting that it was stably folded in the absence of gamma. beta(5) monomer could be assembled with partially purified hexahistidine-tagged gamma(2) in vitro to form a functional dimer that could selectively activate PLC beta2 but not PLC beta3. alpha(o)-GDP inhibited activation of PLC beta2 by beta(5)gamma(2) supporting the idea that beta(5)gamma(2) can bind to alpha(o). beta(5) monomer and beta(5)gamma(2) only supported a small degree of ADP ribosylation of alpha(i) by pertussis toxin (PTX), but beta(5) monomer was able to compete for beta(1)gamma(2) binding to alpha(i) and alpha(o) to inhibit PTX-catalyzed ADP ribosylation. These data indicate that beta(5) functionally interacts with PTX-sensitive GDP alpha subunits and that beta(5) subunits can be assembled with gamma subunits in vitro to reconstitute activity and also support the idea that there are determinants on beta subunits that are selective for even very closely related effectors.     

Constitutive signaling of the human cytomegalovirus-encoded chemokine receptor US28

Previously it was shown that the HHV-8-encoded chemokine receptor ORF74 shows considerable agonist-independent, constitutive activity giving rise to oncogenic transformation (Arvanitakis, L., Geras-Raaka, E., Varma, A., Gershengorn, M. C., and Cesarman, E. (1997) Nature 385, 347-350). In this study we report that a second viral-encoded chemokine receptor, the human cytomegalovirus-encoded US28, also efficiently signals in an agonist-independent manner. Transient expression of US28 in COS-7 cells leads to the constitutive activation of phospholipase C and NF-kappaB signaling via G(q/11) protein-dependent pathways. Whereas phospholipase C activation is mediated via Galpha(q/11) subunits, the activation of NF-kappaB strongly depends on betagamma subunits with a preference for the beta(2)gamma(1) dimer. The CC chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and MCP-1 (monocyte chemotactic protein-1) act as neutral antagonists at US28, whereas the CX(3)C chemokine fractalkine acts as a partial inverse agonist with IC(50) values of 1-5 nm. Our data suggest that a high level of constitutive activity might be a more general characteristic of viral G protein-coupled receptors and that human cytomegalovirus might exploit this G protein-coupled receptor property to modulate the homeostasis of infected cells via the early gene product US28.     

A novel mechanism for JAK2 activation by a G protein-coupled receptor, the CCK2R: implication of this signaling pathway in pancreatic tumor models

To date very few G protein-coupled receptors (GPCRs) have been shown to be connected to the Janus kinase (JAK)/STAT pathway. Thus our understanding of the mechanisms involved in the activation of this signaling pathway by GPCRs remains limited. In addition, little is known about the role of the JAK pathway in the physiological or pathophysiological functions of GPCRs. Here, we described a new mechanism of JAK activation that involves Galpha(q) proteins. Indeed, transfection of a constitutively activated mutant of Galpha(q) (Q209L) in COS-7 cells demonstrated that Galpha(q) is able to associate and activate JAK2. In addition, we showed that this mechanism is used to activate JAK2 by a GPCR principally coupled to G(q), the CCK2 receptor (CCK2R), and involves a highly conserved sequence in GPCRs, the NPXXY motif. In a pancreatic tumor cell line expressing the endogenous CCK2R, we demonstrated the activation of the JAK2/STAT3 pathway by this receptor and the involvement of this signaling pathway in the proliferative effects of the CCK2R. In addition, we showed in vivo that the targeted CCK2R expression in pancreas of Elas-CCK2 mice leads to the activation of JAK2 and STAT3. This process may contribute to the increase of pancreas growth as well as the formation of preneoplastic lesions leading to pancreatic tumor development observed in these transgenic animals.     

Selective regulation of Galpha(q/11) by an RGS domain in the G protein-coupled receptor kinase, GRK2

G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein alpha subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF(4)(-)-dependent binding to G protein alpha subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF(4)(-). This revealed the specific ability of bovine brain Galpha(q/11) to bind to both GRK2 and GRK3 in an AlF(4)(-)-dependent manner. In contrast, Galpha(s), Galpha(i), and Galpha(12/13) did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain Galpha(q/11) could also bind to an N-terminal construct of GRK2, while no binding of Galpha(q/11), Galpha(s), Galpha(i), or Galpha(12/13) to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Galpha(q) revealed significant binding of both Galpha(q) GDP/AlF(4)(-) and Galpha(q)(GTPgammaS), but not Galpha(q)(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Galpha(q)(R183C) but not wild type Galpha(q). In vitro analysis revealed that GRK2 possesses weak GAP activity toward Galpha(q) that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Galpha(q)-mediated activation of phospholipase C-beta both in vitro and in cells, possibly through sequestration of activated Galpha(q). These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.     

RGS protein specificity towards Gq- and Gi/o-mediated ERK 1/2 and Akt activation, in vitro

Extracellular Regulated Kinases (ERK) and Protein Kinase B (Akt) are intermediaries in relaying extracellular growth signals to intracellular targets. Each pathway can become activated upon stimulation of G protein-coupled receptors mediated by G(q) and G(i/o) proteins subjected to regulation by RGS proteins. The goal of the study was to delineate the specificity in which cardiac RGS proteins modulate G(q)and G(i/o)-induced ERK and Akt phosphorylation. To isolate G(q)- and G(i/o)-mediated effects, we exclusively expressed muscarinic M(2) or M(3) receptors in COS-7 cells. Western blot analyses demonstrated increase of phosphorylation of ERK 1.7-/3.3-fold and Akt 2.4-/6-fold in M(2)-/M(3)- expressing cells through carbachol stimulation. In co-expressions, M(3)/G(q)-induced activation of Akt was exclusively blunted through RGS3s/RGS3, whereas activation of ERK was inhibited additionally through RGS2/RGS5. M(2)/G(i/o) induced Akt activation was inhibited by all RGS proteins tested. RGS2 had no effect on M(2)/G(i/o)-induced ERK activation. The high degree of specificity in RGS proteins-depending modulation of G(q)- and G(i/o)-mediated ERK and Akt activation in the muscarinic network cannot merely be attributed exclusively to RGS protein selectivity towards G(q) or G(i/o) proteins. Counter-regulatory mechanisms and inter-signaling cross-talk may alter the sensitivity of GPCR-induced ERK and Akt activation to RGS protein regulation.     

Cannabinoid receptor 1 induces a biphasic ERK activation via multiprotein signaling complex formation of proximal kinases PKCε, Src, and Fyn in primary neurons

Cannabinoid receptors 1 (CB1Rs) play important roles in the regulation of dendritic branching, synapse density, and synaptic transmission through multiple G-protein-coupled signaling systems, including the activation of the extracellular signal-regulated kinases ERK1/2. The proximal signaling interactions leading to ERK1/2 activation by CB1R in CNS remain, however, unclear. Here, we present evidence that the CB1R agonist methanandamide induced a biphasic and sustained activation of ERK1/2 in primary neurons derived from E7 telencephalon. We show that E7 neurons natively express high levels of CB1R message and protein, the majority of which associates with PKC? at basal conditions. We now demonstrate that the first peak of ERK activation by CB1R was mediated by the sequential activation of G(q), PLC, and PKC?, selectively, and that the CB1R-activated PKC? acutely formed transient signaling modules containing activated Src and Fyn. A second pool of CB1Rs, coupled to PTX-sensitive activation of G(i/o), utilized as effectors additional Src and Fyn molecules to generate a second, additional wave of ERK activation at 15 min. Concurrently to these intermolecular signaling interactions, cytoskeleton-associated proteins MARCKS and p120catenin were drastically modified by phosphorylation of PKC and Src, respectively. These receptor-proximal signaling events correlated well with induction of neuritic outgrowth in the long term. Our data provide evidence for multiprotein signaling complex formation in the coupling of CB1R to activation of ERK in CNS neurons, and may elucidate several of the less understood acute effects of cannabinoid drugs.