The time-course of agonist-induced solubilization of trimeric G(q)alpha/G(11)alpha proteins resolved by two-dimensional electrophoresis

Prolonged agonist stimulation results in specific transfer of activated Galpha subunits of G(q)alpha/G(11)alpha family from particulate membrane fraction to soluble (cytosol) cell fraction isolated as 250,000 x g supernatant. In this study, we have used 2D electrophoresis for more defined resolution of Galpha subunits of G(q)alpha/G(11)alpha family and followed the time course of solubilization effect. The small signal of soluble G proteins was already detected in control, hormone-unexposed cells. Hormone stimulation resulted in a slow but continuous increase of both intensity and number of immunoreactive signals/spots of these G proteins (10, 30, 60, 120 and 240 min). At longer times of agonist exposure (>2 hours), a marked increase of G(q)alpha/G(11)alpha proteins was detected. The maximal level of soluble G(q)alpha/G(11)alpha proteins was reached after 16 hours of continuous agonist exposure. At this time interval, eight individual immunoreactive signals of G(q)alpha/G(11)alpha proteins could be resolved. The relative proportion among these spots was 15:42:10:11:7:7:2:5. Solubilization of this class of Galpha proteins was thus observed after prolonged agonist stimulation only, induced by ultra high concentration of hormone and in cells expressing a large number of GPCRs. Our data therefore rather indicate tight/persisting binding of G(q)alpha/G(11)alpha proteins to the membrane.     

Differential effects of RGS proteins on G alpha(q) and G alpha(11) activity

Heterotrimeric G proteins play a pivotal role in GPCR signalling; they link receptors to intracellular effectors and their inactivation by RGS proteins is a key factor in resetting the pathway following stimulation. The precise GPCR:G protein:RGS combination determines the nature and duration of the response. Investigating the activity of particular combinations is difficult in cells which contain multiples of each component. We have therefore utilised a previously characterised yeast system to express mammalian proteins in isolation. Human G alpha(q) and G alpha(11) spontaneously activated the yeast pheromone-response pathway by a mechanism which required the formation of G alpha-GTP. This provided an assay for the specific activity of human RGS proteins. RGS1, RGS2, RGS3 and RGS4 inhibited the spontaneous activity of both G alpha(q) and G alpha(11) but, in contrast, RGS5 and RGS16 were much less effective against G alpha(11) than G alpha(q). Interestingly, RGS2 and RGS3 were able to inhibit signalling from the constitutively active G alpha(q)QL/G alpha(11)QL mutants, confirming the GAP-independent activity of these RGS proteins. To determine if the RGS-G alpha specificity was maintained under conditions of GPCR stimulation, minor modifications to the C-terminus of G alpha(q)/G alpha(11) enabled coupling to an endogenous receptor. RGS2 and RGS3 were effective inhibitors of both G alpha subunits even at high levels of receptor stimulation, emphasising their GAP-independent activity. At low levels of stimulation RGS5 and RGS16 retained their differential G alpha activity, further highlighting that RGS proteins can discriminate between two very closely related G alpha subunits.     

Activation of phospholipase C by the alpha subunits of the Gq and G11 proteins in transfected Cos-7 cells.

High efficiency transient transfection was used to introduce cDNA corresponding to various G protein alpha subunits into Cos-7 cells. The proteins that were subsequently synthesized were detected with specific G protein alpha subunit antipeptide antiserum and were localized in the membrane fraction of the cell. Cells that were prelabeled with the [3H]inositol and transfected with G alpha q and G alpha 11 cDNA showed marked increases in formation of [3H]inositol phosphates after stimulation with aluminum fluoride. Co-transfection with cDNAs corresponding to phosphoinositide specific phospholipase C beta 1 (PI-PLC beta 1) and to G alpha q or G alpha 11 resulted in even higher levels of inositol phosphate formation. The introduction of mutations that convert residue glutamine 209 to leucine in G alpha q and G alpha 11 resulted in persistent activation of PI-PLC and high steady state levels of inositol phosphates. On the other hand, transfection with a variety of other G alpha subunit cDNAs, i.e. G alpha Z, G alpha OA, G alpha OB, transducin, and the glutamine 205 to leucine mutants of G alpha Z and of G alpha OA did not increase inositol phosphate formation. To further test the specificity of G protein activation of PI-PLC, a cell-free system was prepared by using washed membranes of transiently transfected cells and purified PI-PLC beta 1. Membranes derived from G alpha q and G alpha 11, but not G alpha OA transfected cells, showed guanosine 5-O-thiotriphosphate (GTP gamma S)-stimulated PIP2 hydrolysis. The activity seen in the system reconstituted with membranes derived from G alpha 11-transfected cells was blocked by preincubation with specific G alpha 11 antipeptide antibodies. All of these results are consistent with the conclusion that G alpha q and G alpha 11 cDNA encode proteins that in the presence of GTP gamma S specifically activate PI-PLC.     

eat-11 encodes GPB-2, a Gbeta(5) ortholog that interacts with G(o)alpha and G(q)alpha to regulate C. elegans behavior

In C. elegans, a G(o)/G(q) signaling network regulates locomotion and egg laying [1-8]. Genetic analysis shows that activated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is suppressed by perturbations of this network, which include loss of the GOA-1 G(o)alpha, DGK-1 diacylglycerol kinase, EAT-16 G protein gamma subunit-like (GGL)-containing RGS protein, or an unidentified protein encoded by the gene eat-11 [9]. We cloned eat-11 and report that it encodes the Gbeta(5) ortholog GPB-2. Gbeta(5) binds specifically to GGL-containing RGS proteins, and the Gbeta(5)/RGS complex can promote the GTP-hydrolyzing activity of Galpha subunits [10, 11]. However, little is known about how this interaction affects G protein signaling in vivo. In addition to EAT-16, the GGL-containing RGS protein EGL-10 participates in G(o)/G(q) signaling; EGL-10 appears to act as an RGS for the GOA-1 G(o)alpha, while EAT-16 appears to act as an RGS for the EGL-30 G(q)alpha [4, 5]. We have combined behavioral, electrophysiological, and pharmacological approaches to show that GPB-2 is a central member of the G(o)/G(q) network and that GPB-2 may interact with both the EGL-10 and EAT-16 RGS proteins to mediate the opposing activities of G(o)alpha and G(q)alpha. These interactions provide a mechanism for the modulation of behavior by antagonistic G protein networks.     

Protein kinase A-mediated phosphorylation of serine 357 of the mouse prostacyclin receptor regulates its coupling to G(s)-, to G(i)-, and to G(q)-coupled effector signaling.

The prostacyclin receptor (IP) is primarily coupled to G alpha(s)-dependent activation of adenylyl cyclase; however, a number of studies indicate that the IP may couple to other secondary effector systems perhaps in a species-specific manner. In the current study, we investigated the specificity of G protein:effector coupling by the mouse (m) IP overexpressed in human embryonic kidney 293 cells and endogenously expressed in murine erythroleukemia cells. The mIP exhibited efficient G alpha(s) coupling and concentration-dependent increases in cAMP generation in response to the IP agonist cicaprost; however, mIP also coupled to G alpha(i) decreasing the levels of cAMP in forskolin-treated cells. mIP coupling to G alpha(i) was pertussis toxin-sensitive and was dependent on protein kinase (PK) A activation status. In addition, the mIP coupled to phospholipase C (PLC) activation in a pertussis toxin-insensitive, G alpha(i)-, G beta gamma-, and PKC-independent but in a G alpha(q)- and PKA-dependent manner. Whole cell phosphorylation assays demonstrated that the mIP undergoes cicaprost-induced PKA phosphorylation. mIP(S357A), a site-directed mutant of mIP, efficiently coupled to G alpha(s) but failed to couple to G alpha(i) or to efficiently couple to G alpha(q):PLC. Moreover, mIP(S357A) did not undergo cicaprost-induced phosphorylation confirming that Ser(357) is the target residue for PKA-dependent phosphorylation. Finally, co-precipitation experiments permitted the detection of G alpha(s), G alpha(i), and G alpha(q) in the immunoprecipitates of mIP, whereas only G alpha(s) was co-precipitated with mIP(S357A) indicating that Ser(357) of mIP is essential for G alpha(i) and G alpha(q) interaction. Moreover, inhibition of PKA blocked co-precipitation of mIP with G alpha(i) or G alpha(q). Taken together our data indicate that the mIP, in addition to coupling to G alpha(s), couples to G alpha(i) and G alpha(q); however, G alpha(i) and G alpha(q) coupling is dependent on initial cicaprost-induced mIP:G alpha(s) coupling and phosphorylation of mIP by cAMP-dependent PKA where Ser(357) was identified as the target residue for PKA phosphorylation.     

Stable association between G alpha(q) and phospholipase C beta 1 in living cells

Signal transduction through G alpha(q) involves stimulation of phospholipase C beta (PLC beta) that results in increased intracellular Ca2+ and activation of protein kinase C. We have measured complex formation between G alpha(q) and PLC beta1 in vitro and in living PC12 and HEK293 cells by fluorescence resonance energy transfer. In vitro measurements show that PLC beta1 will bind to G alpha(q)(guanosine 5'-3-O-(thio)triphosphate) and also to G alpha(q)(GDP), and the latter association has a different protein-protein orientation. In cells, image analysis of fluorescent-tagged proteins shows that G alpha(q) is localized almost entirely to the plasma membrane, whereas PLC beta1 has a significant cytosolic population. By using fluorescence resonance energy transfer, we found that these proteins are pre-associated in the unstimulated state in PC12 and HEK293 cells. By determining the cellular levels of the two proteins in transfected versus nontransfected cells, we found that under our conditions overexpression should not significantly promote complex formation. G alpha(q)-PLC beta1 complexes are observed in both single cell measurements and measurements of a large (i.e. 10(6)) cell suspension. The high level (approximately 40% maximum) of FRET is surprising considering that G alpha(q) is more highly expressed than PLC beta1 and that not all PLC beta1 is plasma membrane-localized. Our measurements suggest a model in which G proteins and effectors can exist in stable complexes prior to activation and that activation is achieved through changes in intermolecular interactions rather than diffusion and association. These pre-formed complexes in turn give rise to rapid, localized signals.     

Disruption of the plasma membrane integrity by cholesterol depletion impairs effectiveness of TRH receptor-mediated signal transduction via G(q)/G(11)alpha proteins

We monitored the radioligand-binding characteristics of thyrotropin-releasing hormone (TRH) receptors, functional activity of G(q/11)alpha proteins, and functional status of the whole signaling cascade in HEK293 expressing high levels of TRH receptors and G(11)alpha. Our analyses indicated that disruption of plasma membrane microdomains by cholesterol depletion did not markedly influence the binding parameters of TRH receptors, but it altered efficacy of signal transduction. The functional coupling between TRH receptor and G(q/11)alpha was assessed by agonist-stimulated [(35)S]GTPgammaS binding, and results of these measurements pointed out to significantly lower potency of TRH to mediate G protein activation in the plasma membrane fraction isolated from cholesterol-depleted cells; there was a shift in sensitivity by one order of magnitude to the higher concentrations. A markedly lower sensitivity to stimulation with TRH was also observed in our experiments dealing with determination of hormone-induced Ca(2+) response. These data suggest that the intact structure of plasma membranes is an important optimum signal transduction initiated by TRH receptors and mediated by G(q/11)alpha proteins.     

Coordinated agonist regulation of receptor and G protein palmitoylation and functional rescue of palmitoylation-deficient mutants of the G protein G11alpha following fusion to the alpha1b-adrenoreceptor: palmitoylation of G11alpha is not required for interaction with beta*gamma complex.

Transfection of either the alpha(1b)-adrenoreceptor or Galpha(11) into a fibroblast cell line derived from a Galpha(q)/Galpha(11) double knockout mouse failed to produce elevation of intracellular [Ca(2+)] upon the addition of agonist. Co-expression of these two polypeptides, however, produced a significant stimulation. Co-transfection of the alpha(1b)-adrenoreceptor with the palmitoylation-resistant C9S,C10S Galpha(11) also failed to produce a signal, and much reduced and kinetically delayed signals were obtained using either C9S Galpha(11) or C10S Galpha(11). Expression of a fusion protein between the alpha(1b)-adrenoreceptor and Galpha(11) allowed [Ca(2+)](i) elevation, and this was also true for a fusion protein between the alpha(1b)-adrenoreceptor and C9S,C10S Galpha(11), since this strategy ensures proximity of the two polypeptides at the cell membrane. For both fusion proteins, co-expression of transducin alpha, as a beta.gamma-sequestering agent, fully attenuated the Ca(2+) signal. Both of these fusion proteins and one in which an acylation-resistant form of the receptor was linked to wild type Galpha(11) were also targets for agonist-regulated [(3)H]palmitoylation and bound [(35)S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) in an agonist concentration-dependent manner. The potency of agonist to stimulate [(35)S]GTPgammaS binding was unaffected by the palmitoylation potential of either receptor or G protein. These studies provide clear evidence for coordinated, agonist-mediated regulation of the post-translational acylation of both a receptor and partner G protein and demonstrate the capacity of such fusions to bind and then release beta.gamma complex upon agonist stimulation whether or not the G protein can be palmitoylated. They also demonstrate that Ca(2+) signaling in EF88 cells by such fusion proteins is mediated via release of the G protein beta.gamma complex.     

Inhibition of subsets of G protein-coupled receptors by empty mutants of G protein alpha subunits in g(o), G(11), and G(16)

We previously reported that the xanthine nucleotide binding G(o)alpha mutant, G(o)alphaX, inhibited the activation of G(i)-coupled receptors. We constructed similar mutations in G(11)alpha and G(16)alpha and characterized their nucleotide binding and receptor interaction. First, we found that G(11)alphaX and G(16)alphaX expressed in COS-7 cells bound xanthine 5'-O-(thiotriphosphate) instead of guanosine 5'-O-(thiotriphosphate). Second, we found that G(11)alphaX and G(16)alphaX interacted with betagamma subunits in the presence of xanthine diphosphate. These experiments demonstrated that G(11)alphaX and G(16)alphaX were xanthine nucleotide-binding proteins, similar to G(o)alphaX. Third, in COS-7 cells, both G(11)alphaX and G(16)alphaX inhibited the activation of G(q)-coupled receptors, whereas only G(16)alphaX inhibited the activation of G(i)-coupled receptors. Therefore, when in the nucleotide-free state, empty G(11)alphaX and G(16)alphaX appeared to retain the same receptor binding specificity as their wild-type counterparts. Finally, we found that G(o)alphaX, G(11)alphaX, and G(16)alphaX all inhibited the endogenous thrombin receptors and lysophosphatidic acid receptors in NIH3T3 cells, whereas G(11)alphaX and G(16)alphaX, but not G(o)alphaX, inhibited the activation of transfected m1 muscarinic receptor in these cells. We conclude that these empty G protein mutants of G(o)alpha, G(11)alpha, and G(16)alpha can act as dominant negative inhibitors against specific subsets of G protein-coupled receptors.     

Constitutively active alpha subunits of G(q/11) and G(12/13) families inhibit activation of the pro-survival Akt signaling cascade

Accumulating evidence indicates that G protein signaling plays an active role in the regulation of cell survival. Our previous study demonstrated the regulatory effects of G(i/o) proteins in nerve growth factor-induced activation of pro-survival Akt kinase. In the present study we explored the role of various members of the G(s), G(q/11) and G(12/13) subfamilies in the regulation of Akt in cultured mammalian cells. In human embryonic kidney 293 cells transiently expressing constitutively active mutants of G alpha11, G alpha14, G alpha16, G alpha12, or G alpha13 (G alpha11QL, G alpha14QL, G alpha16QL, G alpha12QL and G alpha13QL, respectively), basal phosphorylation of Akt was attenuated, as revealed by western blotting analysis using a phosphospecific anti-Akt immunoglobulin. In contrast, basal Akt phosphorylation was unaffected by the overexpression of a constitutively active G alpha(s) mutant (G alpha(s)QL). Additional experiments showed that G alpha11QL, G alpha14QL, G alpha16QL, G alpha12QL and G alpha13QL, but not G alpha(s)QL, attenuated phosphorylation of the Akt-regulated translation regulator tuberin. Moreover, they were able to inhibit the epidermal growth factor-induced Akt activation and tuberin phosphorylation. The inhibitory mechanism of Gq family members was independent of phospholipase Cbeta activation and calcium signaling because G alpha11QL, G alpha14QL and G alpha16QL remained capable of inhibiting epidermal growth factor-induced Akt activation in cells pretreated with U73122 and the intracellular calcium chelator, BAPTA/AM. Finally, overexpression of the dominant negative mutant of RhoA blocked G alpha12QL- and G alpha13QL-mediated inhibition, suggesting that activated G alpha12 and G alpha13 inhibit Akt signaling via RhoA. Collectively, this study demonstrated the inhibitory effect of activated G alpha11, G alpha14, G alpha16, G alpha12 and G alpha13 on pro-survival Akt signaling.     

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.     

Differential distribution of alpha subunits and beta gamma subunits of heterotrimeric G proteins on Golgi membranes of the exocrine pancreas

Heterotrimeric G proteins are well known to be involved in signaling via plasma membrane (PM) receptors. Recent data indicate that heterotrimeric G proteins are also present on intracellular membranes and may regulate vesicular transport along the exocytic pathway. We have used subcellular fractionation and immunocytochemical localization to investigate the distribution of G alpha and G beta gamma subunits in the rat exocrine pancreas which is highly specialized for protein secretion. We show that G alpha s, G alpha i3 and G alpha q/11 are present in Golgi fractions which are > 95% devoid of PM. Removal of residual PM by absorption on wheat germ agglutinin (WGA) did not deplete G alpha subunits. G alpha s was largely restricted to TGN- enriched fractions by immunoblotting, whereas G alpha i3 and G alpha q/11 were broadly distributed across Golgi fractions. G alpha s did not colocalize with TGN38 or caveolin, suggesting that G alpha s is associated with a distinct population of membranes. G beta subunits were barely detectable in purified Golgi fractions. By immunofluorescence and immunogold labeling, G beta subunits were detected on PM but not on Golgi membranes, whereas G alpha s and G alpha i3 were readily detected on both Golgi and PM. G alpha and G beta subunits were not found on membranes of zymogen granules. These data indicate that G alpha s, G alpha q/11, and G alpha i3 associate with Golgi membranes independent of G beta subunits and have distinctive distributions within the Golgi stack. G beta subunits are thought to lock G alpha in the GDP-bound form, prevent it from activating its effector, and assist in anchoring it to the PM. Therefore the presence of free G alpha subunits on Golgi membranes has several important functional implications: it suggests that G alpha subunits associated with Golgi membranes are in the active, GTP-bound form or are bound to some other unidentified protein(s) which can substitute for G beta gamma subunits. It further implies that G alpha subunits are tethered to Golgi membranes by posttranslational modifications (e.g., palmitoylation) or by binding to another protein(s).     

Agonist-induced tyrosine phosphorylation of Gq/G11 alpha requires the intact structure of membrane domains

Stimulation of receptors coupled to G(q)/G(11) protein may induce phosphorylation on a tyrosine residue of the alpha subunit of this G protein, which is an essential event for G(q)/G(11) activation. Here we observed that in HEK293 cells stably expressing high levels of thyrotropin-releasing hormone (TRH) receptors and G(11)alpha protein the maximal tyrosine phosphorylation of G(q)/G(11)alpha was reached within 10 min of TRH stimulation and then it faded away at longer time periods of agonist exposure. The G(q)/G(11)alpha protein levels did not change during this treatment. Incubation of intact cells with beta-cyclodextrin (beta CD) for 40 min prior to hormone exposure significantly decreased the rapid transient tyrosine phosphorylation. Subsequent replenishment of cholesterol levels reversed the former negative effect of beta CD. Isolation of caveolin-enriched, detergent-resistant membrane domains indicated destruction of these structures in beta CD-treated cells. These data indicate that the preserved integrity of plasma membrane domains/caveolae is required for complete agonist-induced phosphorylation of G(q)/G(11)alpha.     

Ethanol inhibits palmitoylation of G protein G alpha(s)

Neurobiological actions of ethanol have been linked to perturbations in cyclic AMP (cAMP)-dependent signaling processes. Chronic ethanol exposure leads to desensitization of cAMP production in response to physiological ligands (heterologous desensitization). Ethanol-induced alterations in neuronal expression of G proteins G(s) and G(i) have been invoked as a cause of heterologous desensitization. However, effects of ethanol on G protein expression vary considerably among different experimental protocols, various brain regions and diverse neuronal cell types. Dynamic palmitoylation of G protein alpha subunits is critical for membrane localization and protein-protein interactions, and represents a regulatory feature of G protein function. We studied the effect of ethanol on G alpha(s) palmitoylation. In NG108-15 rat neuroblastoma x glioma hybrid cells, acute exposure to pharmacologically relevant concentrations of ethanol (25-100 mm) inhibited basal and prostaglandin E1-stimulated incorporation of palmitate into G alpha(s). Exposure of NG108-15 cells to ethanol for 72 h induced a shift in G alpha(s) to its non-palmitoylated state, coincident with an inhibition of prostaglandin E1-induced cAMP production. Both parameters were restored following 24 h of ethanol withdrawal. Chronic ethanol exposure also induced the depalmitoylation of G alpha(s) in human embryonic kidney (HEK)293 cells that overexpress wild-type G alpha(s) and caused heterologous desensitization of adenylyl cyclase. By contrast, HEK293 cells that express a non-palmitoylated mutant of G alpha(s) were insensitive to heterologous desensitization after chronic ethanol exposure. In summary, the findings identify a novel effect of ethanol on post-translational lipid modification of G alpha(s), and represent a mechanism by which ethanol might affect adenylyl cyclase activity.     

The trimeric GTP-binding protein (G(q)/G(11)) alpha subunit is required for insulin-stimulated GLUT4 translocation in 3T3L1 adipocytes

To investigate the potential role of trimeric GTP-binding proteins regulating GLUT4 translocation in adipocytes, wild type and constitutively active G(q) (G(q)/Q209L), G(i) (G(i)/Q205L), and G(s) (G(s)/Q227L) alpha subunit mutants were expressed in 3T3L1 adipocytes. Although expression of neither the wild type nor G(i)/Q205L and G(s)/Q227L alpha subunit mutants had any effect on the basal or insulin-stimulated translocation of a co-expressed GLUT4-enhanced green fluorescent protein (EGFP) fusion protein, expression of G(q)/Q209L resulted in GLUT4-EGFP translocation in the absence of insulin. In contrast, microinjection of an inhibitory G(q)/G(11) alpha subunit-specific antibody but not a G(i) or G(s) alpha subunit antibody prevented insulin-stimulated endogenous GLUT4 translocation. Consistent with a required role for GTP-bound G(q)/G(11), expression of the regulators of G protein signaling (RGS4 and RGS16) also attenuated insulin-stimulated GLUT4-EGFP translocation. To assess the relationship between G(q)/G(11) function with the phosphatidylinositol 3-kinase dependent pathway, expression of a dominant-interfering p85 regulatory subunit, as well as wortmannin treatment inhibited insulin-stimulated but not G(q)/Q209L-stimulated GLUT4-EGFP translocation. Furthermore, G(q)/Q209L did not induce the in vivo accumulation of phosphatidylinositol-3,4,5-trisphosphate (PIP(3)), whereas expression of the RGS proteins did not prevent the insulin-stimulated accumulation of PIP(3). Together, these data demonstrate that insulin stimulation of GLUT4 translocation requires at least two independent signal transduction pathways, one mediated through the phosphatidylinositol 3-kinase and another through the trimeric GTP-binding proteins G(q) and/or G(11).     

G alpha 11 and G alpha q guanine nucleotide regulatory proteins differentially modulate the response to thyrotropin-releasing hormone in Xenopus oocytes.

Xenopus oocytes that express mouse thyrotropin-releasing hormone receptors (TRH-Rs) after injection if RNA transcribed from TRH-R cDNA respond to THR by a depolarizing current. This response is transduced by activation of phosphoinositide-specific phospholipase C and utilizes an as yet unidentified endogenous guanine nucleotide-binding regulatory (G) protein(s). The alpha subunit of G11 and Gq have recently been shown to couple receptors to activation of phospholipase C. To determine whether there are functional differences between these proteins, we have co-expressed the TRH-R with either alpha 11 or alpha q. alpha 11 potentiated the response to TRH (by 61 +/- 16%), while alpha q inhibited the response (by 37 +/- 9%). The changes in amplitudes were accompanied by inverse changes in response latencies. These data show that alpha 11 and alpha q differentially modulate signal transduction in Xenopus oocytes.     

Selective inhibition of heterotrimeric Gs signaling. Targeting the receptor-G protein interface using a peptide minigene encoding the Galpha(s) carboxyl terminus

The blockade of heptahelical receptor coupling to heterotrimeric G proteins by the expression of peptides derived from G protein Galpha subunits represents a novel means of simultaneously inhibiting signals arising from multiple receptors that share a common G protein pool. Here we examined the mechanism of action and functional consequences of expression of an 83-amino acid polypeptide derived from the carboxyl terminus of Galpha(s) (GsCT). In membranes prepared from GsCT-expressing cells, the peptide blocked high affinity agonist binding to beta(2) adrenergic receptors (AR) and inhibited beta(2)AR-induced [35S]GTPgammaS loading of Galpha(s). GsCT expression inhibited beta(2)AR- and dopamine D(1A) receptor-mediated cAMP production, without affecting the cellular response to cholera toxin or forskolin, indicating that the peptide inhibited receptor-G(s) coupling without impairing G protein or adenylyl cyclase function. [35S]GTPgammaS loading of Galpha(q/11) by alpha(1B)ARs and Galpha(i) by alpha(2A)ARs and G(q/11)- or G(i)-mediated phosphatidylinositol hydrolysis was unaffected, indicating that the inhibitory effects of GsCT were selective for G(s). We next employed the GsCT construct to examine the complex role of G(s) in regulation of the ERK mitogen-activated protein kinase cascade, where activation of the cAMP-dependent protein kinase (PKA) pathway reportedly produces both stimulatory and inhibitory effects on heptahelical receptor-mediated ERK activation. For the beta(2)AR in HEK-293 cells, where PKA activity is required for ERK activation, expression of GsCT caused a net inhibition of ERK activation. In contrast, alpha(2A)AR-mediated ERK activation in COS-7 cells was enhanced by GsCT expression, consistent with the relief of a downstream inhibitory effect of PKA. ERK activation by the G(q/11)-coupled alpha(1B)AR was unaffected by GsCT. These findings suggest that peptide G protein inhibitors can provide insights into the complex interplay between G protein pools in cellular regulation.     

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.     

Leukemia-associated Rho guanine nucleotide exchange factor promotes G alpha q-coupled activation of RhoA

Leukemia-associated Rho guanine-nucleotide exchange factor (LARG) belongs to the subfamily of Dbl homology RhoGEF proteins (including p115 RhoGEF and PDZ-RhoGEF) that possess amino-terminal regulator of G protein signaling (RGS) boxes also found within GTPase-accelerating proteins (GAPs) for heterotrimeric G protein alpha subunits. p115 RhoGEF stimulates the intrinsic GTP hydrolysis activity of G alpha 12/13 subunits and acts as an effector for G13-coupled receptors by linking receptor activation to RhoA activation. The presence of RGS box and Dbl homology domains within LARG suggests this protein may also function as a GAP toward specific G alpha subunits and couple G alpha activation to RhoA-mediating signaling pathways. Unlike the RGS box of p115 RhoGEF, the RGS box of LARG interacts not only with G alpha 12 and G alpha 13 but also with G alpha q. In cellular coimmunoprecipitation studies, the LARG RGS box formed stable complexes with the transition state mimetic forms of G alpha q, G alpha 12, and G alpha 13. Expression of the LARG RGS box diminished the transforming activity of oncogenic G protein-coupled receptors (Mas, G2A, and m1-muscarinic cholinergic) coupled to G alpha q and G alpha 13. Activated G alpha q, as well as G alpha 12 and G alpha 13, cooperated with LARG and caused synergistic activation of RhoA, suggesting that all three G alpha subunits stimulate LARG-mediated activation of RhoA. Our findings suggest that the RhoA exchange factor LARG, unlike the related p115 RhoGEF and PDZ-RhoGEF proteins, can serve as an effector for Gq-coupled receptors, mediating their functional linkage to RhoA-dependent signaling pathways.