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.     

PLC-epsilon: a shared effector protein in Ras-, Rho-, and G alpha beta gamma-mediated signaling

The conceptual segregation of G protein-stimulated cell signaling responses into those mediated by heterotrimeric G proteins versus those promoted by small GTPases of the Ras superfamily is no longer vogue. PLC-epsilon, an isozyme of the phospholipase C (PLC) family, has been identified recently and dramatically extends our understanding of the crosstalk that occurs between heterotrimeric and small monomeric GTPases. Like the widely studied PLC-beta isozymes, PLC-epsilon is activated by Gbetagamma released upon activation of heterotrimeric G proteins. However, PLC-epsilon markedly differs from the PLC-beta isozymes in its capacity for activation by Galpha(12/13) - but not Galpha(q) -coupled receptors. PLC-epsilon contains two Ras-associating domains located near the C terminus, and H-Ras regulates PLC-epsilon as a downstream effector. Rho also activates PLC-epsilon, but in a mechanism independent of the C-terminal Ras-associating domains. Therefore, Ca(2+) mobilization and activation of protein kinase C are signaling responses associated with activation of both H-Ras and Rho. A guanine nucleotide exchange domain conserved in the N terminus of PLC-epsilon potentially confers a capacity for activators of this isozyme to cast signals into additional signaling pathways mediated by GTPases of the Ras superfamily. Thus, PLC-epsilon is a multifunctional nexus protein that senses and mediates crosstalk between heterotrimeric and small GTPase signaling pathways.     

Pasteurella multocida toxin stimulates mitogen-activated protein kinase via G(q/11)-dependent transactivation of the epidermal growth factor receptor

The dermatonecrotic toxin produced by Pasteurella multocida is one of the most potent mitogenic substances known for fibroblasts in vitro. Exposure to recombinant P. multocida toxin (rPMT) causes phospholipase C-mediated hydrolysis of inositol phospholipids, calcium mobilization, and activation of protein kinase C via a poorly characterized mechanism involving G(q/11) family heterotrimeric G proteins. To determine whether the regulation of G protein pathways contributes to the mitogenic effects of rPMT, we have examined the mechanism whereby rPMT stimulates the Erk mitogen-activated protein kinase cascade in cultured HEK-293 cells. Treatment with rPMT resulted in a dose and time-dependent increase in Erk 1/2 phosphorylation that paralleled its stimulation of inositol phospholipid hydrolysis. Both rPMT- and alpha-thrombin receptor- stimulated Erk phosphorylation were selectively blocked by cellular expression of two peptide inhibitors of G(q/11) signaling, the dominant negative mutant G protein-coupled receptor kinase, GRK2(K220R), and the Galpha(q) carboxyl-terminal peptide, Galpha(q)-(305-359). Like alpha-thrombin receptor-mediated Erk activation, the effect of rPMT was insensitive to the protein kinase C inhibitor GF109203X, but was blocked by the epidermal growth factor receptor-specific tyrphostin, AG1478 and by dominant negative mutants of mSos1 and Ha-Ras. These data indicate that rPMT employs G(q/11) family heterotrimeric G proteins to induce Ras-dependent Erk activation via protein kinase C-independent "transactivation" of the epidermal growth factor receptor.     

Dosage-dependent switch from G protein-coupled to G protein-independent signaling by a GPCR

G-protein-coupled receptors (GPCRs) mostly signal through heterotrimeric G proteins. Increasing evidence suggests that GPCRs could function in a G-protein-independent manner. Here, we show that at low concentrations of an agonist, beta(2)-adrenergic receptors (beta(2)-ARs) signal through Galpha(s) to activate the mitogen-activated protein kinase pathway in mouse embryonic fibroblast cells. At high agonist concentrations, signals are also transduced through beta(2)-ARs via an additional pathway that is G-protein-independent but tyrosine kinase Src-dependent. This new dosage-dependent switch of signaling modes of GPCRs has significant implications for GPCR intrinsic properties and desensitization.     

RACK1 regulates specific functions of Gbetagamma

We showed previously that Gbetagamma interacts with Receptor for Activated C Kinase 1 (RACK1), a protein that not only binds activated protein kinase C (PKC) but also serves as an adaptor/scaffold for many signaling pathways. Here we report that RACK1 does not interact with Galpha subunits or heterotrimeric G proteins but binds free Gbetagamma subunits released from activated heterotrimeric G proteins following the activation of their cognate receptors in vivo. The association with Gbetagamma promotes the translocation of RACK1 from the cytosol to the membrane. Moreover, binding of RACK1 to Gbetagamma results in inhibition of Gbetagamma-mediated activation of phospholipase C beta2 and adenylyl cyclase II. However, RACK1 has no effect on other functions of Gbetagamma, such as activation of the mitogen-activated protein kinase signaling pathway or chemotaxis of HEK293 cells via the chemokine receptor CXCR2. Similarly, RACK1 does not affect signal transduction through the Galpha subunits of G(i), G(s), or G(q). Collectively, these findings suggest a role of RACK1 in regulating specific functions of Gbetagamma.     

Heterotrimeric G-protein subunit function in Candida albicans: both the alpha and beta subunits of the pheromone response G protein are required for mating

A pheromone-mediated signaling pathway that couples seven-transmembrane-domain (7-TMD) receptors to a mitogen-activated protein kinase module controls Candida albicans mating. 7-TMD receptors are typically connected to heterotrimeric G proteins whose activation regulates downstream effectors. Two Galpha subunits in C. albicans have been identified previously, both of which have been implicated in aspects of pheromone response. Cag1p was found to complement the mating pathway function of the pheromone receptor-coupled Galpha subunit in Saccharomyces cerevisiae, and Gpa2p was shown to have a role in the regulation of cyclic AMP signaling in C. albicans and to repress pheromone-mediated arrest. Here, we show that the disruption of CAG1 prevented mating, inactivated pheromone-mediated arrest and morphological changes, and blocked pheromone-mediated gene expression changes in opaque cells of C. albicans and that the overproduction of CAG1 suppressed the hyperactive cell cycle arrest exhibited by sst2 mutant cells. Because the disruption of the STE4 homolog constituting the only C. albicans gene for a heterotrimeric Gbeta subunit also blocked mating and pheromone response, it appears that in this fungal pathogen the Galpha and Gbeta subunits do not act antagonistically but, instead, are both required for the transmission of the mating signal.     

Activation of phospholipase C-epsilon by heterotrimeric G protein betagamma-subunits

PLC-epsilon was identified recently as a phosphoinositide-hydrolyzing phospholipase C (PLC) containing catalytic domains (X, Y, and C2) common to all PLC isozymes as well as unique CDC25- and Ras-associating domains. Novel regulation of this PLC isozyme by the Ras oncoprotein and alpha-subunits (Galpha(12)) of heterotrimeric G proteins was illustrated. Sequence analyses of PLC-epsilon revealed previously unrecognized PH and EF-hand domains in the amino terminus. The known interaction of Gbetagamma subunits with the PH domains of other proteins led us to examine the capacity of Gbetagamma to activate PLC-epsilon. Co-expression of Gbeta(1)gamma(2) with PLC-epsilon in COS-7 cells resulted in marked stimulation of phospholipase C activity. Gbeta(2) and Gbeta(4) in combination with Ggamma(1), Ggamma(2), Ggamma(3), or Ggamma(13) also activated PLC-epsilon to levels similar to those observed with Gbeta(1)-containing dimers of these Ggamma-subunits. Gbeta(3) in combination with the same Ggamma-subunits was less active, and Gbeta(5)-containing dimers were essentially inactive. Gbetagamma-promoted activation of PLC-epsilon was blocked by cotransfection with either of two Gbetagamma-interacting proteins, Galpha(i1) or the carboxyl terminus of G protein receptor kinase 2. Pharmacological inhibition of PI3-kinase-gamma had no effect on Gbeta(1)gamma(2)-promoted activation of PLC-epsilon. Similarly, activation of Ras in the action of Gbetagamma is unlikely, because a mutation in the second RA domain of PLC-epsilon that blocks Ras activation of PLC failed to alter the stimulatory activity of Gbeta(1)gamma(2). Taken together, these results reveal the presence of additional functional domains in PLC-epsilon and add a new level of complexity in the regulation of this novel enzyme by heterotrimeric G proteins.     

A unique platform for H-Ras signaling involving clathrin-independent endocytosis

Trafficking of H-Ras was examined to determine whether it can enter cells through clathrin-independent endocytosis (CIE). H-Ras colocalized with the CIE cargo protein, class I major histocompatibility complex, and it was sequestered in vacuoles that formed upon expression of an active mutant of Arf6, Q67L. Activation of Ras, either through epidermal growth factor stimulation or the expression of an active mutant of Ras, G12V, induced plasma membrane ruffling and macropinocytosis, a stimulated form of CIE. Live imaging of cells expressing H-RasG12V and fluorescent protein chimeras with pleckstrin homology domains that recognize specific phosphoinositides showed that incoming macropinosomes contained phosphatidylinositol 4,5-bisphosphate (PIP(2)) and phosphatiylinositol 3,4,5-trisphosphate (PIP(3)). PIP(2) loss from the macropinosome was followed by the recruitment of Rab5, a downstream target of Ras, and then PIP(3) loss. Our studies support a model whereby Ras can signal on macropinosomes that pass through three distinct stages: PIP(2)/PIP(3), PIP(3)/Rab5, and Rab5. Vacuoles that form in cells expressing Arf6Q67L trap Ras signaling in the first stage, recruiting the active form of the Ras effectors extracellular signal-regulated kinase and protein kinase B (Akt) but not Rab5. Arf6 stimulation of macropinocytosis also involves passage through the distinct lipid phases, but recruitment of Akt is not observed.     

Phospholipase C(epsilon): a novel Ras effector

Three classes of mammalian phosphoinositide-specific phospholipase C (PLC) have been characterized, PLCbeta, PLCgamma and PLCdelta, that are differentially regulated by heterotrimeric G-proteins, tyrosine kinases and calcium. Here we describe a fourth class, PLCepsilon, that in addition to conserved PLC domains, contains a GTP exchange factor (GRF CDC25) domain and two C-terminal Ras-binding (RA) domains, RA1 and RA2. The RA2 domain binds H-Ras in a GTP-dependent manner, comparable with the Ras-binding domain of Raf-1; however, the RA1 domain binds H-Ras with a low affinity in a GTP-independent manner. While G(alpha)q, Gbetagamma or, surprisingly, H-Ras do not activate recombinant purified protein in vitro, constitutively active Q61L H-Ras stimulates PLC(epsilon) co-expressed in COS-7 cells in parallel with Ras binding. Deletion of either the RA1 or RA2 domain inhibits this activation. Site-directed mutagenesis of the RA2 domain or Ras demonstrates a conserved Ras-effector interaction and a unique profile of activation by Ras effector domain mutants. These studies identify a novel fourth class of mammalian PLC that is directly regulated by Ras and links two critical signaling pathways.     

Dissecting G protein-coupled receptor signaling pathways with membrane-permeable blocking peptides. Endogenous 5-HT(2C) receptors in choroid plexus epithelial cells

To determine the intracellular signaling mechanism of the 5-HT(2C) receptor endogenously expressed in choroid plexus epithelial cells, we implemented a strategy of targeted disruption of protein-protein interactions. This strategy entails the delivery of conjugated membrane-permeable peptides that disrupt domain interaction at specific steps in the signaling cascade. As proof of concept, two peptides targeted against receptor-G protein interaction domains were examined. Only G(q)CT, which targets the receptor-G(q) protein interacting domain, disrupted 5-HT(2C) receptor-mediated phosphatidylinositide hydrolysis. G(s)CT, targeting the receptor-G(s) protein, disrupted beta2 adrenergic receptor-mediated activation of cAMP but not 5-HT(2C) receptor-mediated phosphatidylinositide hydrolysis. The peptide MPS-PLCbeta1M, mimicking the domain of phospholipase Cbeta1 (PLCbeta1) interacting with active Galpha(q), also blocked 5-HT(2C) receptor activation. In contrast, peptides PLCbeta2M and Phos that bind to and sequester free Gbetagamma subunits were ineffective at blocking 5-HT(2C) receptor-mediated phosphoinositol turnover. However, both peptides disrupted Gbetagamma-mediated alpha(2A) adrenergic receptor activation of mitogen-activated protein kinase. These results provide the first direct demonstration that active Galpha(q) subunits mediate endogenous 5-HT(2C) receptor activation of PLCbeta and that Gbetagamma subunits released from Galpha(q) heterotrimeric proteins are not involved. Comparable results were obtained with metabotropic glutamate receptor 5 expressed in astrocytes. Thus, conjugated, membrane-permeable peptides are effective tools for the dissection of intracellular signals.     

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.     

Very-KIND, a KIND domain containing RasGEF, controls dendrite growth by linking Ras small GTPases and MAP2

The regulation of cytoskeletal components in the dendritic shaft core is critical for dendrite elongation and branching. Here, we report that a brain-specific Ras guanine nucleotide exchange factor (RasGEF) carrying two kinase non-catalytic C-lobe domains (KINDs), very-KIND (v-KIND), regulates microtubule-associated protein 2 (MAP2). v-KIND is expressed in developing mouse brain, predominantly in the cerebellar granule cells. v-KIND not only activates Ras small GTPases via the C-terminal RasGEF domain, but also specifically binds to MAP2 via the second KIND domain (KIND2), leading to threonine phosphorylation of MAP2. v-KIND overexpression suppresses dendritic extension and branching of hippocampal neurons and cerebellar granule cells, whereas knockdown of endogenous v-KIND expression promotes dendrite growth. These findings suggest that v-KIND mediates a signaling pathway that links Ras and MAP2 to control dendrite growth.     

Activation of the mitogen-activated protein kinases Erk1/2 by erythropoietin receptor via a G(i )protein beta gamma-subunit-initiated pathway

We have recently shown that a heterotrimeric G(i) protein is coupled to the erythropoietin (Epo) receptor. The G(i) protein constitutively associates in its heterotrimeric form with the intracellular domain of Epo receptor (EpoR). After Epo stimulation G(i) is released from the receptor and activated. In the present study we have investigated the functional role of the heterotrimeric G(i) protein bound to EpoR. In Chinese hamster ovary cells expressing EpoR, the G(i) inhibitor pertussis toxin blocked mitogen-activated protein kinase (MAPK) Erk1/2 activation induced by Epo. Epo-dependent MAPK activation was also sensitive to the G beta gamma competitive inhibitor beta ARK1-ct (C-terminal fragment of the beta-adrenergic receptor kinase), to the Ras dominant negative mutant RasN17, and to the phosphoinositide 3-kinase (PI3K) inhibitor LY 294002. A region of 7 amino acids (469-475) in the C-terminal end of EpoR was shown to be required for G(i) binding to EpoR in vivo. Deletion of this region in EpoR abolished both MAPK and PI3K activation in response to Epo. We conclude that in Chinese hamster ovary cells, Epo activates MAPK via a novel pathway dependent on G(i) association to EpoR, G beta gamma subunit, Ras, and PI3K. The tyrosine kinase Jak2 also contributes to this new pathway, more likely downstream of beta gamma and upstream of Ras and PI3K. This pathway is similar to the best characterized pathway used by seven transmembrane receptors coupled to G(i) to activate MAPK and may cooperate with other described Epo-dependent MAPK activation pathways in hematopoietic cells.     

Galpha(12) and galpha(13) inhibit Ca(2+)-dependent exocytosis through Rho/Rho-associated kinase-dependent pathway

The release of neurotransmitters is known to be regulated by activation of heterotrimeric G protein-coupled receptors, although precise mechanisms have not yet been elucidated. To assess the role of the G(12) family of heterotrimeric G proteins in the regulation of neurotransmitter release, we established PC12 cell lines that expressed constitutively active Galpha(12) or Galpha(13) using an isopropyl-beta-D-thiogalactoside-inducible expression system. In the cells, expression of constitutively active Galpha(12) or Galpha(13) inhibited the high K(+)-evoked [(3)H]dopamine release without any effect on the high K(+)-induced increase in intracellular Ca(2+) concentration. A Ca(2+) ionophore ionomycin-induced [(3)H]dopamine release was also inhibited by the expression of active Galpha(12) or Galpha(13). These inhibitory effects of Galpha(12) and Galpha(13) on [(3)H]dopamine release were mimicked by the expression of constitutively active RhoA. In addition, Y-27632, and inhibitor of Rho-associated kinase, a downstream Rho effector, completely abolished the inhibition of [(3)H]dopamine release by Galpha(12), Galpha(13), and RhoA. These results indicate that Ca(2+)-dependent exocytosis is regulated by Galpha(12) and Galpha(13) through a Rho/Rho-associated kinase-dependent pathway.     

The function of interdomain interactions in controlling nucleotide exchange rates in transducin

The intramolecular contacts in heterotrimeric G proteins that determine the rates of basal and receptor-stimulated nucleotide exchange are not fully understood. The alpha subunit of heterotrimeric G proteins consists of two domains: a Ras-like domain with structural homology to the monomeric G protein Ras and a helical domain comprised of six alpha-helices. The bound nucleotide lies in a deep cleft between the two domains. Exchange of the bound nucleotide may involve opening of this cleft. Thus interactions between the domains may affect the rate of nucleotide exchange in G proteins. We have tested this hypothesis in the alpha subunit of the rod cell G protein transducin (Galpha(t)). Site-directed mutations were prepared in a series of residues located at the interdomain interface. The proteins were expressed in vitro in a reticulocyte lysate system. The rates of basal and rhodopsin-catalyzed nucleotide exchange were determined using a trypsin digestion assay specifically adapted for kinetic measurements. Charge-altering substitutions of two residues at the interdomain interface, Lys(273) and Lys(276), increased basal nucleotide exchange rates modestly (5-10-fold). However, we found no evidence that interactions spanning the two domains in Galpha(t) significantly affected either basal or rhodopsin-catalyzed nucleotide exchange rates. These results suggest that opening of the interdomain cleft is not an energetic barrier to nucleotide exchange in Galpha(t). Experiments with Galpha(i1) suggest by comparison that the organization and function of the interdomain region differ among various G protein subtypes.     

TAO (thousand-and-one amino acid) protein kinases mediate signaling from carbachol to p38 mitogen-activated protein kinase and ternary complex factors

The TAO (for thousand-and-one amino acids) protein kinases activate p38 mitogen-activated protein (MAP) kinase cascades in vitro and in cells by phosphorylating the MAP/ERK kinases (MEKs) 3 and 6. We found that TAO2 activity was increased by carbachol and that carbachol and the heterotrimeric G protein Galphao could activate p38 in 293 cells. Using dominant interfering kinase mutants, we found that MEKs 3 and 6 and TAOs were required for p38 activation by carbachol or the constitutively active mutant GalphaoQ205L. To explore events downstream of TAOs, the effects of TAO2 on ternary complex factors (TCFs) were investigated. Transfection studies demonstrated that TAO2 stimulates phosphorylation of the TCF Elk1 on the major activating site, Ser383, and that TAO2 stimulates transactivation of Elk1 and the related TCF, Sap1. Reporter activity was reduced by the p38-selective inhibitor SB203580. Taken together, these studies suggest that TAO protein kinases relay signals from carbachol through heterotrimeric G proteins to the p38 MAP kinase, which then activates TCFs in the nucleus.     

Acetylcholine muscarinic m1 receptor regulation of cyclic AMP synthesis controls growth factor stimulation of Raf activity.

Acetylcholine muscarinic m2 receptors (m2R) couple to heterotrimeric Gi proteins and activate the Ras/Raf/mitogen-activated protein kinase pathway and phosphatidylinositol 3-kinase in Rat 1a cells. In contrast to the m2R, stimulation of the acetylcholine muscarinic m1 receptor (m1R) does not activate the Ras/Raf/mitogen-activated protein kinase regulatory pathway in Rat 1a cells but rather causes a pronounced inhibition of epidermal growth factor and platelet-derived growth factor receptor activation of Raf. In Rat 1a cells, m1R stimulation of phospholipase C beta and the marked rise in intracellular calcium stimulated cyclic AMP (cAMP) synthesis, resulting in the activation of protein kinase A. Stimulation of protein kinase A inhibited Raf activation in response to growth factors. Platelet-derived growth factor receptor stimulation of phosphatidylinositol 3-kinase activity was not affected by either m1R stimulation or protein kinase A activation in response to forskolin-stimulated cAMP synthesis. GTP loading of Ras in response to growth factors was unaffected by protein kinase A activation but was partially inhibited by carbachol stimulation of the m1R. Therefore, protein kinase A action at the Ras/Raf activation interface selectively inhibited only one branch of the signal transduction network initiated by tyrosine kinases. Specific adenylyl cyclases responding to different signals, including calcium, with enhanced cAMP synthesis will regulate Raf activation in response to Ras.GTP. Taken together, the data indicate that G protein-coupled receptors can positively and negatively regulate the responsiveness of tyrosine kinase-stimulated mitogenic response pathways.     

Distinct roles for two Galpha-Gbeta interfaces in cell polarity control by a yeast heterotrimeric G protein

Saccharomyces cerevisiae mating pheromones trigger dissociation of a heterotrimeric G protein (Galphabetagamma) into Galpha-guanosine triphosphate (GTP) and Gbetagamma. The Gbetagamma dimer regulates both mitogen-activated protein (MAP) kinase cascade signaling and cell polarization. Here, by independently activating the MAP kinase pathway, we studied the polarity role of Gbetagamma in isolation from its signaling role. MAP kinase signaling alone could induce cell asymmetry but not directional growth. Surprisingly, active Gbetagamma, either alone or with Galpha-GTP, could not organize a persistent polarization axis. Instead, following pheromone gradients (chemotropism) or directional growth without pheromone gradients (de novo polarization) required an intact receptor-Galphabetagamma module and GTP hydrolysis by Galpha. Our results indicate that chemoattractant-induced cell polarization requires continuous receptor-Galphabetagamma communication but not modulation of MAP kinase signaling. To explore regulation of Gbetagamma by Galpha, we mutated Gbeta residues in two structurally distinct Galpha-Gbeta binding interfaces. Polarity control was disrupted only by mutations in the N-terminal interface, and not the Switch interface. Incorporation of these mutations into a Gbeta-Galpha fusion protein, which enforces subunit proximity, revealed that Switch interface dissociation regulates signaling, whereas the N-terminal interface may govern receptor-Galphabetagamma coupling. These findings raise the possibility that the Galphabetagamma heterotrimer can function in a partially dissociated state, tethered by the N-terminal interface.     

Membrane trafficking of heterotrimeric G proteins via the endoplasmic reticulum and Golgi

Membrane targeting of G-protein alphabetagamma heterotrimers was investigated in live cells by use of Galpha and Ggamma subunits tagged with spectral mutants of green fluorescent protein. Unlike Ras proteins, Gbetagamma contains a single targeting signal, the CAAX motif, which directed the dimer to the endoplasmic reticulum. Endomembrane localization of farnesylated Ggamma(1), but not geranylgeranylated Ggamma(2), required carboxyl methylation. Targeting of the heterotrimer to the plasma membrane (PM) required coexpression of all three subunits, combining the CAAX motif of Ggamma with the fatty acyl modifications of Galpha. Galpha associated with Gbetagamma on the Golgi and palmitoylation of Galpha was required for translocation of the heterotrimer to the PM. Thus, two separate signals, analogous to the dual-signal targeting mechanism of Ras proteins, cooperate to target heterotrimeric G proteins to the PM via the endomembrane.