Oligomerization, biogenesis, and signaling is promoted by a glycophorin A-like dimerization motif in transmembrane domain 1 of a yeast G protein-coupled receptor

G protein-coupled receptors (GPCRs) can form dimeric or oligomeric complexes in vivo. However, the functions and mechanisms of oligomerization remain poorly understood for most GPCRs, including the alpha-factor receptor (STE2 gene product) of the yeast Saccharomyces cerevisiae. Here we provide evidence indicating that alpha-factor receptor oligomerization involves a GXXXG motif in the first transmembrane domain (TM1), similar to the transmembrane dimerization domain of glycophorin A. Results of fluorescence resonance energy transfer, fluorescence microscopy, endocytosis assays of receptor oligomerization in living cells, and agonist binding assays indicated that amino acid substitutions affecting the glycine residues of the GXXXG motif impaired alpha-factor receptor oligomerization and biogenesis in vivo but did not significantly impair agonist binding affinity. Mutant receptors exhibited signaling defects that were not due to impaired cell surface expression, indicating that oligomerization promotes alpha-factor receptor signal transduction. Structure-function studies suggested that the GXXXG motif in TM1 of the alpha-factor receptor promotes oligomerization by a mechanism similar to that used by the GXXXG dimerization motif of glycophorin A. In many mammalian GPCRs, motifs related to the GXXXG sequence are present in TM1 or other TM domains, suggesting that similar mechanisms are used by many GPCRs to form dimers or oligomeric arrays.     

The extracellular N-terminal domain and transmembrane domains 1 and 2 mediate oligomerization of a yeast G protein-coupled receptor

G protein-coupled receptors (GPCRs) can form homodimers/oligomers and/or heterodimers/oligomers. The mechanisms used to form specific GPCR oligomers are poorly understood because the domains that mediate such interactions and the step(s) in the secretory pathway where oligomerization occurs have not been well characterized. Here we have used subcellular fractionation and fluorescence resonance energy transfer (FRET) experiments to show that oligomerization of a GPCR (alpha-factor receptor; STE2 gene product) of the yeast Saccharomyces cerevisiae occurs in the endoplasmic reticulum. To identify domains of this receptor that mediate oligomerization, we used FRET and endocytosis assays of oligomerization in vivo to analyze receptor deletion mutants. A mutant lacking the N-terminal extracellular domain and transmembrane (TM) domain 1 was expressed at the cell surface but did not self-associate. In contrast, a receptor fragment containing only the N-terminal extracellular domain and TM1 could self-associate and heterodimerize with wild type receptors. Analysis of other mutants suggested that oligomerization is facilitated by the N-terminal extracellular domain and TM2. Therefore, the N-terminal extracellular domain, TM1, and TM2 appear to stabilize alpha-factor receptor oligomers. These domains may form an interface in contact or domain-swapped oligomers. Similar domains may mediate dimerization of certain mammalian GPCRs.     

The C terminus of the Saccharomyces cerevisiae alpha-factor receptor contributes to the formation of preactivation complexes with its cognate G protein

Binding of the alpha-factor pheromone to its G-protein-coupled receptor (encoded by STE2) activates the mating pathway in MATa yeast cells. To investigate whether specific interactions between the receptor and the G protein occur prior to ligand binding, we analyzed dominant-negative mutant receptors that compete with wild-type receptors for G proteins, and we analyzed the ability of receptors to suppress the constitutive signaling activity of mutant Galpha subunits in an alpha-factor-independent manner. Although the amino acid substitution L236H in the third intracellular loop of the receptor impairs G-protein activation, this substitution had no influence on the ability of the dominant-negative receptors to sequester G proteins or on the ability of receptors to suppress the GPA1-A345T mutant Galpha subunit. In contrast, removal of the cytoplasmic C-terminal domain of the receptor eliminated both of these activities even though the C-terminal domain is unnecessary for G-protein activation. Moreover, the alpha-factor-independent signaling activity of ste2-P258L mutant receptors was inhibited by the coexpression of wild-type receptors but not by coexpression of truncated receptors lacking the C-terminal domain. Deletion analysis suggested that the distal half of the C-terminal domain is critical for sequestration of G proteins. The C-terminal domain was also found to influence the affinity of the receptor for alpha-factor in cells lacking G proteins. These results suggest that the C-terminal cytoplasmic domain of the alpha-factor receptor, in addition to its role in receptor downregulation, promotes the formation of receptor-G-protein preactivation complexes.     

A fluorescent alpha-factor analogue exhibits multiple steps on binding to its G protein coupled receptor in yeast

The yeast alpha-factor receptor encoded by the STE2 gene is a member of the extended family of G protein coupled receptors (GPCRs) involved in a wide variety of signal transduction pathways. We report here the use of a fluorescent alpha-factor analogue [K(7)(NBD), Nle(12)] alpha-factor (Lys(7) (7-nitrobenz-2-oxa-1,3-diazol-4-yl), norleucine(12) alpha-factor) in conjunction with flow cytometry and fluorescence microscopy to study binding of ligand to the receptor. Internalization of the fluorescent ligand following receptor binding can be monitored by fluorescence microscopy. The use of flow cytometry to detect binding of the fluorescent ligand to intact yeast cells provides a sensitive and reproducible assay that can be conducted at low cell densities and is relatively insensitive to fluorescence of unbound and nonspecifically bound ligand. Using this assay, we determined that some receptor alleles expressed in cells lacking the G protein alpha subunit exhibit a higher equilibrium binding affinity for ligand than the same alleles expressed in isogenic cells containing the normal complement of G protein subunits. On the basis of time-dependent changes in the intensity and shape of the emission spectrum of [K(7)(NBD),Nle(12)] alpha-factor during binding, we infer that the ligand associates with receptors via a two-step process involving an initial interaction that places the fluorophore in a hydrophobic environment, followed by a conversion to a state in which the fluorophore moves to a more polar environment.     

Real-time analysis of G protein-coupled receptor reconstitution in a solubilized system

Receptor based signaling mechanisms are the primary source of cellular regulation. The superfamily of G protein-coupled receptors is the largest and most ubiquitous of the receptor mediated processes. We describe here the analysis in real-time of the assembly and disassembly of soluble G protein-coupled receptor-G protein complexes. A fluorometric method was utilized to determine the dissociation of a fluorescent ligand from the receptor solubilized in detergent. The ligand dissociation rate differs between a receptor coupled to a G protein and the receptor alone. By observing the sensitivity of the dissociation of a fluorescent ligand to the presence of guanine nucleotide, we have shown a time- and concentration-dependent reconstitution of the N-formyl peptide receptor with endogenous G proteins. Furthermore, after the clearing of endogenous G proteins, purified Galpha subunits premixed with bovine brain Gbetagamma subunits were also able to reconstitute with the solubilized receptors. The solubilized N-formyl peptide receptor and Galpha(i3) protein interacted with an affinity of approximately 10(-6) m with other alpha subunits exhibiting lower affinities (Galpha(i3) > Galpha(i2) > Galpha(i1) Galpha(o)). The N-formyl peptide receptor-G protein interactions were inhibited by peptides corresponding to the Galpha(i) C-terminal regions, by Galpha(i) mAbs, and by a truncated form of arrestin-3. This system should prove useful for the analysis of the specificity of receptor-G protein interactions, as well as for the elucidation and characterization of receptor molecular assemblies and signal transduction complexes.     

Dynamic integration of alpha-adrenergic and cholinergic signals in the atria: role of G protein-regulated inwardly rectifying K+ channels

Numerous heptahelical receptors use activation of heterotrimeric G proteins to convey a multitude of extracellular signals to appropriate effector molecules in the cell. Both high specificity and correct integration of these signals are required for reliable cell function. Yet the molecular machineries that allow each cell to merge information flowing across different receptors are not well understood. Here we demonstrate that G protein-regulated inwardly rectifying K(+) (GIRK) channels can operate as dynamic integrators of alpha-adrenergic and cholinergic signals in atrial myocytes. Acting at the last step of the cholinergic signaling cascade, these channels are activated by direct interactions with betagamma subunits of the inhibitory G proteins (G betagamma), and efficiently translate M(2) muscarinic acetylcholine receptor (M2R) activation into membrane hyperpolarization. The parallel activation of alpha-adrenergic receptors imposed a distinctive "signature" on the function of M2R-activated GIRK1/4 channels, affecting both the probability of G betagamma binding to the channel and its desensitization. This modulation of channel function was correlated with a parallel depletion of G beta and protein phosphatase 1 from the oligomeric GIRK1 complexes. Such plasticity of the immediate GIRK signaling environment suggests that multireceptor integration involves large protein networks undergoing dynamic changes upon receptor activation.     

The C-terminal tail preceding the CAAX box of a yeast G protein gamma subunit is dispensable for receptor-mediated G protein activation in vivo

The gamma subunits of heterotrimeric G proteins are required for receptor-G protein coupling. The C-terminal domains of Ggamma subunits can contact receptors and influence the efficiency of receptor-G protein coupling in vitro. However, it is unknown whether receptor interaction with the C terminus of Ggamma is required for signaling in vivo. To address this question, we cloned Ggamma homologs with diverged C-terminal sequences from five species of budding yeast. Each Ggamma homolog functionally replaced the Ggamma subunit of the yeast Saccharomyces cerevisiae (STE18 gene product). Mutagenesis of S. cerevisiae Ste18 likewise indicated that specific C-terminal sequence motifs are not required for signaling. Strikingly, an internal in-frame deletion removing sequences preceding the C-terminal CAAX box of Ste18 did not impair signaling by either of its cognate G protein-coupled pheromone receptors. Therefore, receptor interaction with the C-terminal domain of yeast Ggamma is not required for receptor-mediated G protein activation in vivo. Because the mechanism of G protein activation by receptors is conserved from yeast to humans, mammalian receptors may not require interaction with the tail of Ggamma for G protein activation in vivo. However, receptor-Ggamma interaction may modulate the efficiency of receptor-G protein coupling or promote activation of Gbetagamma effectors that co-cluster with receptors.     

A limited spectrum of mutations causes constitutive activation of the yeast alpha-factor receptor

Activation of G protein coupled receptors (GPCRs) by binding of ligand is the initial event in diverse cellular signaling pathways. To examine the frequency and diversity of mutations that cause constitutive activation of one particular GPCR, the yeast alpha-factor receptor, we screened libraries of random mutations for constitutive alleles. In initial screens for mutant receptor alleles that exhibit signaling in the absence of added ligand, 14 different point mutations were isolated. All of these 14 mutants could be further activated by alpha-factor. Ten of the mutants also acquired the ability to signal in response to binding of desTrp(1)?Ala(3)?lpha-factor, a peptide that acts as an antagonist toward normal alpha-factor receptors. Of these 10 mutants, at least eight alleles residing in the third, fifth, sixth, and seventh transmembrane segments exhibit bona fide constitutive signaling. The remaining alleles are hypersensitive to alpha-factor rather than constitutive. They can be activated by low concentrations of endogenous alpha-factor present in MATa cells. The strongest constitutively active receptor alleles were recovered multiple times from the mutational libraries, and extensive mutagenesis of certain regions of the alpha-factor receptor did not lead to recovery of any additional constitutive alleles. Thus, only a limited number of mutations is capable of causing constitutive activation of this receptor. Constitutive and hypersensitive signaling by the mutant receptors is partially suppressed by coexpression of normal receptors, consistent with preferential association of the G protein with unactivated receptors.     

Aromatic residues at the extracellular ends of transmembrane domains 5 and 6 promote ligand activation of the G protein-coupled alpha-factor receptor

The alpha-factor receptor (STE2) stimulates a G protein signaling pathway that promotes mating of the yeast Saccharomyces cerevisiae. Previous random mutagenesis studies implicated residues in the regions near the extracellular ends of the transmembrane domains in ligand activation. In this study, systematic Cys scanning mutagenesis across the ends of transmembrane domains 5 and 6 identified two residues, Phe(204) and Tyr(266), that were important for receptor signaling. These residues play a specific role in responding to alpha-factor since the F204C and Y266C substituted receptors responded to an alternative agonist (novobiocin). To better define the structure of this region, the Cys-substituted mutant receptors were assayed for reactivity with a thiol-specific probe that does not react with membrane-imbedded residues. A drop in reactivity coincided with residues likely to be buried in the membrane. Interestingly, both Phe(204) and Tyr(266) are located very near the interface region. However, these assays predict that Phe(204) is accessible at the surface of the receptor, consistent with the strong defect in binding alpha-factor caused by mutating this residue. In contrast, Tyr(266) was not accessible. This correlates with the ability of Y266C mutant receptors to bind alpha-factor and suggests that this residue is involved in the subsequent triggering of receptor activation. These results highlight the role of aromatic residues near the ends of the transmembrane segments in the alpha-factor receptor, and suggest that similar aromatic residues may play an important role in other G protein-coupled receptors.     

Afr1p regulates the Saccharomyces cerevisiae alpha-factor receptor by a mechanism that is distinct from receptor phosphorylation and endocytosis.

The alpha-factor pheromone receptor activates a G protein signaling pathway that induces the conjugation of the yeast Saccharomyces cerevisiae. Our previous studies identified AFR1 as a gene that regulates this signaling pathway because overexpression of AFR1 promoted resistance to alpha-factor. AFR1 also showed an interesting genetic relationship with the alpha-factor receptor gene, STE2, suggesting that the receptor is regulated by Afr1p. To investigate the mechanism of this regulation, we tested AFR1 for a role in the two processes that are known to regulate receptor signaling: phosphorylation and down-regulation of ligand-bound receptors by endocytosis. AFR1 overexpression diminished signaling in a strain that lacks the C-terminal phosphorylation sites of the receptor, indicating that AFR1 acts independently of phosphorylation. The effects of AFR1 overexpression were weaker in strains that were defective in receptor endocytosis. However, AFR1 overexpression did not detectably influence receptor endocytosis or the stability of the receptor protein. Instead, gene dosage studies showed that the effects of AFR1 overexpression on signaling were inversely proportional to the number of receptors. These results indicate that AFR1 acts independently of endocytosis, and that the weaker effects of AFR1 in strains that are defective in receptor endocytosis were probably an indirect consequence of their increased receptor number caused by the failure of receptors to undergo ligand-stimulated endocytosis. Analysis of the ligand binding properties of the receptor showed that AFR1 overexpression did not alter the number of cell-surface receptors or the affinity for alpha-factor. Thus, Afr1p prevents alpha-factor receptors from activating G protein signaling by a mechanism that is distinct from other known pathways.     

Oligomerization of the yeast alpha-factor receptor: implications for dominant negative effects of mutant receptors

Oligomerization of G protein-coupled receptors is commonly observed, but the functional significance of oligomerization for this diverse family of receptors remains poorly understood. We used bioluminescence resonance energy transfer (BRET) to examine oligomerization of Ste2p, a G protein-coupled receptor that serves as the receptor for the alpha-mating pheromone in the yeast Saccharomyces cerevisiae, under conditions where the functional effects of oligomerization could be examined. Consistent with previous results from fluorescence resonance energy transfer (Overton, M. C., and Blumer, K. J. (2000) Curr. Biol. 10, 341-344), we detected efficient energy transfer between Renilla luciferase and a modified green fluorescent protein individually fused to truncated alpha-factor receptors lacking the cytoplasmic C-terminal tail. In addition, the low background of the BRET system allowed detection of significant, but less efficient, energy transfer between full-length receptors. The reduced efficiency of energy transfer between full-length receptors does not appear to result from different levels of receptor expression. Instead, attachment of fluorescent reporter proteins to the full-length receptors appears to significantly increase the distance between reporters. Mutations that were previously reported to block dimerization of truncated alpha-factor receptors reduce but do not completely eliminate BRET transfer between receptors. Dominant negative effects of mutant alleles of alpha-factor receptors appear to be mediated by receptor oligomerization since these effects are abrogated by introduction of additional mutations that reduce oligomerization. We find that heterodimers of normal and dominant negative receptors are defective in their ability to signal. Thus, signal transduction by oligomeric receptors appears to be a cooperative process requiring an interaction between functional monomers.     

Agonist-dependent dissociation of oligomeric complexes of G protein-coupled cholecystokinin receptors demonstrated in living cells using bioluminescence resonance energy transfer.

Dimerization of some G protein-coupled receptors has recently been demonstrated, but how widespread this phenomenon might be and its functional implications are not yet clear. We have utilized biophysical and biochemical techniques to evaluate whether the type A cholecystokinin (CCK) receptor can form oligomeric complexes in the plasma membrane and the impact of ligand binding and signaling on such complexes. We investigated the possibility of bioluminescence resonance energy transfer (BRET) between receptor constructs that included carboxyl-terminal tags of Renilla luciferase or yellow fluorescent protein. Indeed, co-expression of these constructs in COS cells resulted in the constitutive presence of a significant BRET signal above that in a series of controls, with this signal reduced by co-expression of competing non-tagged CCK receptors. The presence of an oligomeric complex of CCK receptor molecules was confirmed in co-immunoprecipitation experiments. Occupation of CCK receptors with agonist ligands (CCK or gastrin-4) resulted in the rapid reduction in BRET signal in contrast to the enhancement of such a signal reported after agonist occupation of the beta(2)-adrenergic receptor. These effects on CCK receptor oligomerization were concentration-dependent, correlating with the potencies of the agonists. A smaller effect was observed for a partial agonist, and no effect was observed for antagonist occupation of this receptor. Agonist-induced reduction in BRET signal was also observed for pairs of CCK receptors with a donor-acceptor pair situated in other positions within the receptor. Manipulation of the phosphorylation state of CCK receptor using protein kinase C activation with phorbol ester or inhibition with staurosporine had no effect on the basal level or agonist effect on CCK receptor oligomerization. This provides the first evidence for CCK receptor oligomerization in living cells, with insights that the active conformation of this receptor dissociates these complexes in a phosphorylation-independent manner.     

The carboxy-terminal tail of the Ste2 receptor is involved in activation of the G protein in the Saccharomyces cerevisiae alpha-pheromone response pathway

The Ste2 gene encodes the yeast alpha-pheromone receptor that belongs to the superfamily of seven-transmembrane G protein-coupled receptors. Binding of pheromone induces activation of the heterotrimeric G protein triggering growth arrest in G1 phase and induction of genes required for mating. By random PCR-mediated mutagenesis we isolated mutant 8L4, which presents a substitution of an asparagine residue by serine at position 388 of the alpha-factor receptor. The 8L4 mutant strain shows phenotypic defects such as: reduction in growth arrest after pheromone treatment, diminished activation of the Fus1 gene, and impaired mating competence. The asparagine residue lies in the second half of the intracellular protruding C-terminal tail of the receptor, and its replacement by serine affects interaction with both the G(alpha) and Gbeta subunits. Since expression of the receptor as well as its kinetic parameters, i.e., ligand affinity and receptor number, are unaffected in the mutant strain, we propose that association of the C-terminal tail of the receptor with G(alpha) and Gbeta subunits is required for proper activation of the heterotrimeric G protein. Besides its described role in downregulation and in formation of preactivation complex, the results here shown indicate that the C-terminal tail of the receptor plays an active role in transmitting the stimulus of mating pheromone to the heterotrimeric G protein.     

Homo- and hetero-oligomerization of G protein-coupled receptors

G protein-coupled receptors (GPCRs) form homo-oligomeric and hetero-oligomeric complexes. This understanding has prompted a re-evaluation of many aspects of GPCR biology, however the concept of receptor complexes has not been fully integrated into the current thinking about GPCR structure and function. Nevertheless, receptor oligomerization is a pivotal aspect of the structure and function of GPCRs that has been shown to have implications for receptor trafficking, signaling, and pharmacology and more intricate models for understanding the physiological roles of these receptors are emerging. Here, we summarize some of the advances made in understanding the structural basis and the functional roles of homo- and hetero- oligomerization in this important group of receptors. Although this discussion focuses primarily on the dopamine receptors, particularly the D2 dopamine receptor, and the opioid and serotonin receptors, we discuss the principles governing the oligomerization of all rhodopsin-like GPCRs and potentially of the entire superfamily of these receptors.     

Bioluminescence resonance energy transfer assays reveal ligand-specific conformational changes within preformed signaling complexes containing delta-opioid receptors and heterotrimeric G proteins

Heptahelical receptors communicate extracellular information to the cytosolic compartment by binding an extensive variety of ligands. They do so through conformational changes that propagate to intracellular signaling partners as the receptor switches from a resting to an active conformation. This active state has been classically considered unique and responsible for regulation of all signaling pathways controlled by a receptor. However, recent functional studies have challenged this notion and called for a paradigm where receptors would exist in more than one signaling conformation. This study used bioluminescence resonance energy transfer assays in combination with ligands of different functional profiles to provide in vivo physical evidence of conformational diversity of delta-opioid receptors (DORs). DORs and alpha(i1)beta(1)gamma(2) G protein subunits were tagged with Luc or green fluorescent protein to produce bioluminescence resonance energy transfer pairs that allowed monitoring DOR-G protein interactions from different vantage points. Results showed that DORs and heterotrimeric G proteins formed a constitutive complex that underwent structural reorganization upon ligand binding. Conformational rearrangements could not be explained by a two-state model, supporting the idea that DORs adopt ligand-specific conformations. In addition, conformational diversity encoded by the receptor was conveyed to the interaction among heterotrimeric subunits. The existence of multiple active receptor states has implications for the way we conceive specificity of signal transduction.     

Homo-oligomeric complexes of the yeast alpha-factor pheromone receptor are functional units of endocytosis

alpha-Factor receptors from Saccharomyces cerevisiae are G-protein-coupled receptors containing seven transmembrane segments. Receptors solubilized with the detergent n-dodecyl beta-D-maltoside were found to sediment as a single 8S species in glycerol density gradients. When the membranes from cells coexpressing two differentially tagged receptors were solubilized with detergent and subjected to immunoprecipitation, we found that the antibodies specific for either epitope tag resulted in precipitation of both tagged species. Coprecipitation was not a consequence of incomplete detergent extraction because the abundant plasma membrane protein Pma1 did not coprecipitate with the receptors. Moreover, the receptor complexes were present prior to detergent extraction because coimmunoprecipitation was not observed when cells expressing the single tagged species were mixed prior to membrane preparation. Treatment of cultures with alpha-factor had little effect on the extent of oligomerization as judged by the sedimentation behavior of the receptor complexes and by the efficiency of coimmunoprecipitation. The ability of receptor complexes to undergo ligand-mediated endocytosis was evaluated by using membrane fractionation and fluorescence microscopy. Mutant receptors that fail to bind alpha-factor (Ste2-S184R) or lack the endocytosis signal (Ste2-T326) became competent for ligand-mediated endocytosis when they were expressed in cells containing wild-type receptors. Coimmunoprecipitation experiments indicated that the C-terminal cytoplasmic domain and intermolecular disulfide bonds were unnecessary for oligomer formation. We conclude that alpha-factor receptors form homo-oligomers and that these complexes are subject to ligand-mediated endocytosis. Furthermore, we show for the first time that unoccupied receptors participate in these endocytosis-competent complexes.     

G-protein-coupled receptors function as oligomers in vivo

Hormones, sensory stimuli, neurotransmitters and chemokines signal by activating G-protein-coupled receptors (GPCRs) [1]. Although GPCRs are thought to function as monomers, they can form SDS-resistant dimers, and coexpression of two non-functional or related GPCRs can result in rescue of activity or modification of function [2-10]. Furthermore, dimerization of peptides corresponding to the third cytoplasmic loops of GPCRs increases their potency as activators of G proteins in vitro [11], and peptide inhibitors of dimerization diminish beta(2)-adrenergic receptor signaling [3]. Nevertheless, it is not known whether GPCRs exist as monomers or oligomers in intact cells and membranes, whether agonist binding regulates monomer-oligomer equilibrium, or whether oligomerization governs GPCR function. Here, we report that the alpha-factor receptor, a GPCR that is the product of the STE2 gene in the yeast Saccharomyces cerevisiae, is oligomeric in intact cells and membranes. Coexpression of receptors tagged with the cyan or yellow fluorescent proteins (CFP or YFP) resulted in efficient fluorescence resonance energy transfer (FRET) due to stable association rather than collisional interaction. Monomer-oligomer equilibrium was unaffected by binding of agonist, antagonist, or G protein heterotrimers. Oligomerization was further demonstrated by rescuing endocytosis-defective receptors with coexpressed wild-type receptors. Dominant-interfering receptor mutants inhibited signaling by interacting with wild-type receptors rather than by sequestering G protein heterotrimers. We suggest that oligomerization is likely to govern GPCR signaling and regulation.     

Regulation of the G-protein-coupled alpha-factor pheromone receptor by phosphorylation.

The alpha-factor pheromone receptor activates a G protein signaling cascade that stimulates MATa yeast cells to undergo conjugation. The cytoplasmic C terminus of the receptor is not necessary for G protein activation but instead acts as a regulatory domain that promotes adaptation to alpha-factor. The role of phosphorylation in regulating the alpha-factor receptor was examined by mutating potential phosphorylation sites. Mutation of the four most distal serine and threonine residues in the receptor C terminus to alanine caused increased sensitivity to alpha-factor and a delay in recovering from a pulse of alpha-factor. 32PO4 labeling experiments demonstrated that the alanine substitution mutations decreased the in vivo phosphorylation of the receptor. Phosphorylation apparently alters the regulation of G protein activation, since neither receptor number nor affinity for ligand was significantly altered by mutation of the distal phosphorylation sites. Furthermore, mutation of the distal phosphorylation sites in a receptor mutant that fails to undergo ligand-stimulated endocytosis caused increased sensitivity to alpha-factor, which suggests that regulation by phosphorylation can occur at the cell surface and is independent of endocytosis. Mutation of the distal serine and threonine residues of the receptor also caused a slight defect in alpha-factor-induced morphogenesis, but the defect was not as severe as the morphogenesis defect caused by truncation of the cytoplasmic C terminus of the receptor. These distal residues in the C terminus play a special role in receptor regulation, since mutation of the next five adjacent serine and threonine residues to alanine did not affect the sensitivity to alpha-factor. Altogether, these results indicate that phosphorylation plays an important role in regulating alpha-factor receptor function.     

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.