Phospholipase C binds to the receptor-like GPR1 protein and controls pseudohyphal differentiation in Saccharomyces cerevisiae.

The hormone receptor-like protein Gpr1p physically interacts with phosphatidylinositol-specific phospholipase C (Plc1p) and with the Galpha protein Gpa2p, as shown by two-hybrid assays and co-immune precipitation of epitope-tagged proteins. Plc1p binds to Gpr1p in either the presence or absence of Gpa2, whereas the Gpr1p/Gpa2p association depends on the presence of Plc1p. Genetic interactions between the null mutations plc1Delta, gpr1Delta, gpa2Delta, and ras2Delta suggest that Plc1p acts together with Gpr1p and Gpa2p in a growth control pathway operating in parallel to the Ras2p function. Diploid cells lacking Gpr1p, Plc1p, or Gpa2p fail to form pseudohyphae upon nitrogen depletion, and the filamentation defect of gpr1Delta and plc1Delta strains is rescued by activating a mitogen-activated protein kinase pathway via STE11-4 or by activating a cAMP pathway via overexpressed Tpk2p. Plc1p is also required for efficient expression of the FG(TyA)::lacZ reporter gene under nitrogen depletion. In conclusion, we have identified two physically interacting proteins, Gpr1p and Plc1p, as novel components of a nitrogen signaling pathway controlling the developmental switch from yeast-like to pseudohyphal growth. Our data suggest that phospholipase C modulates the interaction of the putative nutrient sensor Gpr1p with the Galpha protein Gpa2p as a downstream effector of filamentation control.     

Galpha subunit Gpa2 recruits kelch repeat subunits that inhibit receptor-G protein coupling during cAMP-induced dimorphic transitions in Saccharomyces cerevisiae

All eukaryotic cells sense extracellular stimuli and activate intracellular signaling cascades via G protein-coupled receptors (GPCR) and associated heterotrimeric G proteins. The Saccharomyces cerevisiae GPCR Gpr1 and associated Galpha subunit Gpa2 sense extracellular carbon sources (including glucose) to govern filamentous growth. In contrast to conventional Galpha subunits, Gpa2 forms an atypical G protein complex with the kelch repeat Gbeta mimic proteins Gpb1 and Gpb2. Gpb1/2 negatively regulate cAMP signaling by inhibiting Gpa2 and an as yet unidentified target. Here we show that Gpa2 requires lipid modifications of its N-terminus for membrane localization but association with the Gpr1 receptor or Gpb1/2 subunits is dispensable for membrane targeting. Instead, Gpa2 promotes membrane localization of its associated Gbeta mimic subunit Gpb2. We also show that the Gpa2 N-terminus binds both to Gpb2 and to the C-terminal tail of the Gpr1 receptor and that Gpb1/2 binding interferes with Gpr1 receptor coupling to Gpa2. Our studies invoke novel mechanisms involving GPCR-G protein modules that may be conserved in multicellular eukaryotes.     

Gpr1, a putative G-protein-coupled receptor, regulates morphogenesis and hypha formation in the pathogenic fungus Candida albicans

In response to various extracellular signals, the morphology of the human fungal pathogen Candida albicans switches from yeast to hypha form. Here, we report that GPR1 encoding a putative G-protein-coupled receptor and GPA2 encoding a Galpha subunit are required for hypha formation and morphogenesis in C. albicans. Mutants lacking Gpr1 (gpr1/gpr1) or Gpa2 (gpa2/gpa2) are defective in hypha formation and morphogenesis on solid hypha-inducing media. These phenotypic defects in solid cultures are suppressed by exogenously added dibutyryl-cyclic AMP (dibutyryl-cAMP). Biochemical studies also reveal that GPR1 and GPA2 are required for a glucose-dependent increase in cellular cAMP. An epistasis analysis indicates that Gpr1 functions upstream of Gpa2 in the same signaling pathway, and a two-hybrid assay reveals that the carboxyl-terminal tail of Gpr1 interacts with Gpa2. Moreover, expression levels of HWP1 and ECE1, which are cAMP-dependent hypha-specific genes, are reduced in both mutant strains. These findings support a model that Gpr1, as well as Gpa2, regulates hypha formation and morphogenesis in a cAMP-dependent manner. In contrast, GPR1 and GPA2 are not required for hypha formation in liquid fetal bovine serum (FBS) medium. Furthermore, the gpr1 and the gpa2 mutant strains are fully virulent in a mouse infection. These findings suggest that Gpr1 and Gpa2 are involved in the glucose-sensing machinery that regulates morphogenesis and hypha formation in solid media via a cAMP-dependent mechanism, but they are not required for hypha formation in liquid medium or during invasive candidiasis.     

A Saccharomyces cerevisiae G-protein coupled receptor, Gpr1, is specifically required for glucose activation of the cAMP pathway during the transition to growth on glucose

In the yeast Saccharomyces cerevisiae the accumulation of cAMP is controlled by an elaborate pathway. Only two triggers of the Ras adenylate cyclase pathway are known. Intracellular acidification induces a Ras-mediated long-lasting cAMP increase. Addition of glucose to cells grown on a non-fermentable carbon source or to stationary-phase cells triggers a transient burst in the intracellular cAMP level. This glucose-induced cAMP signal is dependent on the G alpha-protein Gpa2. We show that the G-protein coupled receptor (GPCR) Gpr1 interacts with Gpa2 and is required for stimulation of cAMP synthesis by glucose. Gpr1 displays sequence homology to GPCRs of higher organisms. The absence of Gpr1 is rescued by the constitutively activated Gpa2Val-132 allele. In addition, we isolated a mutant allele of GPR1, named fil2, in a screen for mutants deficient in glucose-induced loss of heat resistance, which is consistent with its lack of glucose-induced cAMP activation. Apparently, Gpr1 together with Gpa2 constitute a glucose-sensing system for activation of the cAMP pathway. Deletion of Gpr1 and/or Gpa2 affected cAPK-controlled features (levels of trehalose, glycogen, heat resistance, expression of STRE-controlled genes and ribosomal protein genes) specifically during the transition to growth on glucose. Hence, an alternative glucose-sensing system must signal glucose availability for the Sch9-dependent pathway during growth on glucose. This appears to be the first example of a GPCR system activated by a nutrient in eukaryotic cells. Hence, a subfamily of GPCRs might be involved in nutrient sensing.     

The G protein-coupled receptor Gpr1 and the Galpha protein Gpa2 act through the cAMP-protein kinase A pathway to induce morphogenesis in Candida albicans

We investigated the role in cell morphogenesis and pathogenicity of the Candida albicans GPR1 gene, encoding the G protein-coupled receptor Gpr1. Deletion of C. albicans GPR1 has only minor effects in liquid hypha-inducing media but results in strong defects in the yeast-to-hypha transition on solid hypha-inducing media. Addition of cAMP, expression of a constitutively active allele of the Galpha protein Gpa2 or of the catalytic protein kinase A subunit TPK1 restores the wild-type phenotype of the CaGPR1-deleted strain. Overexpression of HST7, encoding a component of the mitogen-activated protein kinase pathway, does not suppress the defect in filamentation. These results indicate that CaGpr1 functions upstream in the cAMP-protein kinase A (PKA) pathway. We also show that, in the presence of glucose, CaGpr1 is important for amino acid-induced transition from yeast to hyphal cells. Finally, as opposed to previous reports, we show that CaGpa2 acts downstream of CaGpr1 as activator of the cAMP-PKA pathway but that deletion of neither CaGpr1 nor CaGpa2 affects glucose-induced cAMP signaling. In contrast, the latter is abolished in strains lacking CaCdc25 or CaRas1, suggesting that the CaCdc25-CaRas1 rather than the CaGpr1-CaGpa2 module mediates glucose-induced cAMP signaling in C. albicans.     

The G protein-coupled receptor gpr1 is a nutrient sensor that regulates pseudohyphal differentiation in Saccharomyces cerevisiae

Pseudohyphal differentiation in the budding yeast Saccharomyces cerevisiae is induced in diploid cells in response to nitrogen starvation and abundant fermentable carbon source. Filamentous growth requires at least two signaling pathways: the pheromone responsive MAP kinase cascade and the Gpa2p-cAMP-PKA signaling pathway. Recent studies have established a physical and functional link between the Galpha protein Gpa2 and the G protein-coupled receptor homolog Gpr1. We report here that the Gpr1 receptor is required for filamentous and haploid invasive growth and regulates expression of the cell surface flocculin Flo11. Epistasis analysis supports a model in which the Gpr1 receptor regulates pseudohyphal growth via the Gpa2p-cAMP-PKA pathway and independently of both the MAP kinase cascade and the PKA related kinase Sch9. Genetic and physiological studies indicate that the Gpr1 receptor is activated by glucose and other structurally related sugars. Because expression of the GPR1 gene is known to be induced by nitrogen starvation, the Gpr1 receptor may serve as a dual sensor of abundant carbon source (sugar ligand) and nitrogen starvation. In summary, our studies reveal a novel G protein-coupled receptor senses nutrients and regulates the dimorphic transition to filamentous growth via a Galpha protein-cAMP-PKA signal transduction cascade.     

The kelch proteins Gpb1 and Gpb2 inhibit Ras activity via association with the yeast RasGAP neurofibromin homologs Ira1 and Ira2

The G protein-coupled receptor Gpr1 and associated Galpha subunit Gpa2 govern dimorphic transitions in response to extracellular nutrients by signaling coordinately with Ras to activate adenylyl cyclase in the yeast Saccharomyces cerevisiae. Gpa2 forms a protein complex with the kelch Gbeta mimic subunits Gpb1/2, and previous studies demonstrate that Gpb1/2 negatively control cAMP-PKA signaling via Gpa2 and an unknown second target. Here, we define these targets of Gpb1/2 as the yeast neurofibromin homologs Ira1 and Ira2, which function as GTPase activating proteins of Ras. Gpb1/2 bind to a conserved C-terminal domain of Ira1/2, and loss of Gpb1/2 results in a destabilization of Ira1 and Ira2, leading to elevated levels of Ras2-GTP and unbridled cAMP-PKA signaling. Because the Gpb1/2 binding domain on Ira1/2 is conserved in the human neurofibromin protein, an analogous signaling network may contribute to the neoplastic development of neurofibromatosis type 1.     

Activation state of the Ras2 protein and glucose-induced signaling in Saccharomyces cerevisiae

The activity of adenylate cyclase in the yeast Saccharomyces cerevisiae is controlled by two G-protein systems, the Ras proteins and the Galpha protein Gpa2. Glucose activation of cAMP synthesis is thought to be mediated by Gpa2 and its G-protein-coupled receptor Gpr1. Using a sensitive GTP-loading assay for Ras2 we demonstrate that glucose addition also triggers a fast increase in the GTP loading state of Ras2 concomitant with the glucose-induced increase in cAMP. This increase is severely delayed in a strain lacking Cdc25, the guanine nucleotide exchange factor for Ras proteins. Deletion of the Ras-GAPs IRA2 (alone or with IRA1) or the presence of RAS2Val19 allele causes constitutively high Ras GTP loading that no longer increases upon glucose addition. The glucose-induced increase in Ras2 GTP-loading is not dependent on Gpr1 or Gpa2. Deletion of these proteins causes higher GTP loading indicating that the two G-protein systems might directly or indirectly interact. Because deletion of GPR1 or GPA2 reduces the glucose-induced cAMP increase the observed enhancement of Ras2 GTP loading is not sufficient for full stimulation of cAMP synthesis. Glucose phosphorylation by glucokinase or the hexokinases is required for glucose-induced Ras2 GTP loading. These results indicate that glucose phosphorylation might sustain activation of cAMP synthesis by enhancing Ras2 GTP loading likely through inhibition of the Ira proteins. Strains with reduced feedback inhibition on cAMP synthesis also display elevated basal and induced Ras2 GTP loading consistent with the Ras2 protein acting as a target of the feedback-inhibition mechanism.     

The RACK1 ortholog Asc1 functions as a G-protein beta subunit coupled to glucose responsiveness in yeast

According to the prevailing paradigm, G-proteins are composed of three subunits, an alpha subunit with GTPase activity and a tightly associated betagamma subunit complex. In the yeast Saccharomyces cerevisiae there are two known Galpha proteins (Gpa1 and Gpa2) but only one Gbetagamma, which binds only to Gpa1. Here we show that the yeast ortholog of RACK1 (receptor for activated protein kinase C1) Asc1 functions as the Gbeta for Gpa2. As with other known Gbeta proteins, Asc1 has a 7-WD domain structure, interacts directly with the Galpha in a guanine nucleotide-dependent manner, and inhibits Galpha guanine nucleotide exchange activity. In addition, Asc1 binds to the effector enzyme adenylyl cyclase (Cyr1), and diminishes the production of cAMP in response to glucose stimulation. Thus, whereas Gpa2 promotes glucose signaling through elevated production of cAMP, Asc1 has opposing effects on these same processes. Our findings reveal the existence of an unusual Gbeta subunit, one having multiple functions within the cell in addition to serving as a signal transducer for cell surface receptors and intracellular effectors.     

Dominant-negative inhibition of pheromone receptor signaling by a single point mutation in the G protein alpha subunit

In yeast, two different constitutive mutants of the G protein alpha subunit have been reported. Gpa1(Q323L) cannot hydrolyze GTP and permanently activates the pheromone response pathway. Gpa1(N388D) was also proposed to lack GTPase activity, yet it has an inhibitory effect on pheromone responsiveness. We have characterized this inhibitory mutant (designated Galpha(ND)) and found that it binds GTP, interacts with G protein betagamma subunits, and exhibits full GTPase activity in vitro. Although pheromone leads to dissociation of the receptor from wild-type G protein, the same treatment promotes stable association of the receptor with Galpha(ND). We conclude that agonist binding to the receptor promotes the formation of a nondissociable complex with Galpha(ND), and in this manner prevents activation of the endogenous wild-type G protein. Dominant-negative mutants may be useful in matching specific receptors and their cognate G proteins and in determining mechanisms of G protein signaling specificity.     

G-protein-coupled receptor Gpr1 and G-protein Gpa2 of cAMP-dependent signaling pathway are involved in glucose-induced pexophagy in the yeast Saccharomyces cerevisiae

In yeast cell, glucose induces various changes of cellular metabolism on genetic and metabolic levels. One of such changes is autophagic degradation of dispensable peroxisomes (pexophagy) which occurs in vacuoles. We have found that in Saccharomyces cerevisiae, defect of G-protein-coupled receptor Gpr1 and G-protein Gpa2, both the components of cAMP-signaling pathway, strongly suppressed glucose-induced degradation of matrix peroxisomal protein thiolase. We conclude that proteins Gpr1 and Gpa2 are involved in glucose sensing and signal transduction during pexophagy process in yeast.     

A comprehensive structure-function map of the intracellular surface of the human C5a receptor. II. Elucidation of G protein specificity determinants

Within any given cell many G protein-coupled receptors are expressed in the presence of multiple G proteins, yet most receptors couple to a specific subset of G proteins to elicit their programmed response. Numerous studies demonstrate that the carboxyl-terminal five amino acids of the Galpha subunits are a major determinant of specificity, however the receptor determinants of specificity are less clear. We have used a collection of 133 functional mutants of the C5a receptor obtained in a mutagenesis screen targeting the intracellular loops and the carboxyl terminus (Matsumoto, M. L., Narzinski, K., Kiser, P. D., Nikiforovich, G. V., and Baranski, T. J. (2007) J. Biol. Chem. 282, 3105-3121) to investigate how specificity is encoded. Each mutant, originally selected for its ability to signal through a nearly full-length Galpha(i) in yeast, was tested to see whether it could activate three versions of chimeric Galpha subunits consisting of Gpa1 fused to the carboxyl-terminal five amino acids of Galpha(i), Galpha(q), or Galpha(s) in yeast. Surprisingly the carboxyl-terminal tail of the C5a receptor is the most important specificity determinant in that nearly all mutants in this region showed a gain in coupling to Galpha(q) and/or Galpha(s). More than half of the receptors mutated in the second intracellular loop also demonstrated broadened G protein coupling. Given a lack of selective advantage for this broadened signaling in the initial screen, we propose a model in which the carboxyl-terminal tail acts together with the intracellular loops to generate a specificity filter for receptor-G protein interactions that functions primarily to restrict access of incorrect G proteins to the receptor.     

G protein signaling governing cell fate decisions involves opposing Galpha subunits in Cryptococcus neoformans

Communication between cells and their environments is often mediated by G protein-coupled receptors and cognate G proteins. In fungi, one such signaling cascade is the mating pathway triggered by pheromone/pheromone receptor recognition. Unlike Saccharomyces cerevisiae, which expresses two Galpha subunits, most filamentous ascomycetes and basidiomycetes have three Galpha subunits. Previous studies have defined the Galpha subunit acting upstream of the cAMP-protein kinase A pathway, but it has been unclear which Galpha subunit is coupled to the pheromone receptor and response pathway. Here we report that in the pathogenic basidiomycetous yeast Cryptococcus neoformans, two Galpha subunits (Gpa2, Gpa3) sense pheromone and govern mating. gpa2 gpa3 double mutants, but neither gpa2 nor gpa3 single mutants, are sterile in bilateral crosses. By contrast, deletion of GPA3 (but not GPA2) constitutively activates pheromone response and filamentation. Expression of GPA2 and GPA3 is differentially regulated: GPA3 expression is induced by nutrient-limitation, whereas GPA2 is induced during mating. Based on the phenotype of dominant active alleles, Gpa2 and Gpa3 signal in opposition: Gpa2 promotes mating, whereas Gpa3 inhibits. The incorporation of an additional Galpha into the regulatory circuit enabled increased signaling complexity and facilitated cell fate decisions involving choice between yeast growth and filamentous asexual/sexual development.     

Scanning mutagenesis of regions in the Galpha protein Gpa1 that are predicted to interact with yeast mating pheromone receptors.

The mechanism by which receptors activate heterotrimeric G proteins was examined by scanning mutagenesis of the Saccharomyces cerevisiae pheromone-responsive Galpha protein (Gpa1). The juxtaposition of high-resolution structures for rhodopsin and its cognate G protein transducin predicted that at least six regions of Galpha are in close proximity to the receptor. Mutagenesis was targeted to residues in these domains in Gpa1, which included four loop regions (beta2-beta3, alpha2-beta4, alpha3-beta5, and alpha4-beta6) as well as the N and C termini. The mutants displayed a range of phenotypes from nonsignaling to constitutive activation of the pheromone pathway. The constitutive activity of some mutants could be explained by decreased production of Gpa1, which permits unregulated signaling by Gbetagamma. However, the constitutive activity caused by the F344C and E335C mutations in the alpha2-beta4 loop and F378C in the alpha3-beta5 loop was not due to decreased protein levels, and was apparently due to defects in sequestering Gbetagamma. The strongest loss of the function mutant, which was not detectably induced by a pheromone, was caused by a K314C substitution in the beta2-beta3 loop. Several other mutations caused weak signaling phenotypes. Altogether, these results suggest that residues in different interface regions of Galpha contribute to activation of signaling.     

Directly from Galpha to protein kinase A: the kelch repeat protein bypass of adenylate cyclase

One major class of G proteins typically functions as heterotrimeric complexes consisting of Galpha, Gbeta and Ggamma subunits. However, recent work in yeast has identified an atypical Galpha protein, Gpa2p, which functions without cognate Gbetagamma subunits. Two novel kelch repeat protein binding partners of Gpa2p, Krh1p and Krh2p, do not function as alternative Gbeta subunits, as initially thought, but rather as Gpa2p effectors. They directly link Gpa2p to protein kinase A, thus forming an adenylate cyclase bypass pathway that enables inputs other than cellular cAMP concentration to affect protein kinase A activity. Because mammalian protein kinase A expressed in yeast is also subject to control by the same bypass pathway, it is exciting to postulate that a functionally similar mechanism might exist in mammalian cells, and that other Galpha proteins could exhibit similar characteristics to Gpa2p.     

The yeast G protein alpha subunit Gpa1 transmits a signal through an RNA binding effector protein Scp160

In yeast Saccharomyces cerevisiae the G protein betagamma subunits (Ste4/Ste18) have long been known to transmit the signal required for mating. Here we demonstrate that GTPase-deficient mutants of Galpha (Gpa1) directly activate the mating response pathway. We also show that signaling by activated Gpa1 requires direct coupling to an RNA binding protein Scp160. These findings suggest an additional role for Gpa1 and reveal Scp160 as a component of the mating response pathway in yeast.     

Molecular characterization of a G protein alpha-subunit-encoding gene from Mucor circinelloides

Genes encoding the Galpha subunit were cloned from Mucor circinelloides, a zygomycete dimorphic fungus. There are at least four genes that encode for Galpha subunits, gpa1, gpa2, gpa3, and gpa4. The genes gpa1 and gpa3 were isolated and characterized, and their predicted products showed 36%-67% identity with Galpha subunits from diverse fungi. Northern blot analysis of gpa3 showed that it is present in spores and constitutively expressed during mycelium development and during yeast-mycelium and mycelium-yeast transitions. However, during yeast cell growth, decreased levels of mRNA were observed. Sequence analysis of gpa3 cDNA revealed that Gpa3 encodes a polypeptide of 356 amino acids with a calculated molecular mass of 40.8 kDa. The deduced sequence of Gpa3 protein contains all the consensus regions of Galpha subunits of the Galpha(i/o/t) subfamily except the cysteine near the C terminus for potential ADP-ribosylation by pertussis toxin. This cDNA was expressed in Escherichia coli and purified by affinity chromatography. Based on its electrophoretic mobility in SDS-PAGE, the molecular mass of the His6-tagged Gpa3 was 45 kDa. The recombinant protein was recognized by a polyclonal antibody against a fragment of a human Galpha(i/o/t). Furthermore, the recombinant Gpa3 was ADP-ribosylated by activated cholera toxin and [32P]NAD but not by pertussis toxin. These results indicate that in M. circinelloides the Galpha subunit Gpa3 is expressed constitutively during differentiation.     

Glucose-induced cAMP signalling in yeast requires both a G-protein coupled receptor system for extracellular glucose detection and a separable hexose kinase-dependent sensing process

In Saccharomyces cerevisiae, glucose activation of cAMP synthesis requires both the presence of the G-protein-coupled receptor (GPCR) system, Gpr1-Gpa2, and uptake and phosphorylation of the sugar. In a hxt-null strain that lacks all physiologically important glucose carriers, glucose transport as well as glucose-induced cAMP signalling can be restored by constitutive expression of the galactose permease. Hence, the glucose transporters do not seem to have a regulatory function but are only required for glucose uptake. We established a system in which the GPCR-dependent glucose-sensing process is separated from the glucose phosphorylation process. It is based on the specific transport and hydrolysis of maltose providing intracellular glucose in the absence of glucose transport. Preaddition of a low concentration (0.7 mM) of maltose to derepressed hxt-null cells and subsequent addition of glucose restored the glucose-induced cAMP signalling, although there was no glucose uptake. Addition of a low concentration of maltose itself does not increase the cAMP level but enhances Glu6P and apparently fulfils the intracellular glucose phosphorylation requirement for activation of the cAMP pathway by extracellular glucose. This system enabled us to analyse the affinity and specificity of the GPCR system for fermentable sugars. Gpr1 displayed a very low affinity for glucose (apparent Ka = 75 mM) and responded specifically to extracellular alpha and beta D-glucose and sucrose, but not to fructose, mannose or any glucose analogues tested. The presence of the constitutively active Gpa2val132 allele in a wild-type strain bypassed the requirement for Gpr1 and increased the low cAMP signal induced by fructose and by low glucose up to the same intensity as the high glucose signal. Therefore, the low cAMP increases observed with fructose and low glucose in wild-type cells result only from the low sensitivity of the Gpr1-Gpa2 system and not from the intracellular sugar kinase-dependent process. In conclusion, we have shown that the two essential requirements for glucose-induced activation of cAMP synthesis can be fulfilled separately: an extracellular glucose detection process dependent on Gpr1 and an intracellular sugar-sensing process requiring the hexose kinases.