G protein-coupled receptor Gpr4 senses amino acids and activates the cAMP-PKA pathway in Cryptococcus neoformans

The Galpha protein Gpa1 governs the cAMP-PKA signaling pathway and plays a central role in virulence and differentiation in the human fungal pathogen Cryptococcus neoformans, but the signals and receptors that trigger this pathway were unknown. We identified seven putative proteins that share identity with known G protein-coupled receptors (GPCRs). One protein, Gpr4, shares limited sequence identity with the Dictyostelium discoideum cAMP receptor cAR1 and the Aspergillus nidulans GPCR protein GprH and also shares structural similarity with the Saccharomyces cerevisiae receptor Gpr1. gpr4 mutants exhibited reduced capsule production and mating defects, similar to gpa1 mutants, and exogenous cAMP suppressed both gpr4 mutant phenotypes. Epistasis analysis provides further evidence that Gpr4 functions upstream of the Galpha subunit Gpa1. Gpr4-Gpr4 homomeric interactions were observed in the yeast two-hybrid assay, and Gpr4 was shown to physically interact with Gpa1 in the split-ubiquitin system. A Gpr4::DsRED fusion protein was localized to the plasma membrane and methionine was found to trigger receptor internalization. The analysis of intracellular cAMP levels showed that gpr4 mutants still respond to glucose but not to certain amino acids, such as methionine. Amino acids might serve as ligands for Gpr4 and could contribute to engage the cAMP-PKA pathway. Activation of the cAMP-PKA pathway by glucose and amino acids represents a nutrient coincidence detection system shared in other pathogenic fungi.     

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.     

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.     

Phospholipase C interacts with Sgd1p and is required for expression of <Emphasis Type="Italic">GPD1</Emphasis> and osmoresistance in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The Saccharomyces cerevisiae PLC1 gene encodes a homolog of the delta isoform of mammalian phosphoinositide-specific phospholipase C. Cells deleted for PLC1 ( plc1Delta) are viable, but display several phenotypes, including osmotic, temperature, and nocodazole sensitivity. We have used a two-hybrid screen to identify Plc1p-interacting proteins. One of the interacting proteins found was Sgd1p, a recently identified, essential, nuclear protein. The SGD1 gene was originally cloned by complementation of an osmostress-sensitive mutant. The Plc1p-Sgd1p interaction was confirmed biochemically by affinity chromatography. SGD1 interacts genetically with both PLC1 and HOG1 (which encodes an osmosensing mitogen-activated protein kinase). Overexpression of Sgd1p suppresses the temperature sensitivity of cells bearing the plc1-4 allele, and the double mutant strain plc1Delta sgd1-1 displays enhanced temperature and nocodazole sensitivity. The plc1Delta hog1Delta strain displays increased osmosensitivity, and has a synthetic defect in glycerol synthesis and the expression of GPD1 (which encodes the enzyme glycerol 3-phosphate dehydrogenase that is involved in glycerol biosynthesis), suggesting that Plc1p and Hog1p function in independent pathways. The hog1Delta sgd1-1 double mutant displays enhanced osmosensitivity relative to that of either single mutant. The triple mutant plc1Delta hog1Delta sgd1-1 is inviable, while the plc1Delta hog1Delta sgd1-2 strain grows extremely slowly and is more osmosensitive than the plc1Delta hog1Delta or hog1Delta sgd1-2 strain. These results are consistent with a model in which Plc1p and Hog1p function in parallel pathways affecting osmoregulation, and signals from both these pathways converge, at least partly, on Sgd1p.     

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.     

Phospholipase C is involved in kinetochore function in Saccharomyces cerevisiae

The budding yeast PLC1 gene encodes a homolog of the delta isoform of mammalian phosphoinositide-specific phospholipase C. Here, we present evidence that Plc1p associates with the kinetochore complex CBF3. This association is mediated through interactions with two established kinetochore proteins, Ndc10p and Cep3p. We show by chromatin immunoprecipitation experiments that Plc1p resides at centromeric loci in vivo. Deletion of PLC1, as well as plc1 mutations which abrogate the interaction of Plc1p with the CBF3 complex, results in a higher frequency of minichromosome loss, nocodazole sensitivity, and mitotic delay. Overexpression of Ndc10p suppresses the nocodazole sensitivity of plc1 mutants, implying that the association of Plc1p with CBF3 is important for optimal kinetochore function. Chromatin extracts from plc1Delta cells exhibit reduced microtubule binding to minichromosomes. These results suggest that Plc1p associates with kinetochores and regulates some aspect of kinetochore function and demonstrate an intranuclear function of phospholipase C in eukaryotic cells.     

Plc1p is required for SAGA recruitment and derepression of Sko1p-regulated genes

In Saccharomyces cerevisiae, many osmotically inducible genes are regulated by the Sko1p-Ssn6p-Tup1p complex. On osmotic shock, the MAP kinase Hog1p associates with this complex, phosphorylates Sko1p, and converts it into an activator that subsequently recruits Swi/Snf and SAGA complexes. We have found that phospholipase C (Plc1p encoded by PLC1) is required for derepression of Sko1p-Ssn6p-Tup1p-controlled osmoinducible genes upon osmotic shock. Although plc1Delta mutation affects the assembly of the preinitiation complex after osmotic shock, it does not affect the recruitment of Hog1p and Swi/Snf complex at these promoters. However, Plc1p facilitates osmotic shock-induced recruitment of the SAGA complex. Like plc1Delta cells, SAGA mutants are osmosensitive and display compromised expression of osmotically inducible genes. The reduced binding of SAGA to Sko1p-Ssn6p-Tup1p-repressed promoters in plc1Delta cells does not correlate with reduced histone acetylation. However, SAGA functions at these promoters to facilitate recruitment of the TATA-binding protein. The results thus provide evidence that Plc1p and inositol polyphosphates affect derepression of Sko1p-Ssn6p-Tup1p-controlled genes by a mechanism that involves recruitment of the SAGA complex and TATA-binding protein.     

Genetic evidence for a role of phospholipase C at the budding yeast kinetochore

Chromosome segregation during mitosis requires kinetochores, specialized organelles that mediate chromosome attachment to spindle microtubules. We have shown previously that in budding yeast, Plc1p (phosphoinositide-specific phospholipase C) localizes to centromeric loci, associates with the kinetochore proteins Ndc10p and Cep3p, and affects the function of kinetochores. Deletion of PLC1 results in nocodazole sensitivity, mitotic delay, and a higher frequency of chromosome loss. We report here that despite the nocodazole sensitivity of plc1Delta cells, Plc1p is not required for the spindle checkpoint. However, plc1Delta cells require a functional BUB1/BUB3-dependent spindle checkpoint for viability. PLC1 displays strong genetic interactions with genes encoding components of the inner kinetochore, including NDC10, SKP1, MIF2, CEP1, CEP3, and CTF13. Furthermore, plc1Delta cells display alterations in chromatin structure in the core centromere. Chromatin immunoprecipitation experiments indicate that Plc1p localizes to centromeric loci independently of microtubules, and accumulates at the centromeres during G(2)/M stage of cell cycle. These results are consistent with the view that Plc1p affects kinetochore function, possibly by modulating the structure of centromeric chromatin.     

Diacylglycerol and its formation by phospholipase C regulate Rab- and SNARE-dependent yeast vacuole fusion

Although diacylglycerol (DAG) can trigger liposome fusion, biological membrane fusion requires Rab and SNARE proteins. We have investigated whether DAG and phosphoinositide-specific phospholipase C (PLC) have a role in the Rab- and SNARE-dependent homo-typic vacuole fusion in Saccharomyces cerevisiae. Vacuole fusion was blocked when DAG was sequestered by a recombinant C1b domain. DAG underwent ATP-dependent turnover during vacuole fusion, but was replenished by the hydrolysis of phosphatidylinositol 4,5-bisphosphate to DAG by PLC. The PLC inhibitors 3-nitrocoumarin and U73122 blocked vacuole fusion in vitro, whereas their inactive homologues did not. Plc1p is the only known PLC in yeast. Yeast cells lacking the PLC1 gene have many small vacuoles, indicating defects in protein trafficking to the vacuole or vacuole fusion, and purified Plc1p stimulates vacuole fusion. Docking-dependent Ca(2+) efflux is absent in plc1Delta vacuoles and was restored only upon the addition of both Plc1p and the Vam7p SNARE. However, vacuoles purified from plc1Delta strains still retain PLC activity and significant 3-nitrocoumarin- and U73122-sensitive fusion, suggesting that there is another PLC in S. cerevisiae with an important role in vacuole fusion.     

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.     

Phosphoinositide-specific phospholipase C forms a complex with 14-3-3 proteins and is involved in expression of UV resistance in fission yeast

The fission yeast plc1 ⁺ gene encodes phosphoinositide-specific phospholipase C. The two- hybrid interaction assay with plexA-plc1 ⁺ as a bait revealed that Plc1p interacted with the 14-3-3 proteins Rad24p and Rad25p. Formation of a complex containing Plc1p and Rad24p in vivo was confirmed by an immunological method. As predicted from the fact that rad24 null mutant cells are hypersensitive to UV irradiation, plc1 null mutant cells were almost as sensitive to UV irradiation as rad24 null mutant cells. In addition, deletion of rad24 in the plc1 null mutant cells did not enhance the UV sensitivity, indicating that plc1 ⁺ and rad24 ⁺ belong to the same epistasis group with respect to UV sensitivity. Whereas Rad24p has been reported to be involved in the DNA damage checkpoint pathway, the delay to mitosis after UV irradiation was not defective either in rad24 null mutant cells or in plc1 null mutant cells in our analysis. Thus, Plc1p is responsible for resistance to UV irradiation, but not for the DNA damage checkpoint pathway, in cooperation with 14-3-3 proteins.     

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.     

Phosphoinositide-specific phospholipase C forms a complex with 14-3-3 proteins and is involved in expression of UV resistance in fission yeast

The fission yeast plc1 ⁺ gene encodes phosphoinositide-specific phospholipase C. The two- hybrid interaction assay with plexA-plc1 ⁺ as a bait revealed that Plc1p interacted with the 14-3-3 proteins Rad24p and Rad25p. Formation of a complex containing Plc1p and Rad24p in vivo was confirmed by an immunological method. As predicted from the fact that rad24 null mutant cells are hypersensitive to UV irradiation, plc1 null mutant cells were almost as sensitive to UV irradiation as rad24 null mutant cells. In addition, deletion of rad24 in the plc1 null mutant cells did not enhance the UV sensitivity, indicating that plc1 ⁺ and rad24 ⁺ belong to the same epistasis group with respect to UV sensitivity. Whereas Rad24p has been reported to be involved in the DNA damage checkpoint pathway, the delay to mitosis after UV irradiation was not defective either in rad24 null mutant cells or in plc1 null mutant cells in our analysis. Thus, Plc1p is responsible for resistance to UV irradiation, but not for the DNA damage checkpoint pathway, in cooperation with 14-3-3 proteins. Received: 10 July 1997 / Accepted: 15 December 1997     

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.     

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.