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Results for “Receptors, Mating Factor”

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At least 19 recordsLinked to original sources

PubMed2001

Probing the binding domain of the Saccharomyces cerevisiae alpha-mating factor receptor with rluorescent ligands.

Three analogues of the alpha-mating factor pheromone of Saccharomyces cerevisiae containing the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group were synthesized that had high binding affinity to the receptor and retained biological activity. The fluorescence emission maximum of the NBD group in [K7(NBD),Nle(12)]-alpha-factor was blue shifted by 35 nm compared to buffer when the pheromone bound to its receptor. Fluorescence quenching experiments revealed that the NBD group in [K7(NBD),Nle(12)]-alpha-factor bound to the receptor was shielded from collision with iodide anion when in aqueous buffer. In contrast, the emission maximum of NBD in [K7(ahNBD),Nle(12)]-alpha-factor or [Orn7(NBD),Nle(12)]-alpha-factor was not significantly shifted and iodide anion efficiently quenched the fluorescence of these derivatives when they were bound to receptor. The fluorescence investigation suggests that when the alpha-factor is bound to its receptor, K7 resides in an environment that has both hydrophobic and hydrophilic groups within a few angstroms of each other.

4-Chloro-7-nitrobenzofurazan↗

PubMed1992

Yeast alpha-mating factor receptor and G-protein-linked adenylyl cyclase inhibition requires RAS2 and GPA2 activities.

Saccharomyces cerevisiae expresses two RAS gene products (RAS1 and RAS2) highly homologous to mammalian p21ras which mediate glucose-stimulated cyclic-AMP formation. Mating pheromone inhibits RAS-linked adenylyl cyclase activation and this is dependent upon the alpha-factor receptor (STE2) and its associated G-protein beta-subunit (STE4). We now show that this pheromone effect is independent of mating pathway signalling components "downstream" of STE4 but displays an absolute requirement for an additional G-protein alpha-subunit encoded by GPA2. alpha-mating factor effects also involve a specific suppression of normal RAS2 activity as the constitutively activated mutant RAS2vall9 as well as wild type. RAS1 are insensitive to inhibition. Interaction between GPA2, STE4-STE18, RAS2 and adenylyl cyclase in yeast could give important insight into signalling pathways controlling normal and oncogenic p21ras activity in man.

Adenylyl Cyclase Inhibitors↗

PubMed2001

ATR-FTIR study of the structure and orientation of transmembrane domains of the Saccharomyces cerevisiae alpha-mating factor receptor in phospholipids.

The structures of seven synthetic transmembrane domains (TMDs) of the alpha-factor receptor (Ste2p) from Saccharomyces cerevisiae were studied in phospholipid multilayers by transmission Fourier transform infrared (FTIR) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopies. Peptide conformation assumed in multilayers depended on the method of sample preparation. Amide proton H/D exchange experiments showed that 60-80% of the NH bonds in these TMDs did not exchange with bulk water in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) multilayers. FTIR results showed that peptides corresponding to TMDs one, two, and seven were mostly alpha-helical in DMPC multilayers. Peptides corresponding to TMDs three and six assumed predominantly beta-sheet structures, whereas those corresponding to TMDs four and five were a mixture of alpha-helices and beta-sheets. ATR-FTIR showed that in DMPC the alpha-helices of TMDs two and five oriented with tilt angles of 34 degrees and 32 degrees, respectively, with respect to the multilayer normal. Similar results were obtained for six of the transmembrane domains in DMPC/DMPG (4:1) multilayers. In a mixture [POPC/POPE/POPS/PI/ergosterol (30:20:5:20:25)] which mimicked the lipid composition of the S. cerevisiae cell membrane, the percentage of alpha-helical structures found for TMDs one and five increased compared to those in DMPC and DMPC/DMPG (4:1) multilayers, and TMD six exhibited a mixture of beta-sheet ( approximately 60%) and alpha-helical ( approximately 40%) structure. These experiments provide biophysical evidence that peptides representing the seven transmembrane domains in Ste2p assume different structures and tilt angles within a membrane multilayer.

Amides↗

PubMedDate not supplied

Signal transduction in yeast mating: receptors, transcription factors, and the kinase connection.

In the yeast Saccharomyces cerevisiae, peptide mating pheromones activate a signal transduction pathway that leads to cellular differentiation and cell division cycle arrest. It is now possible to trace the major events of this pathway. binding of pheromone to G-protein-coupled receptors activates a cascade of serine/threonine protein kinases that ultimately phosphorylate and increase the activity of an identified transcription factor.

Cell Cycle↗

PubMed1989

The STE4 and STE18 genes of yeast encode potential beta and gamma subunits of the mating factor receptor-coupled G protein.

The STE4 and STE18 genes are required for haploid yeast cell mating. Sequencing of the cloned genes revealed that the STE4 polypeptide shows extensive homology to the beta subunits of mammalian G proteins, while the STE18 polypeptide shows weak similarity to the gamma subunit of transducin. Null mutations in either gene can suppress the haploid-specific cell-cycle arrest caused by mutations in the SCG1 gene (previously shown to encode a protein with similarity to the alpha subunit of G proteins). We propose that the products of the STE4 and STE18 genes comprise the beta and gamma subunits of a G protein complex coupled to the mating pheromone receptors. The genetic data suggest pheromone-receptor binding leads to the dissociation of the alpha subunit from beta gamma (as shown for mammalian G proteins), and the free beta gamma element initiates the pheromone response.

Amino Acid Sequence↗

PubMed2001

Mutational analysis of the role of N-glycosylation in alpha-factor receptor function.

The alpha-factor mating pheromone receptor (encoded by STE2) activates a G protein signaling pathway that stimulates the conjugation of Saccharomyces cerevisiae yeast cells. The alpha-factor receptor is known to undergo several forms of post-translational modification, including phosphorylation, mono-ubiquitination, and N-linked glycosylation. Since phosphorylation and mono-ubiquitination have been shown previously to play key roles in regulating the signaling activity and membrane trafficking of the alpha-factor receptors, the role of N-linked glycosylation was investigated in this study. The Asn residues in the five consensus sites for N-linked glycosylation present in the extracellular regions of the receptor protein were mutated to prevent carbohydrate attachment at these sites. Mutation of two sites near the receptor N-terminus (N25Q and N32Q) diminished the degree of receptor glycosylation, and the corresponding double mutant was not detectably N-glycosylated. The nonglycosylated receptors displayed normal function and subcellular localization, indicating that glycosylation is not important for wild-type receptor activity. However, mutation of the glycosylation sites resulted in improved plasma membrane localization for the Ste2-3 mutant receptors that are normally retained intracellularly at elevated temperatures. These results suggest that N-glycosylation may be involved in the sorting process for misfolded Ste2 proteins, and may similarly affect certain mutant receptors whose altered trafficking is implicated in human diseases.

Amino Acid Sequence↗

PubMed1991

Yeast alpha-mating factor receptor-linked G-protein signal transduction suppresses Ras-dependent activity.

Homologues of mammalian Ras conserved in Saccharomyces cerevisiae mediate glucose-stimulated cyclic AMP formation and we used this response to test for regulation of yeast Ras activity by the alpha-mating factor signal transduction pathway. alpha-Mating factor suppresses glucose-stimulated cyclic AMP formation by up to 57 +/- 12.6% (n = 5) and similar inhibition was observed in four different yeast strains (MATa cells). Moreover, this response is potent (IC50 = 0.14 +/- 0.19 microM (n = 4)), rapid (maximal within 1-2 min), and displays an absolute requirement for both the alpha-mating factor receptor (STE2) and associated G-protein beta-subunit (STE4). Inhibition appears independent of both phosphodiesterase activation and alpha-mating factor-stimulated cytoplasmic alkalinization. Also, basal cyclic AMP levels are unaffected by pheromone. This is the first demonstration that a cell-surface receptor linked to a heterotrimeric G-protein can suppress Ras-dependent activity and could provide important insight into mechanisms controlling p21ras in man. Inhibition of Ras-dependent cyclic AMP formation could also be a key event facilitating responses characteristic of yeast mating.

Cyclic AMP↗

PubMed2002

Mutagenic mapping of helical structures in the transmembrane segments of the yeast alpha-factor receptor.

The alpha-mating pheromone receptor encoded by the yeast STE2 gene is a G protein coupled receptor that initiates signaling via a MAP kinase pathway that prepares haploid cells for mating. To establish the range of allowed amino acid substitutions within transmembrane segments of this receptor, we conducted extensive random mutagenesis of receptors followed by screening for receptor function. A total of 157 amino acid positions in seven different mutagenic libraries corresponding to the seven predicted transmembrane segments were analyzed, yielding 390 alleles that retain at least 60 % of normal signaling function. These alleles contained a total of 576 unique amino acid substitutions, including 61 % of all the possible amino acid changes that can arise from single base substitutions. The receptor exhibits a surprising tolerance for amino acid substitutions. Every amino acid in the mutagenized regions of the transmembrane regions could be substituted by at least one other residue. Polar amino acids were tolerated in functional receptors at 115 different positions (73 % of the total). Hydrophobic amino acids were tolerated in functional receptors at all mutagenized positions. Substitutions introducing proline residues were recovered at 53 % of all positions where they could be brought about by single base changes. Residues with charged side-chains could also be tolerated at 53 % of all positions where they were accessible through single base changes. The spectrum of allowed amino acid substitutions was characterized in terms of the hydrophobicity, radius of gyration, and charge of the allowed substitutions and mapped onto alpha-helical structures. By comparing the patterns of allowed substitutions with the recently determined structure of rhodopsin, structural features indicative of helix-helix interactions can be discerned in spite of the extreme sequence divergence between these two proteins.

Alleles↗

PubMed1999

Heterologous gene expression in a membrane-protein-specific system.

We have constructed an expression system for heterologous proteins which uses the molecular machinery responsible for the high level production of bacteriorhodopsin in Halobacterium salinarum. Cloning vectors were assembled that fused sequences of the bacterio-opsin gene (bop) to coding sequences of heterologous genes and generated DNA fragments with cloning sites that permitted transfer of fused genes into H. salinarum expression vectors. Gene fusions include: (i) carboxyl-terminal-tagged bacterio-opsin; (ii) a carboxyl-terminal fusion with the catalytic subunit of the Escherichia coli aspartate transcarbamylase; (iii) the human muscarinic receptor, subtype M1; (iv) the human serotonin receptor, type 5HT2c; and (v) the yeast alpha mating factor receptor, Ste2. Characterization of the expression of these fusions revealed that the bop gene coding region contains previously undescribed molecular determinants which are critical for high level expression. For example, introduction of immunogenic and purification tag sequences into the C-terminal coding region significantly decreased bop gene mRNA and protein accumulation. The bacteriorhodopsin-aspartate transcarbamylase fusion protein was expressed at 7 mg per liter of culture, demonstrating that E. coli codon usage bias did not limit the system's potential for high level expression. The work presented describes initial efforts in the development of a novel heterologous protein expression system, which may have unique advantages for producing multiple milligram quantities of membrane-associated proteins.

Amino Acid Sequence↗

PubMed1997

Ubiquitin-dependent internalization and down-regulation of plasma membrane proteins.

The modification of cytosolic proteins with polyubiquitin chains targets them for recognition and degradation by the multisubunit proteolytic particle, the 26S proteasome. Membrane proteins are also substrates for ubiquitination. Integral membrane proteins of the endoplasmic reticulum are ubiquitinated and destroyed by the proteasome. However, it has been shown recently that the ubiquitination of Saccharomyces cerevisiae plasma membrane proteins signals their degradation by the proteolytic system in the lysosome-like vacuole. Ubiquitination of several different classes of cell surface proteins serves as a signal for their entry into the endocytic pathway; this leads to their transport to the vacuole, where they are permanently inactivated by degradation. In yeast, ubiquitin has been implicated as an internalization signal for most, if not all, endogenous plasma membrane proteins that are known to be endocytosed. Ubiquitin-dependent internalization has been best characterized for two proteins: the mating pheromone alpha-factor receptor and the uracil permease. Some mammalian cell surface receptors are also ubiquitinated at the plasma membrane. Ubiquitination machinery is required for ligand-induced endocytosis of the growth hormone receptor, suggesting that ubiquitin-dependent endocytosis and sorting is also an important regulatory process in mammalian cells. Mammalian receptors may also be down-regulated through the degradation of their cytosolic domains by a proteasome-dependent pathway.

Animals↗

PubMed2001

Functional analysis of the C-terminal cytoplasmic region of the M-factor receptor in fission yeast.

BACKGROUND: Yeast mating-pheromone receptors facilitate the study of G protein-coupled signal transduction. To date, molecular dissection of the budding yeast alpha-factor receptor has been done extensively, but little analysis has been performed with pheromone receptors of fission yeast, another genetically tractable yeast species. RESULTS: We analysed the fission yeast M-factor receptor Map3p. Truncation of the C-terminal 54 amino acids made Map3p dominant-negative over the wild-type. This form, called Map3-dn9p, was competent in the induction of pheromone-dependent gene expression, although it could not direct proper conjugation. Map3-dn9p failed both to provoke the orientated projection of conjugation tubes and to induce adaptation to the pheromone signal associated with endocytosis of the receptor. Deletion and substitution analyses suggested that the integrity of the C-terminal region, rather than a specific subgroup of amino acid residues therein, was vital for the respective Map3p activities. Ubiquitination of the C-terminus was not absolutely essential for Map3p function. CONCLUSIONS: The C-terminal region of Map3p is dispensable for the pheromone signalling per se, but is pivotal for adaptation and pheromone-induced conjugation tube formation, as is true with the budding yeast alpha-factor receptor. However, the mechanisms which induce adaptation appear to differ between fission and budding yeast concerning the necessity of ubiquitination.

Alleles↗

PubMed1989

GPA1Val-50 mutation in the mating-factor signaling pathway in Saccharomyces cerevisiae.

The GPA1 gene of Saccharomyces cerevisiae encodes a protein that is highly homologous to the alpha subunit of mammalian hetrotrimeric G proteins and is essential for haploid cell growth. A mutation of the GPA1 protein, GPA1Val-50, in which Gly-50 was replaced by valine, could complement the growth defect of a GPA1 disruption, gpal::HIS3. However, cells with gpa1::HIS3 expressing the GPA1Val-50 protein were supersensitive to alpha-factor in a short-term incubation but resumed growth after long-term incubation even after exposure to high concentrations of alpha-factor. The former phenotype associated with GPA1Val-50 is recessive, and the latter phenotype is dominant to GPA1+. The supersensitivity of GPA1Val-50 to alpha-factor was dependent on STE2 and STE4, which demonstrates that this GPA1Val-50-produced phenotype requires the mating-factor receptor and the beta subunit of the G protein. The double mutant of sst2-1 GPA1Val-50 recovered from division arrest, which suggested that SST2 is not required for recovery of the GPA1Val-50 mutant.

Amino Acid Sequence↗

PubMed2004

A microdomain formed by the extracellular ends of the transmembrane domains promotes activation of the G protein-coupled alpha-factor receptor.

The alpha-factor receptor (Ste2p) that promotes mating in Saccharomyces cerevisiae is similar to other G protein-coupled receptors (GPCRs) in that it contains seven transmembrane domains. Previous studies suggested that the extracellular ends of the transmembrane domains are important for Ste2p function, so a systematic scanning mutagenesis was carried out in which 46 residues near the ends of transmembrane domains 1, 2, 3, 4, and 7 were replaced with cysteine. These mutants complement mutations constructed previously near the ends of transmembrane domains 5 and 6 to analyze all the extracellular ends. Eight new mutants created in this study were partially defective in signaling (V45C, N46C, T50C, A52C, L102C, N105C, L277C, and A281C). Treatment with 2-([biotinoyl] amino) ethyl methanethiosulfonate, a thiol-specific reagent that reacts with accessible cysteine residues but not membrane-embedded cysteines, identified a drop in the level of reactivity over a consecutive series of residues that was inferred to be the membrane boundary. An unusual prolonged zone of intermediate reactivity near the extracellular end of transmembrane domain 2 suggests that this region may adopt a special structure. Interestingly, residues implicated in ligand binding were mainly accessible, whereas residues involved in the subsequent step of promoting receptor activation were mainly inaccessible. These results define a receptor microdomain that provides an important framework for interpreting the mechanisms by which functionally important residues contribute to ligand binding and activation of Ste2p and other GPCRs.

Amino Acid Sequence↗

PubMed1994

Biophysical studies on fragments of the alpha-factor receptor protein.

The receptor for the alpha-factor mating pheromone of the yeast Saccharomyces cerevisiae consists of 431 amino acid residues and is a member of a family of membrane proteins predicted to have seven transmembrane helices. Fragments of the receptor corresponding to two of the transmembrane helices [residues 246-269 (M6) and 273-302 (M7)], two of the interhelical loops [residues 107-125 (E2) and 191-206 (E3)], and to a portion of the carboxyl terminus [residues 350-372 (CT)] were synthesized using solid-phase methodologies and purified to near homogeneity. CD was used to characterize the secondary structure of these peptides in trifluoroethanol (TFE), in TFE/water mixtures, in sodium dodecyl sulfate (SDS), and in the presence of dimyristoyl phosphatidylcholine (DMPC) liposomes. In TFE, M6 and M7 exhibited CD spectra consistent with highly helical peptides, whereas CT was partially helical. In contrast, E2 and E3 were either disordered or aggregated in this solvent. M6 did not partition well into DMPC vesicles whereas M7 remained helical. Both M6 and M7 assumed helical conformations in 25 mM SDS. The loop peptides and the carboxyl terminus peptide were either in a beta-structure or disordered in the presence of lipid. These findings represent the first biophysical evidence for conformations assumed by specific segments of the STE2 receptor protein.

Amino Acid Sequence↗

PubMed1997

Roles of Wee1 and Nim1 protein kinases in regulating the switch from mitotic division to sexual development in Schizosaccharomyces pombe.

In self-fertile strains of the fission yeast Schizosaccharomyces pombe, nitrogen starvation initiates a program of sexual development in which cells express mating pheromones and receptors, arrest cell cycle progression in G1, and conjugate. This process is dependent on Rum1, an inhibitor of the Cdc2-Cdc13 and Cdc2-Cig2 cyclin B kinases. The M-phase induction activity of Cdc2-Cdc13 is inhibited by Wee1 tyrosine kinase, which phosphorylates Cdc2 on tyrosine-15. We report here that Wee1 activity is also important for mating. This discovery arose from studies of Nim1, a kinase which promotes mitosis by inhibiting Wee1. Nim1 was previously thought to have an important role in promoting mitosis during nitrogen starvation, but our studies revealed that Nim1 protein drops to an undetectable level within 15 min of nitrogen depletion. In contrast, Wee1 remains abundant, and tyrosine-phosphorylated Cdc2 is detected for at least 4 h after resuspension of cells in nitrogen-free medium. This suggested that maintenance of Wee1 activity may be important during the early stages of nitrogen starvation, a proposal confirmed by the observation that mating efficiency is reduced ca. fivefold in wee1- cells. Transcriptional induction of genes encoding mating factors and receptors is also delayed in wee1- cells. The wee1- mating defect is suppressed by deletion of cig2+, which encodes a B-type cyclin that promotes the onset of S and inhibits conjugation. These findings indicate that Wee1 and Rum1 act jointly to inhibit Cdc2 and promote sexual development in nitrogen-starved cells.

Ammonium Chloride↗

PubMed2000

Synthesis and biophysical analysis of transmembrane domains of a Saccharomyces cerevisiae G protein-coupled receptor.

The Ste2p receptor for alpha-factor, a tridecapeptide mating pheromone of the yeast Saccharomyces cerevisiae, belongs to the G protein-coupled family of receptors. In this paper we report on the synthesis of peptides corresponding to five of the seven transmembrane domains (M1-M5) and two homologues of the sixth transmembrane domain corresponding to the wild-type sequence and a mutant sequence found in a constitutively active receptor. The secondary structures of all new transmembrane peptides and previously synthesized peptides corresponding to domains 6 and 7 were assessed using a detailed CD analysis in trifluoroethanol, trifluoroethanol-water mixtures, sodium dodecyl sulfate micelles, and dimyristoyl phosphatidyl choline bilayers. Tryptophan fluorescence quenching experiments were used to assess the penetration of the membrane peptides into lipid bilayers. All peptides were predominantly (40-80%) helical in trifluoroethanol and most trifluoroethanol-water mixtures. In contrast, two of the peptides M3-35 (KKKNIIQVLLVASIETSLVFQIKVIFTGDNFKKKG) and M6-31 (KQFDSFHILLINleSAQSLLVPSIIFILAYSLK) formed stable beta-sheet structures in both sodium dodecyl sulfate micelles and DMPC bilayers. Polyacrylamide gel electrophoresis showed that these two peptides formed high molecular aggregates in the presence of SDS whereas all other peptides moved as monomeric species. The peptide (KKKFDSFHILLIMSAQSLLVLSIIFILAYSLKKKS) corresponding to the sequence in the constitutive mutant was predominantly helical under a variety of conditions, whereas the homologous wild-type sequence (KKKFDSFHILLIMSAQSLLVPSIIFILAYSLKKKS) retained a tendency to form beta-structures. These results demonstrate a connection between a conformational shift in secondary structure, as detected by biophysical techniques, and receptor function. The aggregation of particular transmembrane domains may also reflect a tendency for intermolecular interactions that occur in the membrane environment facilitating formation of receptor dimers or multimers.

Amino Acid Sequence↗

PubMed2001

Peptide fragments as models to study the structure of a G-protein coupled receptor: the alpha-factor receptor of Saccharomyces cerevisiae.

The alpha-factor tridecapeptide initiates mating in Saccharomyces cerevisiae upon interaction with Ste2p, its cognate G-protein coupled receptor (GPCR). This interaction is being used as a paradigm for understanding the structure and mechanism of activation of GPCRs by medium-sized peptides. In this article, the use of fragments of Ste2p to study its structure is reviewed. Methods of synthesis of peptides corresponding to both extramembranous and transmembrane domains of Ste2p are evaluated and problems that are encountered during synthesis and purification are described. The results from conformational analyses of the peptide fragments using fluorescence spectroscopy, CD, infrared spectroscopy, and NMR spectroscopy in organic-aqueous mixtures and in the presence of detergent micelles and lipid bilayers are critically reviewed. The data obtained to date provide biophysical evidence for the structure of different domains of Ste2p and indicate that peptides corresponding to these domains have unique biophysical tendencies. The studies carried out on Ste2p fragments indicate that valuable information concerning the structure of the intact receptor can be obtained by studying peptide fragments corresponding to domains of these polytopic integral membrane proteins.

Amino Acid Sequence↗

PubMed2004

The alpha-factor mating pheromone of Saccharomyces cerevisiae: a model for studying the interaction of peptide hormones and G protein-coupled receptors.

Mating in Saccharomyces cerevisiae is initiated by the secretion of diffusible peptide pheromones that are recognized by G protein-coupled receptors (GPCR). This review summarizes the use of the alpha-factor (WHWLQLKPGQPMY)--GPCR (Ste2p) interaction as a paradigm to understand the recognition between medium-sized peptide hormones and their cognate receptors. Studies over the past 15 years have indicated that the alpha-factor is bent around the center of the pheromone and that residues near the amine terminus play a central role in triggering signal transduction. The bend in the center appears not to be rigid and this flexibility is likely necessary for conformational changes that occur as the receptor switches from the inactive to active state. The results of synthetic, biological, biochemical, molecular biological, and biophysical analyses have led to a preliminary model for the structure of the peptide bound to its receptor. Antagonists for Ste2p have changes near the N-terminus of alpha-factor, and mutated forms of Ste2p were discovered that appear to favor binding of these antagonists relative to agonists. Many features of this yeast recognition system are relevant to and have counterparts in mammalian cells.

Alanine↗