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1.
Biochem Biophys Res Commun ; 329(1): 281-7, 2005 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15721304

RESUMO

G-protein-coupled receptors (GPCRs) are membrane-embedded cell signaling devices transducing ligand binding to activation of heterotrimeric G-proteins, providing a paradigm for signaling for yeast and mammals alike. Probing the extent to which yeast GPCRs may couple to mammalian G-proteins has been problematic. In the current work, we explored conditions that enable the cell-surface expression of a yeast alpha-factor pheromone receptor (Ste2). When expressed in human HEK293 cells, Ste2 is shown to bind its ligand alpha-factor, to be functional and catalyze activation of the mitogen-activated protein kinase cascade, and to demonstrate agonist-induced internalization. In response to agonist Ste2 as maintained intracellularly for several hours and avoids the degradation process observed for Ste2 in yeast cells. This is the first successful demonstration of the ability to express a functional yeast GPCR in mammalian cells.


Assuntos
Bioquímica/métodos , Receptores de Peptídeos/biossíntese , Fatores de Transcrição/biossíntese , Animais , Células CHO , Catálise , Linhagem Celular , Cricetinae , Proteínas de Fluorescência Verde/metabolismo , Humanos , Immunoblotting , Ligantes , Sistema de Sinalização das MAP Quinases , Microscopia Confocal , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Receptores Adrenérgicos beta 2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Fator de Acasalamento , Transdução de Sinais , Fatores de Tempo
2.
Biochemistry ; 44(4): 1278-87, 2005 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-15667221

RESUMO

The alpha-factor receptor (Ste2p) stimulates mating of the yeast Saccharomyces cerevisiae. Ste2p belongs to the large family of G protein-coupled receptors that are characterized by seven transmembrane alpha-helices. Receptor activation is thought to involve changes in the packing of the transmembrane helix bundle. To identify residues that contribute to Ste2p activation, second-site suppressor mutations were isolated that restored function to defective receptors carrying either an F204S or Y266C substitution which affect residues at the extracellular ends of transmembrane domains 5 and 6, respectively. Thirty-five different suppressor mutations were identified. On their own, these mutations caused a range of phenotypes, including hypersensitivity, constitutive activity, altered ligand binding, and loss of function. The majority of the mutations affected residues in the transmembrane segments that are predicted to face the helix bundle. Many of the suppressor mutations caused constitutive receptor activity, suggesting they improved receptor function by partially restoring the balance between the active and inactive states. Analysis of mutations in transmembrane domain 7 implicated residues Ala281 and Thr282 in receptor activation. The A281T and T282A mutants were supersensitive to S. cerevisiae alpha-factor, but were defective in responding to a variant of alpha-factor produced by another species, Saccharomyces kluyveri. The A281T mutant also displayed 8.7-fold enhanced basal signaling. Interestingly, Ala281 and Thr282 are situated in approximately the same position as Lys296 in rhodopsin, which is covalently linked to retinal. These results suggest that transmembrane domain 7 plays a role in receptor activation in a wide range of G protein-coupled receptors from yeast to humans.


Assuntos
Mutagênese , Peptídeos/química , Receptores Acoplados a Proteínas G/genética , Receptores de Peptídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Alanina/genética , Sequência de Aminoácidos , Cisteína/genética , Análise Mutacional de DNA , Genes Supressores , Testes Genéticos , Fator de Acasalamento , Dados de Sequência Molecular , Peptídeos/fisiologia , Fenilalanina/genética , Receptores Acoplados a Proteínas G/fisiologia , Receptores de Fator de Acasalamento , Receptores de Peptídeos/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Serina/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Treonina/genética , Fatores de Transcrição/genética , Tirosina/genética
3.
Mol Cell Biol ; 24(5): 2041-51, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14966283

RESUMO

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.


Assuntos
Estrutura Secundária de Proteína , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição , Sequência de Aminoácidos , Sítios de Ligação , Biotina/química , Biotina/metabolismo , Divisão Celular/fisiologia , Cisteína/metabolismo , Genes Reporter , Ligantes , Mesilatos/química , Mesilatos/metabolismo , Modelos Moleculares , Mutação , Fenótipo , Feromônios/metabolismo , Ligação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores de Fator de Acasalamento , Receptores de Peptídeos/química , Receptores de Peptídeos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
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