Accessibility of cysteine residues substituted into the cytoplasmic regions of the alpha-factor receptor identifies the intracellular residues that are available for G protein interaction.
The yeast alpha-factor pheromone receptor (Ste2) belongs to the family of G protein-coupled receptors (GPCRs) that contain seven transmembrane domains. To define the residues that are accessible to the cytoplasmic G protein, Cys scanning mutagenesis was carried out in which each of the residues that span the intracellular loops and the cytoplasmic end of transmembrane domain 7 was substituted with Cys. The 90 different Cys-substituted residues were then assayed for reactivity with MTSEA-biotin [[2-[(biotinoyl)amino]ethyl]methanethiosulfonate], which reacts with solvent-accessible sulfhydryl groups. As part of these studies we show that adding free Cys to stop the MTSEA-biotin reactions has potential pitfalls in that Cys can rapidly undergo disulfide exchange with the biotinylated receptor proteins at pH >or=7. The central regions of the intracellular loops of Ste2 were all highly accessible to MTSEA-biotin. Residues near the ends of the loops typically exhibited a drop in the level of reactivity over a consecutive series of residues that was inferred to be the membrane boundary. Interestingly, these boundary residues were enriched in hydrophobic residues, suggesting that they may form a hydrophobic pocket for interaction with the G protein. Comparison with accessibility data from a previous study of the extracellular side of Ste2 indicates that the transmembrane domains vary in length, consistent with some transmembrane domains being tilted relative to the plane of the membrane as they are in rhodopsin. Altogether, these results define the residues that are accessible to the G protein and provide an important structural framework for the interpretation of the role of Ste2 residues that function in G protein activation.
Choi
Yunsook
Y
Graduate Program in Physiology and Biophysics, State University of New York, Stony Brook, New York 11794-5222, USA.
Konopka
James B
JB
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics*; D003593:Cytoplasm / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D007424:Intracellular Fluid / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D025941:Protein Interaction Mapping; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051964:Receptors, Mating Factor / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D051964:Receptors, Mating Factor; C498031:STE2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D020962:Heterotrimeric GTP-Binding Proteins; D003545:Cysteine