Interacting residues in an activated state of a G protein-coupled receptor.
Ste2p, the G protein-coupled receptor (GPCR) for the tridecapeptide pheromone alpha-factor of Saccharomyces cerevisiae, was used as a model GPCR to investigate the role of specific residues in the resting and activated states of the receptor. Using a series of biological and biochemical analyses of wild-type and site-directed mutant receptors, we identified Asn(205) as a potential interacting partner with the Tyr(266) residue. An N205H/Y266H double mutant showed pH-dependent functional activity, whereas the N205H receptor was non-functional and the Y266H receptor was partially active indicating that the histidine 205 and 266 residues interact in an activated state of the receptor. The introduction of N205K or Y266D mutations into the P258L/S259L constitutively active receptor suppressed the constitutive activity; in contrast, the N205K/Y266D/P258L/S259L quadruple mutant was fully constitutively active, again indicating an interaction between residues at the 205 and 206 positions in the receptor-active state. To further test this interaction, we introduced the N205C/Y266C, F204C/Y266C, and N205C/A265C double mutations into wild-type and P258L/S259L constitutively active receptors. After trypsin digestion, we found that a disulfide-cross-linked product, with the molecular weight expected for a receptor fragment with a cross-link between N205C and Y266C, formed only in the N205C/Y266C constitutively activated receptor. This study represents the first experimental demonstration of an interaction between specific residues in an active state, but not the resting state, of Ste2p. The information gained from this study should contribute to an understanding of the conformational differences between resting and active states in GPCRs.
Lee
Yong-Hun
YH
Department of Microbiology, University of Tennessee, Nashville, TN 37996, USA.
Naider
Fred
F
Becker
Jeffrey M
JM
D000409:Alanine / Q000737:chemistry; D001216:Asparagine / Q000737:chemistry; D001681:Biological Assay; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005799:Genes, Dominant; D017930:Genes, Reporter; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007700:Kinetics; D007763:Lac Operon; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D010675:Pheromones / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000378:metabolism; D051964:Receptors, Mating Factor / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D015398:Signal Transduction; D014357:Trypsin / Q000494:pharmacology; D014443:Tyrosine / Q000737:chemistry
D003432:Cross-Linking Reagents; D004220:Disulfides; D010675:Pheromones; D043562:Receptors, G-Protein-Coupled; D051964:Receptors, Mating Factor; C498031:STE2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014443:Tyrosine; D006639:Histidine; D001216:Asparagine; D014357:Trypsin; D000409:Alanine