The third cytoplasmic loop of a yeast G-protein-coupled receptor controls pathway activation, ligand discrimination, and receptor internalization.
To identify functional domains of G-protein-coupled receptors that control pathway activation, ligand discrimination, and receptor regulation, we have used as a model the alpha-factor receptor (STE2 gene product) of the yeast Saccharomyces cerevisiae. From a collection of random mutations introduced in the region coding for the third cytoplasmic loop of Ste2p, six ste2sst alleles were identified by genetic screening methods that increased alpha-factor sensitivity 2.5- to 15-fold. The phenotypic effects of ste2sst and sst2 mutations were not additive, consistent with models in which the third cytoplasmic loop of the alpha-factor receptor and the regulatory protein Sst2p control related aspects of pheromone response and/or desensitization. Four ste2sst mutations did not dramatically alter cell surface expression or agonist binding affinity of the receptor; however, they did permit detectable responses to an alpha-factor antagonist. One ste2sst allele increased receptor binding affinity for alpha-factor and elicited stronger responses to antagonist. Results of competition binding experiments indicated that wild-type and representative mutant receptors bound antagonist with similar affinities. The antagonist-responsive phenotypes caused by ste2sst alleles were therefore due to defects in the ability of receptors to discriminate between agonist and antagonist peptides. One ste2sst mutation caused rapid, ligand-independent internalization of the receptor. These results demonstrate that the third cytoplasmic loop of the alpha-factor receptor is a multifunctional regulatory domain that controls pathway activation and/or desensitization and influences the processes of receptor activation, ligand discrimination, and internalization.
Stefan
C J
CJ
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Blumer
K J
KJ
D000483:Alleles; D000595:Amino Acid Sequence; D005800:Genes, Fungal; D005838:Genotype; D007700:Kinetics; D000072235:Mating Factor; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000494:pharmacology; D010675:Pheromones / Q000494:pharmacology; D010957:Plasmids; D017433:Protein Structure, Secondary; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012440:Saccharomyces / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000378:metabolism
D010455:Peptides; D010675:Pheromones; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D014157:Transcription Factors; D000072235:Mating Factor