title: Amplification of agonist stimulation of human G-protein-coupled receptor signaling in yeast.
abstract: G-protein-coupled receptors (GPCRs) are considered as important targets for drug discovery. The yeast Saccharomyces cerevisiae is an attractive host for high-throughput screening of agonistic ligands for human GPCRs because it can simplify the complicated signaling pathways that are present in mammalian cell lines. Unfortunately, many human GPCRs induce only partial signal activation in yeast cells depending on their coupling efficiency with yeast G-proteins. This problem often results in unsatisfactory detection sensitivity, thereby resulting in a limitation to yeast-based detection systems. Here we introduce a new highly sensitive detection method that provides robust agonist detection of human GPCRs. Our strategy is designed to invoke feedback activation of signals within yeast G-protein signaling pathways. Briefly, agonist stimulation of human GPCRs triggers expression of an artificial signal activator that amplifies signaling. We chose human somatostatin receptor subtype 5 (hSSTR5) as a model of a human GPCR. Investigation of the response of hSSTR5-expressing yeast to various concentrations of somatostatin demonstrated that feedback activation of the signal can successfully improve the detection limit and the maximum level of signaling. This novel approach will enhance the usefulness of yeast-based screening of agonistic ligands for a variety of human GPCRs.
authors: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Nada-ku, Kobe 657-8501, Japan.
authors: Nobuo
authors: N
authors: Fukuda
authors: Jun
authors: J
authors: Ishii
authors: Misato
authors: M
authors: Kaishima
authors: Akihiko
authors: A
authors: Kondo
mesh_terms: D025461:Feedback, Physiological; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D057166:High-Throughput Screening Assays; D006801:Humans; D008024:Ligands; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000235:genetics; D051964:Receptors, Mating Factor / Q000235:genetics; D017481:Receptors, Somatostatin / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D015398:Signal Transduction; D013004:Somatostatin / Q000378:metabolism
chemical_list: D044387:GTP-Binding Protein beta Subunits; D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D051964:Receptors, Mating Factor; D017481:Receptors, Somatostatin; C498031:STE2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C067576:Ste4 protein, S cerevisiae; D013004:Somatostatin; C094573:somatostatin receptor 5