title: Simultaneous method for analyzing dimerization and signaling of G-protein-coupled receptor in yeast by dual-color reporter system.
abstract: Understanding the role of G-protein-coupled receptor (GPCR) dimerization in cellular function has now become a major research focus. The potentially large functional and physiological diversity of dimerization among GPCRs is expected to provide opportunities for novel drug discovery. However, there is currently a lack of cell-based assays capable of specific profiling for the functional consequences of dimerization linked to ligand-mediated signaling. Here, we present an advanced method to simultaneously analyze the dimerization and ligand response of GPCRs using two yeast-based systems for split-ubiquitin two-hybrid assay and G-protein signaling assay. To permit simultaneous detection, we established a two-color (dualcolor) fluorescence reporter gene assay using enhanced green fluorescent protein (EGFP) and a far-red derivative of the tetrameric fluorescent protein DsRed-Express2 (E2-Crimson). In the present study, we tested our method first by analyzing dimerization and ligand-mediated signaling by the yeast endogenous pheromone receptor (Ste2p). Second, we showed that the system facilitated mutational analysis of domains involved in dimerization and signaling by Ste2p. Third, we successfully demonstrated that the system could simultaneously monitor homo- and hetero-dimerization and somatostatin-induced signaling in the test case of the human SSTR5 somatostatin receptor. Our strategy is expected to provide a useful tool for the elucidation of molecular biological functions of GPCR dimers and for the screening of GPCR dimer-specific agonistic ligands.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Nakamura
authors.forename: Norika
authors.initials: N
authors.lastname: Takemoto
authors.forename: Jun
authors.initials: J
authors.lastname: Ishii
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kondo
mesh_terms: D003116:Color; D005453:Fluorescence; D019204:GTP-Binding Proteins / Q000378:metabolism*; D017930:Genes, Reporter; D005827:Genetics, Microbial / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008967:Molecular Biology / Q000379:methods; D055503:Protein Multimerization; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D008164:Luminescent Proteins; D043562:Receptors, G-Protein-Coupled; C485184:enhanced green fluorescent protein; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins; D019204:GTP-Binding Proteins