title: Dual-color reporter switching system to discern dimer formations of G-protein-coupled receptors using Cre/loxP site-specific recombination in yeast.
abstract: G-protein-coupled receptors (GPCRs) are physiologically important membrane proteins that represent major molecular targets in pharmaceutical and medicinal fields. Many GPCRs have been shown to form not only homodimers, but also heterodimers that can confer large functional and physiological diversity and are therefore expected to offer new opportunities for the discovery of new drugs. Yeast is a useful host organism that can be used to investigate the interactions between eukaryotic protein pairs, as demonstrated by the yeast two-hybrid (Y2H) system. Previously, we established reporter gene assay systems to screen for GPCR dimer pairs based on the split-ubiquitin membrane Y2H (mY2H) system. However, conventional systems only induce reporter gene expressions from the OFF to ON states. In this study, we therefore designed a reporter switching system that can switch the expressions between two reporter genes (one from ON to OFF and the other from OFF to ON) in response to the Y2H readout. To invoke reporter switching, we took advantage of Cre/loxP site-specific recombination. Through optimization of Cre-mediated reporter gene recombination using the split-ubiquitin mY2H system, we built a dual-color reporter switching system to discern the formations of GPCR dimers. This system enabled monitoring of the formations of homodimers and heterodimers of human serotonin 1A receptor or β2 -adrenergic receptor as well as homodimers of the yeast endogenous pheromone receptor (Ste2p) in yeast cells. Our reporter switching system may be a useful tool for identifying potential molecular targets among GPCR dimers, and is also applicable to other reporter gene assay systems. Biotechnol. Bioeng. 2016;113: 2178-2190. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Nakamura
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan.
authors.forename: Takamichi
authors.initials: T
authors.lastname: Hashimoto
authors.affiliation: Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan. junjun@port.kobe-u.ac.jp.
authors.forename: Jun
authors.initials: J
authors.lastname: Ishii
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kondo
mesh_terms: D017930:Genes, Reporter / Q000235:genetics*; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D055503:Protein Multimerization / Q000502:physiology*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic / Q000235:genetics; D020798:Two-Hybrid System Techniques
chemical_list: D043562:Receptors, G-Protein-Coupled; C045073:Cre recombinase; D019426:Integrases
keywords: Cre/loxP recombination; G-protein-coupled receptor; Saccharomyces cerevisiae; dimerization; flow cytometry; split-ubiquitin membrane yeast two-hybrid system