Dual-color reporter switching system to discern dimer formations of G-protein-coupled receptors using Cre/loxP site-specific recombination in yeast.

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 ß2 -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.

Stabilization of androgen receptor protein is induced by agonist, not by antagonists.

The action of nuclear receptor ligands in target tissues is specified mainly by the expression levels of their cognate nuclear receptors. The expression levels of these receptors are controlled through transcriptional and post-transcriptional events. Among post-transcriptional events, the effect of ligand on nuclear receptor protein turnover still remains largely unknown. Therefore, we studied the effects of agonist and antagonists on the turnover of the human androgen receptor (hAR) protein in stably transformed Chinese hamster ovary cells expressing exogenous hAR. Western blot analysis showed that the most potent androgen, dihydrotestosterone (DHT), stabilizes hAR with the induction of the transactivation function of hAR. However, this androgen-induced stabilization of hAR protein was abrogated by well-known androgen antagonists, hydroxyflutamide and bicalutamide (BIC), with inhibition of the transactivation function of hAR. Thus, the present study suggests that androgen antagonists exert their effects through, at least in part, abrogating the agonist-induced stabilization of hAR protein as well as blocking the ligand-induced transactivation function of hAR.