The Improvement of Human β2 Adrenergic Receptor Signal Output in Yeast Mating Pathway by Addition of Signal Sequence

Applying G protein coupled receptor（GPCR） to test and screen specific ligand has always been the hot spot in scientific research. Research which was aimed to find out the optimal experimental conditions to improve the β2 AR（β2 adrenergic receptor） signal transduction in yeast by reducing the testing time would lay the foundation for ligand screening. First, we appended mammalian GPCR with yeast α factor prepro sequence, pre sequence, endogenous GPCR Ste2, invertase Suc2 N terminus as signal sequence. Fluorescent microscope was used to check the location of GPCR. Second, we constructed a yeast strain in which mating pathway related genes have been deleted and endogenous G protein α subunit was substituted by Gpa1-Gαs-transplant and mammalian Gαs. In addition to this, pFUS1-GFP and pFUS1-Rluc reporter systems were transformed into it and isoproterenol was applied as ligand to analyze the ligand-induced signal. Finally, the engineered yeast strains which included ligand induced signaling pathway have been successfully constructed. This experiment made a conclusion that plasma membrane located β2 AR could improve the signal transduction. Furthermore, this approach provided the optimal condition including 12 h ligand incubation time and 100 μmol/L ligand concentration, which will have an important influence on the heterogenous expression of GPCR in yeast.