People Bradykinin Receptor (b <sub> 2 </ Sub> R), And Endothelial Cell Differentiation Factor Receptor (edg Is 1) The Functional Expression And Localization Studies In Yeast

G protein-coupled receptors(GPCRs) are the largest class of targets for modern drugs by virtue of their roles in the regulation of cellular functions. Heterologous production of GPCRs in a defined host background has been established as an important and valuable tool for the pharmacological and biochemical analysis of defined receptor subtypes, as well as chimeric or mutant receptors. Furthermore, the analysis of G protein interaction and subsequent signal transduction, the detection of 'lead' compounds in the search for new drugs and of ligands for orphan GPCRs by high-throughput screens, as well as the purification of receptor protein suitable for biochemical, biophysical and/or structural studies are important goals. Here, due to their ability of high level production, easy manipulation, and low costs, yeast-based expression systems have proven very attractive. Unlike Escherichia coli, yeasts have the potential to perform eukaryotic post-translational modifications, such as N-glycosylation, which may affect receptor function. The methylotropic yeast strain, Pichia pastoris, has a strong inducible promoter and it is less prone to hyperglycosylation than Saccharomyces cerevisiae. Therefore, recombinant P. pastoris has been developed as an excellent host for the expression of foreign GPCRs.The human endothelial differentiation gene l(edg-l) and bradykinin B2 receptor (B₂R) belong to the large family of G-protein-coupled receptors. In order to develop a convenient and rapid assay for testing compounds which might be effective as edg-1 and B₂R receptors agonists or antagonists, the gene of edg-1 and B₂R receptors were expressed in the methylotrophic yeast Pichia pastoris.The expression plasmids were constructed in which the edg-1 or B₂R gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase 1 gene, the green fluorescent protein(GFP) gene were introduced at the C-terminal of the edg-1 or B₂R gene to permit easy detection.Fluorescence activated cell sorting (FACS) analysis and Western blotanalysis revealed that the edg-1 and B₂R recombinant receptor proteins were distinctly expressed. The results were further confirmed by Confocal microscopy which produced bright green fluorescence.The localization of the recombinant edg-1 or B₂R receptor proteins were proved by immunofluorescence microscopy which indicated a distinct expression of the edg-1 or B₂R in the plasma membrane of the transformed yeast cells.The binding of bradykinin ligand to the B₂R recombinant receptor proteins were also proved by immunofluorescence microscopy which exhibited a high affinities of bradykinin to the GS115-B2-EGFP cell membrane.In radioligand binding analysis, the edg-1 recombinant receptor proteins also showed high affinities of S— 1 -³²P.Exogenous SPP treatment, inhibited GS115-edg-1-EGFP cells proliferation.In conclusion, we provide compelling evidence that the edg-1 and B₂R receptor were functionally expressed in P. pastoris and localization on the plasma membrane for the first time. These results strongly suggest that the GS115-B2-EGFP or the GS115-edg-1-EGFP cell line is a useful tool to study the effect of potential pharmacological agents that may modulate the function of edg-1 or B₂R receptor.