Multiplex G Protein–Coupled Receptor Screen Reveals Reliably Acting Agonists and a Gq-Phospholipase C Coupling Mode of GPR30/GPER1

G protein-coupled receptors (GPCR) constitute the most versatile family of pharmacological target proteins. For some "orphan" GPCR, no ligand or drug-like modulator is known. In this study, we have established and applied a parallelized assay to co-screen 29 different human GPCR. Three compounds, chlorhexidine, Lys-05, and 9-aminoacridine triggered transient Ca²⁺ signals linked to the expression of GPR30. GPR30, also named G protein-coupled estrogen receptor 1 (GPER1), was reported to elicit increases in cAMP in response to 17b-estradiol, 4-hydroxytamoxifen, or G-1. These findings could, however, not be reproduced by other groups, and the de-orphanisation of GPR30 is, therefore, intensely disputed. The unbiased screen and following experiments in transiently or stably GPR30-overexpressing HEK293 cells did not show responses to 17b-estradiol, 4-hydroxytamoxifen or G-1. A thorough analysis of the activated signalling cascade revealed a canonical Gq-coupled pathway, including phospholipase C, protein kinase C and ERK activation, receptor internalisation, and sensitivity to the Gq inhibitor YM-254890. When expressed in different cell lines, the localisation of a fluorescent GPR30 fusion protein appeared variable. An efficient integration into the plasma membrane and stronger functional responses were found in HEK293 and in MCF-7 cells, whereas GPR30 appeared mostly retained in endomembrane compartments in Cos-7 or HeLa cells. Thus, conflicting findings may result from the use of different cell lines. The newly identified agonists and the finding that GPR30 couples to Gq are expected to serve as starting point for identifying physiological responses that are controlled by this GPCR. Significance Statement We have identified and thoroughly characterized novel and reliably acting agonists of the G protein-coupled receptor GPER1/GPR30. Applying these agonists, we demonstrate that GPR30 couples to the canonical Gq-phospholipase C pathway and is rapidly internalized upon continuous exposure to the agonists.