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

Abstract

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 Ca 2+ 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 17βestradiol, 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 17β-estradiol, 4-hydroxytamoxifen or G-1. A thorough analysis of the activated signalling cascade revealed a canonical G q -coupled pathway, including phospholipase C, protein kinase C and ERK activation, receptor internalisation, and sensitivity to the G q 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 G q are expected to serve as starting point for identifying physiological responses that are controlled by this GPCR. Significance StatementWe 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 G q -phospholipase C pathway and is rapidly internalized upon continuous exposure to the agonists.

Proteomic Analyses of the G Protein-Coupled Estrogen Receptor GPER1 Reveal Constitutive Links to Endoplasmic Reticulum, Glycosylation, Trafficking, and Calcium Signaling

“…An important conclusion from our investigations is that the activity assay and the GPER1 interactome are not affected by estrogen. In agreement with some previous publications, we must conclude that GPER1 has a constitutive, ligand-independent activity [24,25,29]. Formally, we cannot exclude that much higher E2 concentrations than the ones we have used (100 nM) would elicit responses beyond the constitutive ones that we have observed, but responses at pharmacological doses even further above the highest concentrations of about 1.5 nM during the menstrual cycle are unlikely to be physiologically relevant.…”

“…In contrast with previous reports, this group provided strong evidence for ligand-independent activity of GPER1 with a MAPK activity assay and using an assay exploiting the conformational changes of a Rac1 sensor. Moreover, in a multiplexed screen for novel ligands of GPCRs, HEK293T cells transiently or stably expressing GPER1 showed no responses to E2, the anti-estrogen 4-hydroxytamoxifen, or G-1 [25].…”

Tamoxifen Activates Transcription Factor EB and Triggers Protective Autophagy in Breast Cancer Cells by Inducing Lysosomal Calcium Release: A Gateway to the Onset of Endocrine Resistance

“…Nevertheless, the mechanism whereby GPER1 activation leads to intracellular Ca 2+ signaling has never been clearly elucidated. Intriguingly, a recent investigation demonstrated that GPER1 may also signal the InsP 3 -dependent increase in [Ca 2+ ] i by coupling to phospholipase C through G q proteins [ 57 ]). Here, we find for the first time that Tam elicits ER Ca 2+ release through InsP 3 Rs and lysosomal Ca 2+ release through TPCs and TRPML1.…”

“…Early studies showed that G s -coupled receptors, such as β1-adrenergic receptors, are coupled to NAADP production [ 61 ]. Therefore, Tam could stimulate GPER1 to recruit both G s - and G q -dependent signaling pathways, as shown in [ 57 ]. By contrast, the signal transduction pathway coupling GPER1 to TRPML1 activation remains unclear, since the mechanisms by which G protein-coupled receptors are coupled to PIKfyve are yet to be elucidated [ 60 ].…”

The G protein-coupled oestrogen receptor GPER in health and disease: an update

“…The discovery of GPER-selective antagonists G15 and G36 complemented the use of G-1 as an agonist in understanding the roles of GPER in cell biology and physiology 68 , 69 . Some reports suggest that the activity of these compounds can vary depending on the system employed 70 , 71 . Other reported GPER-selective ligands include the agonists GPERL1 and GPERL2 (ref.…”