TY - JOUR T1 - <strong>Ligand-independent G protein-coupled Estrogen Receptor (GPER)/GPR30 Activity: Lack of receptor-dependent effects of G-1 and 17β-estradiol.</strong> JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/molpharm.121.000259 SP - MOLPHARM-AR-2021-000259 AU - Julia Tutzauer AU - Ernesto Gonzalez de Valdivia AU - Karl Swärd AU - Ioannis Alexandrakis Eilard AU - Stefan Broselid AU - Robin Kahn AU - Björn Olde AU - L. M. Fredrik Leeb-Lundberg Y1 - 2021/01/01 UR - http://molpharm.aspetjournals.org/content/early/2021/07/30/molpharm.121.000259.abstract N2 - GPR30 is a membrane receptor reported to bind 17β-estradiol (E2) and mediate rapid non-genomic estrogen responses, hence also named G protein-coupled estrogen receptor (GPER). G-1 is a proposed GPR30-specific agonist that has been used to implicate the receptor in several pathophysiological events. However, controversy surrounds the role of GPR30 in G-1- and E2 responses. We investigated GPR30 activity in the absence and presence of G-1 and E2 in several eukaryotic systems ex vivo and in vitro in the absence and presence of the receptor. Ex vivo activity was addressed using the caudal artery from wild-type (WT) and GPR30 knock-out (KO) mice, and in vitro activity was addressed using a HeLa cell line stably expressing a synthetic multifunctional promoter (NF-κB, STAT, AP-1)-luciferase construct (HFF11 cells) and a human GPR30-inducible T-REx system (T-REx HFF11 cells), HFF11 and HEK293 cells transiently expressing WT GPR30 and GPR30 lacking the C-terminal PDZ (PSD-95/Discs-large/ZO-1 homology) motif -SSAV (GPR30DSSAV), and yeast S. cerevisiae transformed to express GPR30. WT and KO arteries exhibited similar contractile responses to 60 mM KCl and 0.3 mM cirazoline, and G-1 relaxed both arteries with the same potency and efficacy. Furthermore, expression of GPR30 did not introduce any responses to 1 μM G-1 and 0.1 μM E2 in vitro. On the other hand, receptor expression caused considerable ligand-independent activity in vitro, which was receptor PDZ motif-dependent in mammalian cells. We conclude from these results that GPR30 exhibits ligand-independent activity in vitro but no G-1- or E2-stimulated activity in any of the systems used. Significance Statement Much controversy surrounds E2 and G-1 as GPR30 agonists. We used several recombinant eukaryotic systems ex vivo and in vitro with and without GPR30 expression to address the role of this receptor in responses to these proposed agonists. Our results show that GPR30 exhibits considerable ligand-independent activity in vitro but no G-1- or E2-stimulated activity in any of the systems used. Thus, classifying GPR30 as an estrogen receptor and G-1 as a specific GPR30 agonist is unfounded. ER -