The G protein‐coupled estrogen receptor (GPER)/GPR30 C‐terminal PDZ motif regulates receptor plasma membrane retention and constitutive Gαi/o‐independent inhibition of cAMP production (662.13)

Abstract

G protein‐coupled estrogen receptor (GPER), or GPR30, is a G protein‐coupled receptor reported to bind 17β‐estradiol (E2), couple Gαs, and mediate non‐genomic estrogen responses. However, there remains a debate about the receptor pharmacological profile, effector coupling, and membrane trafficking. We addressed receptor membrane trafficking and effects on cAMP production in well‐defined model systems, HEK293 cells and CHO cells, and MDCK cells containing a Cre reporter, transfected to express GPER. We also addressed the role of the receptor C‐terminal type‐I PDZ motif (‐SSAV) by deleting the motif (GPERΔSSAV). E2 and G‐1, a reported GPER agonist, did not stimulate or inhibit cAMP production either in the absence or in the presence of wild‐type (WT) GPER or GPERΔSSAV. However, WT GPER constitutively inhibited forskolin (FSK)‐ and agonist‐stimulated cAMP production in a PDZ‐dependent and pertussis toxin‐insensitive manner. Consistent with this, attenuation of native receptor expression with siRNA increased FSK‐stimulated cAMP production in MDCK cells. Confocal microscopy and cell sorting of HEK293 cells stained with antibody live revealed that both WT GPER and GPERΔSSAV matured to the plasma membrane, but that GPERΔSSAV was subject to much higher constitutive endocytosis. Thus, GPER depends on a PDZ motif for plasma membrane retention and constitutive Gαi/o‐independent inhibition of cAMP production.