G protein‐coupled Receptor 30 (GPR30) PDZ‐dependently and Constitutively Increases ERK1/2 Signaling Through Calcineurin and Kinase Suppressor of Ras 2 (KSR2)

Abstract

The objective of the present study was to map the mechanism whereby G protein‐coupled receptor 30 (GPR30), also called G protein‐coupled estrogen receptor (GPER), stimulates extracellular signal‐regulated kinase (ERK)1/2 signaling. GPR30 plays important roles in cancer and cardiometabolic regulation. We showed recently that GPR30 forms a plasma membrane complex through its C‐terminal type I PSD‐95/Discs‐large/ZO‐1 homology (PDZ) motif with a membrane‐associated guanylate kinase (MAGUK) and protein kinase A (PKA)‐anchoring protein 5 (AKAP5), and AKAP5‐anchored PKA regulatory subunit RII suppresses receptor endocytosis and enables the receptor to constitutively inhibit cAMP production. Here, we investigated if this PDZ‐dependent GPR30 complex also regulates ERK1/2 signaling. To do so, human and mouse GPR30 were ectopically expressed in HEK293 cells and MDCK cells, and receptors and effectors were monitored by immunoblotting, immunoprecipitation, confocal immunofluorescence microscopy, split luciferase reporter techniques, and reporter gene assays. We found that GPR30 constitutively increased extracellular signal‐regulated kinase (ERK) 1/2 activity in several cell systems. The response was dependent on an intact receptor PDZ motif. Furthermore, knocking down AKAP5 or inhibiting calcineurin with FK506 inhibited the receptor response. GPR30 PDZ‐dependently inhibited basal NFAT signaling, consistent with the receptor favoring AKAP5‐anchoring of calcineurin. The calcineurin substrate kinase suppressor of Ras 2 (KSR2), a mitogen‐activated protein kinase (MAPK) scaffold, enhanced the GPR30‐promoted response, also dependently on the receptor PDZ motif. GPR30 also PDZ‐dependently favored a membrane KSR2 complex at the expense of the monomeric form. On the other hand, disrupting AKAP5‐PKA RII interaction with St‐Ht31, or inhibiting protein kinase C (PKC) with GF109203X or epidermal growth factor receptor (EGFR) tyrosine kinase with AG1478 had no effect on the GPR30‐stimulated response. FK506 also increased the amount of GPR30 in the plasma membrane, thus acting opposite to St‐Ht31, which increased receptor endocytosis. We conclude that the PDZ‐dependent GPR30 complex with AKAP5 includes calcineurin, which favors GPR30 endocytosis and enables the receptor to constitutively increase ERK1/2 signaling through KSR2.Support or Funding InformationSwedish Cancer Foundation