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Activation of GPER (G protein‐coupled estrogen receptor) increases Gs activity via forming a plasma membrane complex with AKAP/SAP97
Author(s) -
Yu Xuan,
Stallone John N.,
Heaps Cristine L.,
Han Guichun
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.1069.9
Subject(s) - gper , hek 293 cells , chemistry , agonist , microbiology and biotechnology , estrogen receptor , medicine , estrogen , endocrinology , receptor , biology , biochemistry , cancer , breast cancer
Evidence suggest that G protein‐coupled estrogen receptor (GPER) is emerging as a therapeutic target in cardiovascular diseases. Activation of GPER elicits many cardiovascular protective effects, including lowering blood pressure, reducing ischemia/reperfusion‐induced cardiac infarct size and decreasing the process of atherosclerosis in animal models. However, the understanding of the mechanisms underlying GPER‐mediated actions is still limited. The objective of this study is to test the hypothesis that activation of GPER by selective agonist G‐1 increases Gs activation by forming a plasma membrane complex with AKAP/SAP97. Isometric tension studies, Gs activity assays, immunoprecipitation studies and Western blots were performed by using porcine coronary arteries, primary cultured porcine coronary artery smooth muscle cells (PCASMCs) and HA‐tagged GPER gene stably transfected HEK293 cells (GPER‐HEK293). First, we measured the Gs activity in both GPER‐HEK293 cells and PCASMCs with selective GPER agonist G‐1 (1 μM); and observed that treatment with G‐1 significantly elevated the Gs‐GTP level. Next, we explored the association of GPER with Gs in immunoprecipitation studies of both GPER‐HEK293 cells and PCASMCs. Similar to Gs activity assays, activation of GPER with G‐1 promoted the association of activated GPER with Gs, and it was in a concentration dependent manner in PCASMCs. We further observed effects of GPER activation on cAMP production in PCASMCs and demonstrated that G‐1 significantly stimulated cAMP production. Last, we investigated the interaction of both Protein Kinase A‐anchoring Protein (AKAP5) and membrane‐associated guanylate kinase SAP97 to GPER by applying FMP‐API‐1 (100 μM) in isometric tension studies to disrupt the interaction between AKAPs and PKA, and detecting AKAP and SAP97 in immunoprecipitation studies in PCASMCs. Pretreatment with FMP‐API‐1 attenuated G‐1‐induced relaxation (10 – 3,000 nM) of endothelium‐denuded coronary arteries preconstricted with PGF2α (1 μM). In immunoprecipitation studies, G‐1 clearly enhanced the interaction of both AKAP5 and SAP97 to GPER. Therefore, we conclude that activation of GPER with G‐1 stimulates the production of cAMP, which is due to GPER coupling to Gs via forming a plasma membrane signaling complex with AKAP/SAP97. Support or Funding Information AHA, Diversity of G Protein