Abstract
G protein-coupled receptor 30 [G protein-coupled estrogen receptor 1 (GPER1)], has been introduced as a membrane estrogen receptor and a candidate cancer biomarker and therapeutic target. However, several questions surround the subcellular localization and signaling of this receptor. In native cells, including mouse myoblast C2C12 cells, Madin-Darby canine kidney epithelial cells, and human ductal breast epithelial tumor T47-D cells, G-1, a GPER1 agonist, and 17β-estradiol stimulated GPER1-dependent cAMP production, a defined plasma membrane (PM) event, and recruitment of β-arrestin2 to the PM. Staining of fixed and live cells showed that GPER1 was localized both in the PM and on intracellular structures. One such intracellular structure was identified as cytokeratin (CK) intermediate filaments, including those composed of CK7 and CK8, but apparently not endoplasmic reticulum, Golgi, or microtubules. Reciprocal coimmunoprecipitation of GPER1 and CKs confirmed an association of these proteins. Live staining also showed that the PM receptors constitutively internalize apparently to reach CK filaments. Receptor localization was supported using FLAG- and hemagglutinin-tagged GPER1. We conclude that GPER1-mediated stimulation of cAMP production and β-arrestin2 recruitment occur in the PM. Furthermore, the PM receptors constitutively internalize and localize intracellularly on CK. This is the first observation that a G protein-coupled receptor is capable of associating with intermediate filaments, which may be important for GPER1 regulation in epithelial cells and the relationship of this receptor to cancer.
Footnotes
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This work was supported in part by funds from European Foundation for the Study of Diabetes/Servier, Swedish Research Council, Alfred Österlund Foundation, The Swedish Diabetes Association, Konsul Thure Carlssons Minne Foundation, Magnus Bergvalls Foundation, Diabetes Association in Malmö, Syskonen Svenssons Foundation, Anders Otto Swärds Foundation, Royal Physiographic Society in Lund, and the Faculty of Medicine at Lund University through the Vascular Wall prioritized program.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.069500.
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ABBREVIATIONS:
- GPER1
- G protein-coupled estrogen receptor 1
- B2R
- B2 bradykinin receptor
- ERα
- estrogen receptor α
- ERβ
- estrogen receptor β
- CK
- cytokeratin
- E2
- 17β-estradiol
- ER
- endoplasmic reticulum
- PM
- plasma membrane
- HEK
- human embryonic kidney
- MDCK
- Madin-Darby canine kidney
- GPCR
- G protein-coupled receptor
- FBS
- fetal bovine serum
- PCR
- polymerase chain reaction
- PBS
- phosphate-buffered saline
- PNGase F
- peptide:N-glycanase
- HA
- hemagglutinin
- ATCC
- American Type Culture Collection
- GFP
- green fluorescent protein
- siRNA
- short interfering RNA.
- Received October 19, 2010.
- Accepted December 13, 2010.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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