G protein-coupled receptor 30 is an estrogen receptor in the plasma membrane,☆☆

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Abstract

Recently, GPR30 was reported to be a novel estrogen receptor; however, its intracellular localization has remained controversial. To investigate the intracellular localization of GPR30 in vivo, we produced four kinds of polyclonal antibodies for distinct epitopes on GPR30. Immunocytochemical observations using anti-GPR30 antibody and anti-FLAG antibody show that FLAG-GPR30 localizes to the plasma membrane 24 h after transfection. Treatment with estrogen (17β-estradiol or E2) causes an elevation in the intracellular Ca2+ concentration ([Ca2+]i) within 10 s in HeLa cells expressing FLAG-GPR30. In addition, E2 induces the translocation of GPR30 from the plasma membrane to the cytoplasm by 1 h after stimulation. Immunohistochemical analysis shows that GPR30 exists on the cell surface of CA2 pyramidal neuronal cells. The images on transmission electron microscopy show that GPR30 is localized to a particular region associated with the plasma membranes of the pyramidal cells. These data indicate that GPR30, a transmembrane receptor for estrogen, is localized to the plasma membrane of CA2 pyramidal neuronal cells of the hippocampus in rat brain.

Section snippets

Materials and methods

Materials. Anti-FLAG mouse monoclonal antibody was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Lipofectamine 2000 was from Invitrogen (Carlsbad, CA, USA). p3xFLAG-CMV10 was from Sigma-Aldrich Co. (St. Louis, MO, USA). All other chemicals were available commercially.

Plasmid construction. The full-length GPR30 fragment was confirmed by DNA sequencing and cloned into p3xFLAG-CMV10 or pCMV-GFP as described previously [16].

Production of polyclonal antibody. Rabbit polyclonal antibodies

Production of anti-GPR30 antibody and observation of the intracellular localization of GPR30

To examine the localization of GPR30 in vivo, we produced anti-GPR30 antibodies using four kinds of synthetic peptides corresponding to amino acid sequences in GPR30. In immunoblotting experiments on HeLa cells transfected with FLAG-GPR30, among the antibodies produced, that produced by the synthetic peptide for amino acid residues 352–370 clearly recognized a protein with a molecular mass of approximately 40 kDa, which is almost equal to the calculated molecular mass of GPR30, and the band

Discussion

We conclude that plasma membrane GPR30 is activated by E2 for the following reasons. First, GPR30 exists in the plasma membrane of HeLa cells transfected with FLAG-GPR30 and translocates from the plasma membrane to the cytoplasm in response to stimulation by physiological concentrations (10 nM–1 μM) of E2. Second, an elevation in [Ca2+]i was observed within several seconds after the addition of E2 in cells expressing GPR30. No elevation in [Ca2+]i was observed in HeLa cells transfected with empty

Acknowledgment

We thank Mr. C. Matsuo (Yamaguchi University School of Medicine) for technical support with immunohistochemistry.

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      Citation Excerpt :

      GPER1 subcellular location is still controversial: as a member of the GCPRs superfamily and being characterized by the hepta-helical transmembrane domain, it was expected to be found at the plasma membrane (Cheng et al., 2011), but, surprisingly, a large fraction of GPER1 has been also observed in the endoplasmic reticulum, the trans-Golgi (Revankar et al., 2005) and mitochondria (Revankar et al., 2005). In the brain, GPER1 plasma membrane staining has been detected in hippocampal pyramidal neurons (Akama et al., 2013; Funakoshi et al., 2006; Llorente et al., 2020), hypothalamic neurons (Lagunas et al., 2019; Qiu et al., 2006) and dorsal striatal neurons (Almey et al., 2012). However, cytoplasmic localization has been also described, for example in the ARC nucleus, where the cells displayed GPER1 immunoreactivity either at the plasma membrane or in the cytoplasm, in a Golgi apparatus-like arrangement (Marraudino et al., 2020).

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    Abbreviations: GPR30, G protein-coupled receptor 30; GPCR, G protein-coupled receptor; E2, estrogen or 17β-estradiol; [Ca2+]i, intracellular Ca2+ concentrations; PBS, phosphate-buffered saline.

    ☆☆

    This work was supported in part by Grants from Grants-in-Aid for Scientific Research on Priority Areas (Nos. 17052016 and 16601002) by the Ministry of Education, Culture, Sports, Science and Technology (MEXT).

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