Proteomic Analyses of the G Protein-Coupled Estrogen Receptor GPER1 Reveal Constitutive Links to Endoplasmic Reticulum, Glycosylation, Trafficking, and Calcium Signaling.

The G protein-coupled estrogen receptor 1 (GPER1) has been proposed to mediate rapid responses to the steroid hormone estrogen. However, despite a strong interest in its potential role in cancer, whether it is indeed activated by estrogen and how this works remain controversial. To provide new tools to address these questions, we set out to determine the interactome of exogenously expressed GPER1. The combination of two orthogonal methods, namely APEX2-mediated proximity labeling and immunoprecipitation followed by mass spectrometry, gave us high-confidence results for 73 novel potential GPER1 interactors. We found that this GPER1 interactome is not affected by estrogen, a result that mirrors the constitutive activity of GPER1 in a functional assay with a Rac1 sensor. We specifically validated several hits highlighted by a gene ontology analysis. We demonstrate that CLPTM1 interacts with GPER1 and that PRKCSH and GANAB, the regulatory and catalytic subunits of α-glucosidase II, respectively, associate with CLPTM1 and potentially indirectly with GPER1. An imbalance in CLPTM1 levels induces nuclear association of GPER1, as does the overexpression of PRKCSH. Moreover, we show that the Ca2+ sensor STIM1 interacts with GPER1 and that upon STIM1 overexpression and depletion of Ca2+ stores, GPER1 becomes more nuclear. Thus, these new GPER1 interactors establish interesting connections with membrane protein maturation, trafficking, and calcium signaling.

miR-27a and miR-214 exert opposite regulatory roles in Th17 differentiation via mediating different signaling pathways in peripheral blood CD4+ T lymphocytes of patients with relapsing-remitting multiple sclerosis.

Multiple sclerosis (MS) is one of the most prevalent autoimmune diseases, which involves the central nervous system. In this illness, Treg/Th17 cell imbalance causes the defect. Several studies revealed that T helper 17 (Th17) cells play a crucial role in pathogenesis, inflammation, and autoimmunity of several autoimmune diseases such as MS. In the present study, we assessed transcript levels of miR-27a and miR-214, in purified CD4+ T cells of MS patients, during relapsing and remitting phases in inducing differentiation of T naïve cells to Th17 cells. Forty RR-MS patient samples including those in relapsing (n=20) and remitting (n=20) phases were participated in this study. In addition, transcript levels of IL-17A, RORγt, IL-23R, Foxp3, and TGF-ß in purified CD4+ T cells of patients in relapsing and remitting phases of RRMS patients were compared to healthy controls. Expression levels of miR-27a and miR-214 were measured by RT-qPCR and compared to healthy control group (n=10). Data indicated upregulation of miR27a in relapsing phase of multiple sclerosis compared to remitting phase and healthy volunteers while miR-214 downregulated in relapsing phase of MS compared to remitting phase and healthy volunteers. In silico studies demonstrated pathways which miR-27a and miR-214 could effect on CD4+ T cell lineage fate including TGF-ß and mTOR signaling, respectively. Our data suggest that miR-27a may probably inhibit negative regulators of Th17 cell differentiation, thus promoting its differentiation while miR-214 has an adverse effect.