Endocrine Abstracts

During the menstrual cycle, selection of the dominant follicle is characterized by changes of gonadotropin and steroid hormone receptor expression. We aim to evaluate whether co-existing follicle-stimulating hormone receptor (FSHR) and G protein-coupled estrogen receptor (GPER) form heterodimers, suggesting their role in ovarian follicle selection. FSHR-GPER dimerization was evaluated in transfected HEK293 cells in vitro by bioluminescence resonance energy transfer (BRET). To this purpose, a 3D model of FSHR-GPER heterodimer was predicted by structural bioinformatics tools, which identified the sixth and seventh transmembrane segments (T6, T7) of both receptors as carriers of contact residues. Seven T6 and eight T7 amino acid residues from GPER were identified as putative interaction interfaces with FSHR and mutated to alanine (mutGPER). WT GPER, FSHR (wtGPER; wt FSHR) and mutGPER were C-terminally tagged with either the BRET donor Rluc8 or acceptor yFP. Increasing amount of yFP-tagged FSHR or GPER were expressed in HEK293 cells, together with a fixed amount of Rluc8-tagged GPER or FSHR and protein-protein interaction was evaluated by BRET technique, in the presence of the luciferase substrate coelenterazine H. Data were represented as donor:acceptor emission ratio (net BRET) and plotted against the ratio of the donor:acceptor concentration. Heterodimer formation was evaluated after data interpolation using non-linear regression. In transfected HEK293 cells, expressing the wtGPER-yFP and wtFSHR-Rluc8, heterodimer formation was demonstrated, revealing a BRET max (donor:acceptor proximity) and BRET50 (interaction affinity) of 0.71±0.04 and 0.05±0.02 ng yFP:Rluc8 cDNA/well respectively; P<0.0001; mean±S.E.M.). In contrast, HEK293 cells transfected with mutGPER, exhibited no specific BRET interaction indicating an inability of heterodimerization between wtFSHR and mutGPER. Experiments assessing heterodimerization in cells expressing exogenous receptors fused with exchanged biosensors, i.e. wtFSHR-yFP and wt/mutGPER-Rluc8, did not reveal any saturation in BRET signal. Since heterodimers were demonstrated by wtFSHR-Rluc8 and wtGPER-yFP, receptor with exchanged BRET tags are functional but not suitable for evaluating dimerization, likely due to unproper molecular conformation not allowing biosensor-biosensor contact and energy exchange. We demonstrated for the first time FSHR-GPER physical interaction in vitro, and identified a molecular interface mediating this interaction, which could be exploited to understand the role this heterodimer may play in granulosa cell physiology.