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Endocrine Abstracts (2020) 70 OC9.4 | DOI: 10.1530/endoabs.70.OC9.4

ECE2020 Oral Communications Reproductive and Developmental Endocrinology (7 abstracts)

Demonstration of follicle-stimulating hormone receptor and G protein-coupled estrogen receptor heteromers in vitro via BRET and super-resolution imaging

Clara Lazzaretti 1,2 , Niamh Sayers 3 , Elia Paradiso 1,2 , Samantha Sperduti 1,4 , Francesca Fanelli 5,6 , Manuela Simoni 1,4,7,8 , Aylin Hanyaloglu 3 & Livio Casarini 1,4

1University of Modena and Reggio Emilia, Unit of Endocrinology, Department of Biomedical, Metabolic and Neural Sciences, Baggiovara, Italy; 2University of Modena and Reggio Emilia , International PhD School in Clinical and Experimental Medicine (CEM), Modena, Italy; 3Imperial College London, Department of Metabolism, Digestion and Reproduction, London, United Kingdom; 4University of Modena and Reggio Emilia , Center for Genomic Research, Modena, Italy; 5University of Modena and Reggio Emilia , Department of Life Sciences, Modena, Italy; 6Center for Neuroscience and Neurotechnology, Modena, Italy; 7Azienda Ospedaliero-Universitaria di Modena, Department of Medical Specialties, Baggiovara, Italy; 8Institut National de la Recherche Agronomique, Unitè Physiologie de la Reproduction et des Comportements, Nouzilly, France

Introduction: The developmental stages of the ovarian follicle are characterized by changes of gonadotropin and steroid hormone receptor expression and their potential interactions on the surface of granulosa cells. In this study in vitro, we aim to evaluate whether the co-existing follicle-stimulating hormone receptor (FSHR) and G protein-coupled estrogen receptor (GPER) form heteromers, which may play role in selecting the dominant ovarian follicle.

Methods: FSHR-GPER interactions was evaluated in transfected HEK293 cells by bioluminescence resonance energy transfer (BRET) and photo-activated localization microscopy using photoactivatable dyes (PD-PALM). Molecular modelling of FSHR-GPER heterodimers identified the sixth and seventh transmembrane segments (T6, T7) of both receptors as a potential heteromer interface. Seven T6 and eight T7 GPER putative interaction amino acid residues were mutated to alanine (mutGPER). Intracellular Ca2+ levels were measured by aequorin-dependent calcium assay (AEQ-GFP) and BRET.

Results: In order to evaluate its functionality and compare it to the wild-type receptor (wtGPER), wt or mutGPER were transiently expressed in HEK293 cells and demonstrated to both mediate estradiol (E2)-induced intracellular Ca2+ increase (two-way ANOVA; P < 0.0001; n = 8; mean ± S.E.M). FSHR-wtGPER dimerization was demonstrated by BRET in HEK293 cells transiently transfected with a fixed amount of Rluc8-tagged FSHR and increasing doses of wtGPER-Venus. The BRET signal logarithmically increased together with the acceptor concentration (non-linear regression; r2 = 0.876; n = 4; mean ± S.E.M), revealing a specific heretomeric interaction between the two receptors. Further confirmation of the FSHR-wtGPER complexes at the cell surface was obtained by super-resolution PD-PALM imaging in cells co-expressing HA-tagged FSHR and FLAG-tagged wtGPER. We found FSHR and wtGPER both form monomers and homomers in equivalent amounts, however, while FSHR formed both dimers and a range of low to higher order homo-oligomers, GPER-GPER associations were primarily homodimer. FSHR-wtGPER heteromers were also observed with heterodimers as the predominant form (15% +/− 2.2, Mean ± S.E.M; n = 8). The ability of mutGPER to associate with FSHR was first determined by BRET, which demonstrated no specific BRET interaction (linear regression; r2 = 0.014; n = 4; mean ± S.E.M). Furthermore, a fourfold decrease in the percentage of heterodimeric structures in the cell surface were found by PALM (n = 3), indicating the failure of heteromeric assembly between FSHR and mutGPER at the plasma membrane.

Conclusion: We demonstrated for the first time the physical interaction of FSHR-GPER in vitro, and identified a molecular heteromer interface, which could be exploited to understand the role of this heteromer in granulosa cell physiology.

Volume 70

22nd European Congress of Endocrinology

05 Sep 2020 - 09 Sep 2020

European Society of Endocrinology 

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