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Endocrine Abstracts (2023) 90 OC4.5 | DOI: 10.1530/endoabs.90.OC4.5

ECE2023 Oral Communications Oral Communications 4: Reproductive and Developmental Endocrinology (6 abstracts)

Reprogramming of reproductive signals via human luteinizing hormone/choriogonadotropin receptor (LHCGR)/G protein-coupled estrogen receptor (GPER) heteromers

Clara Lazzaretti 1 , Elia Paradiso 1 , Samantha Sperduti 1,2 , Niamh Sayers 3 , Ginevra Pelagatti 1 , Sara D’Alessandro 1,4 , Carmela Perri 1 , Lara Baschieri 1,4 , Elisa Mascolo 1 , Neena Roy 1 , Manuela Simoni 1,2,5 , Aylin Hanyaloglu 3 & Livio Casarini 1,2

1Unit of Endocrinology- University of Modena and Reggio Emilia, Department of Biomedical, Metabolic and Neural Sciences, Modena, Italy; 2University of Modena and Reggio Emilia , Center for Genomic Research, Modena,; 3Institute of Reproductive and Developmental Biology-Imperial College London, Department of Metabolism, Digestion and Reproduction, London, United Kingdom; 4University of Modena and Reggio Emilia, International PhD School in Clinical and Experimental Medicine (CEM), Modena, Italy; 5Azienda Ospedaliero-Universitaria of Modena, Department of Medical Specialties, Modena, Italy

In the ovary, the G protein-coupled estrogen receptor (GPER) forms heteromeric complexes with the follicle-stimulating hormone receptor (FSHR), reprogramming FSH-induced signals and determining the follicular fate. Based on the structural similarity, we evaluated whether GPER interacts with the luteinizing hormone (LH)/choriogonadotropin (hCG) receptor (LHCGR) modulating gonadotropin-dependent signals. LHCGR-GPER heteromers were evaluated in transiently transfected HEK293 cells co-expressing specifically tagged receptors and biosensors, and signals were collected by bioluminescence resonance energy transfer (BRET) and photo-activated localization microscopy using photoactivatable dyes (PD-PALM). Cells were treated by LH/hCG and signalling analyses were performed by evaluating G proteins coupling and displacement, intracellular Ca2+, cAMP and inositol monophosphate (IP1) increase by BRET and homogeneous time-resolved fluorescence (HTRF). Activation of gene transcription regulated by nuclear factor of activated T-cells (NFAT) and cAMP response element (CRE) promoters was performed using a reporter system. Data were analysed by non-linear regression or Kruskal-Wallis test and Dunn’s post-hoc test (P<0.05; n=4 to 6), as appropriate. Super-resolution microscopy revealed that about 20% of FLAG-GPER and HA-LHCGR form heteromers on the cell surface, and results were confirmed by BRET detecting yFP-GPER and rluc-LHCGR interaction (r2=0.91; n=6). The presence of GPER induces concentration-dependent displacement of Gαq to LHCGR, resulting in depletion of hCG-induced Ca2+ response under GPER/lHCGR co-expression (AUC LHCGR= 5169±506 vs AUC LHCGR+GPER= 3621±277; P<0.05; n=6). Consistently, all other Gαq-dependent events, i.e. IP1 production (LH= 2.72±0.49 nM; hCG= 10.89±2.55 nM; p≥0.05; n=4) and NFAT promoter activation (LHCGR LH= 2.4±0.3 vs LHCGR+GPER LH= 0.6±0.1; LHCGR hCG= 2.5±0.3 vs LHCGR+GPER hCG= 0.7±0.1; n=5; P<0.05), were inhibited in GPER/lHCGR co-expressing cells treated with LH/hCG. This is not in cells expressing LHCGR alone, where gonadotropins induced Gαq-dependent signalling (P<0.05; n=6). Interestingly, GPER-LHCGR complexes have no impact on LH/hCG-induced cAMP/protein kinase A (PKA) pathway activation (p>0.05; n=6), suggesting that GPER specifically inhibits Gαq-, but not Gαs-mediated signals. Control experiments were performed using a biosensor-tagged mutant GPER (GPERmut) unable to form heteromers with LHCGR. PD-PALM and BRET methods revealed the absence of GPERmut/lHCGR complexes and the lack of receptor-receptor interaction (r2=0.05; n=6). Under these conditions, cell treatment with LH/hCG induced IP1 accumulation and gene transcription (LHCGR vs LHCGR+GPER; P>0.05; n=5). In conclusion, GPER interacts with LHCGR in the cell surface, biasing LH/hCG-induced signals via specific inhibition of Gαq-dependent cascades. These data suggest that reproductive functions may be modulated by GPER/lHCGR heteromers in the ovary.

Volume 90

25th European Congress of Endocrinology

Istanbul, Turkey
13 May 2023 - 16 May 2023

European Society of Endocrinology 

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