Background: Follicle-stimulating hormone receptor (FSHR) and G protein-coupled estrogen receptor (GPER) are expressed on the surface of granulosa cells, where they form heteromeric complexes, shifting FSH-induced signals to survival pathways and determining the follicular fate.
Objectives: We evaluated whether GPER can interact with the luteinizing hormone (LH)/choriogonadotropin (hCG) receptor (LHCGR) and modulate the LH/hCG/dependent signalling pathways.
Methods: LHCGR-GPER heteromers were evaluated in transiently transfected HEK293 cells by bioluminescence resonance energy transfer (BRET) and photo-activated localization microscopy using photoactivatable dyes (PD-PALM). The LH/hCG-dependent signalling analyses were performed by evaluating LHCGR-coupling to G proteins, intracellular Ca2+, cAMP and inositol monophosphate (IP1) increase by BRET and homogeneous time-resolved fluorescence (HTRF). Activation of LH/hCG-dependent gene transcription was evaluated using a reporter system. Data were analysed by non-linear regression or KruskalWallis test and Dunns post-hoc test (P<0.05; n=4 to 6), as appropriate.
Results: Super-resolution microscopy and BRET data revealed that LHCGR and GPER form heteromers and interact on the cell surface, causing a displacement of Gαq to LHCGR. Consistently, under GPER/LHCGR co-expression, hCG-induced Ca2+ response is inhibited as well as all other Gαq-dependent events, i.e. IP1 production and NFAT promoter activation, whereas LH and hCG activate the Gαq-dependent signalling in LHCGR-expressing cells. Conversely, GPER-LHCGR complexes have no impact on LH/hCG-induced cAMP/protein kinase A (PKA) pathway activation, suggesting that GPER specifically inhibits Gαq-, but not Gαs-mediated signals. Control experiments were performed using a mutant GPER (GPERmut) unable to interact with LHCGR, as confirmed by PD-PALM and BRET methods, demonstrating that LH/hCG induce IP1 accumulation and gene transcription in GPERmut/LHCGR-expressing cells.
Conclusion: GPER and LHCGR can form heteromers at the cell surface, biasing LH/hCG-induced signals via specific inhibition of Gαq-dependent cascades and suggesting their potential role in the regulation of reproductive functions in the ovary.