Endocrine Abstracts

JOINT3438

The G protein-coupled estrogen receptor (GPER) forms heteromeric complexes with the gonadotropin receptors, reprogramming proliferative and anti-apoptotic signals. In the transfected HEK293 cell line, co-expression of GPER and luteinizing hormone (LH)/choriogonadotropin (hCG) receptor (LHCGR) specifically prevents LH- and hCG-dependent activation of Gq-mediated pathway and inhibits cell proliferation. In this study, we evaluated whether LHCGR/GPER heteromers modulate gonadotropin-dependent signals in gonadal cells endogenously expressing the two receptors. Expression level of genes coding G proteins (GNAS, GNAQ), and receptor (LHCGR and GPER) were quantified by digital droplet PCR, in native and GPER/Gper-coding cDNA transfected murine Leydig tumor cell line 1 (mLTC-1) and primary human granulosa cells (hGLCs). Receptor-receptor interactions were evaluated by proximity ligation assay (PLA). Cells were treated with LH/hCG, and signaling analysis was performed to evaluate intracellular calcium ion (Ca²⁺), cAMP, inositol monophosphate (IP1) increase, and steroid production by bioimaging methods, and homogeneous time-resolved fluorescence (HTRF). Data was analyzed by Kruskal-Wallis test and Dunn’s post-hoc test (P < 0.05; n = 4 to 6), as appropriate. Gene expression analysis confirmed that both cell lines have LHCGR and GPER transcripts, whose levels increased in transfected cells. PLA revealed the presence of LHCGR/GPER heteromers on mLTC-1 and hGLC cell surface. Receptor co-expression did not impair LH/hCG-dependent Gq-mediated pathway, as gonadotropins failed to activate it. In mLTC-1 and hGLC cells, Gs expression levels are higher than those of Gq, with LHCGR:G protein 10:1 e 0.5:1 ratio respectively, suggestive of preferential receptor occupancy by Gs, and only marginal Gq coupling. Therefore, LH/hCG-treatment activates intracellular cAMP response and steroid production but fails to trigger Ca²⁺/IP1 activation in both gonadal cell types. IP1 response is restored in tranfected cells overexpressing Gq protein. In conclusion, endogenous GPER naturally interacts with LHCGR in Leydig and granulosa cells. However, GPER has no effect on Gq pathway, which is switched off by preferential LHCGR coupling to Gs.