Pituitary luteinizing hormone (LH) and placental human choriogonadotropin (hCG) are two heterodimeric glycoprotein hormones regulating reproduction. They bind the same receptor (LHCGR) expressed in gonadal cells, activating hormone-specific G protein- and β-arrestins-dependent signaling cascades, before LHCGR internalization. LH induces preferential proliferative signals, while hCG activates mainly the steroidogenic pathway, reflecting their physiological roles. In this study,we compared the kinetics of LH- versus hCG-mediatedG proteins and β-arrestin 2 activation in vitro. The HEK293 cell line was transiently transfected with the LHCGR-encoding plasmid, together with that encoding G proteins- or β-arrestin 2-tagged bioluminescence resonance energy transfer (BRET) biosensors. Cells were treated by various hCG and LH concentrations (pM-nM range) and the activation of Gαs, Gαq, Gαi protein and β-arrestins was evaluated by BRET, under native conditions or after inhibition of LHCGR internalization by Dynasore. We demonstrated that LH/hCG binding to LHCGR mediates the activation of G proteins and recruitment of β-arrestin 2 in a hormone-specific manner. LHCGR-Gαs protein interaction increased upon 10 nM hCG, but not LH treatment (one-way ANOVA; P < 0.05; n = 8), reflecting the previously demonstrated preferential activation of intracellular cAMP signaling by the placental hormone [doi:10.1210/er.2018-00065]. These data are confirmed by kinetics analysis intracellular cAMP increase over 120-min, which achieved higher levels upon treatment by hCG than LH when hormones are administrated to cells at the 50% effective concentration (EC50; hCG = 100 pM; LH = 500 pM; Kruskal-Wallis test, P < 0.05; n = 4). On the other hand, LH was more potent than hCG in promoting LHCGR-Gαi protein and LHCGR-β-arrestin 2 interactions, thus confirming preferential activation of proliferative signals (means ± SEM one-way ANOVA; P < 0.05; n = 8) [doi:10.1210/er.2018-00065]. Interestingly, LH and hCG mediated similar kinetics of intracellular cAMP increase between 30–120 min, when LHCGR internalization is blocked by Dynasore, while short-term (0-30) min hCG-induced cAMP levels are anyway higher than those LH-mediated (Kruskal-Wallis test, P < 0.05; n = 4). These results suggest that endosomal compartmentalization of LHCGR is involved in the differentiation of the long-term LH/hCG-induced cAMP signaling.
We have demonstrated that LH and hCG drive LHCGR in hormone-specific interaction with Gαs and i proteins, as well as β-arrestin 2 and endosomal compartments, and resulting in different intracellular signaling patterns.
05 Sep 2020 - 09 Sep 2020