Temporal reprogramming of calcium signalling via crosstalk of gonadotrophin receptors that associate as functionally asymmetric heteromers

Abstract

Signal crosstalk between distinct G protein-coupled receptors (GPCRs) is one mechanism that underlies pleiotropic signalling. Such crosstalk is also pertinent for GPCRs activated by gonadotrophic hormones; follicle-stimulating hormone (FSH) and luteinising hormone (LH), with specific relevance to female reproduction. Here, we demonstrate that gonadotrophin receptor crosstalk alters LH-induced Gαq/11-calcium profiles. LH-induced calcium signals in both heterologous and primary human granulosa cells were prolonged by FSHR coexpression via influx of extracellular calcium in a receptor specific manner. LHR/FSHR crosstalk involves Gαq/11 activation as a Gαq/11 inhibitor abolished calcium responses. Interestingly, the enhanced LH-mediated calcium signalling induced by FSHR co-expression was dependent on intracellular calcium store release and involved Gβγ. Biophysical analysis of receptor and Gαq interactions indicated that ligand-dependent association between LHR and Gαq was rearranged in the presence of FSHR, enabling FSHR to closely associate with Gαq following LHR activation. This suggests that crosstalk may occur via close associations as heteromers. Super-resolution imaging revealed that LHR and FSHR formed constitutive heteromers at the plasma membrane. Intriguingly, the ratio of LHR:FSHR in heterotetramers was specifically altered following LH treatment. We propose that functionally significant FSHR/LHR crosstalk reprograms LH-mediated calcium signalling at the interface of receptor-G protein via formation of asymmetric complexes.

Driving gonadotrophin hormone receptor signalling: the role of membrane trafficking

“…Although rodent LHR displays distinct properties in terms of internalisation kinetics and post-endocytic sorting pathways from human LHCGR, perhaps the trafficking properties of rodent LHR may be distinct in the ovary compared to heterologous cells, not only due the distinct intracellular environment of these cell types, but also interaction with other GPCRs, including the FSHR. Heteromers of LHCGR and FSHR have been reported by several groups (Feng et al 2013, Mazurkiewicz et al 2015, Jonas et al 2018, and given both human FSHR and rodent FSHR exhibit similar trafficking properties, perhaps interactions of LHR with FSHR may enable greater internalisation and spatial control of signalling in response to LH. An additional possibility is that the distinct trafficking profiles of human LHCGR and rodent LHR may enable differential spatial control of signal pathways, which then translates to distinct downstream functions, as indicated by known differences in phenotypes of certain disease-causing mutations in human gonadotrophin hormone receptors compared to animal models harbouring the same mutations (Huhtaniemi 2006).…”

Membrane estrogen receptor (GPER) and follicle-stimulating hormone receptor heteromeric complexes promote human ovarian follicle survival

“…We reasoned that the opposing nature of FSHR activity, proliferative and pro-apoptotic, may rely on the cooperation with other factors modulating FSHR signaling. In particular, as described for other structurally similar GPCRs [35][36][37][38] , FSHR signaling may be regulated by forming heteromers with other receptors 10 . Here , we demonstrate that GPER and FSHR interactions reprogram FSHR-related death into life signals, upregulating the viability of FSHR/GPER expressing cells including human ovarian granulosa cells.…”

“…Further evidence of FSHR-GPER heteromer assembly at the plasma membrane was provided by photo-activated localization microscopy with photoactivatable dyes (PD-PALM) ( Fig. 3G-I), a super-resolution imaging approach we have previously employed to quantitate 14 protomer composition within asymmetric heteromer complexes between LHR mutants and between FSHR and LHR 38,42 . HEK 293 cells expressing HA-tagged FSHR and FLAG-tagged GPER individually, or together, were labeled with CAGE500-conjugated anti-HA and CAGE552-conjugated anti-FLAG antibodies and fixed for PD-PALM imaging ( Fig.…”

G Protein-Coupled Receptor Signaling

“…We have employed PD-PALM to study the organization of complexes between the gonadotrophin hormone Class A GPCRs, specifically luteinising hormone receptor (LHR) di/oligomers and LHR heteromers with follicle-stimulating hormone receptor (FSHR) (23). Our prior studies, and reviews, in this area have discussed the physiological importance of these interactions to reproduction (22)(23)(24).…”