Endocrine Abstracts

The G protein-coupled receptor, follicle-stimulating hormone receptor (FSHR), is essential for reproduction. A key drug target of IVF, understanding the mechanisms modulating FSHR functions remains of high importance. The endogenous ligand of the FSHR, FSH, is a heterodimeric glycoprotein hormone with two predominant glycoforms identified. Partially glycosylated FSH21 has faster binding kinetics to the FSHR and more potent at activating cAMP-dependent signal pathways, in comparison to fully glycosylated FSH24. An important mechanism of regulating GPCR function is the formation of dimers and oligomers. The FSHR can self-associate, yet how FSH glycosylation regulates FSHR oligomerization remains unknown. The aim of this study was to determine if modulation of FSHR oligomers mediated the differential signalling displayed by FSH glycoforms. Using a modified super-resolution imaging technique (PD-PALM) to assess FSHR complexes, HEK293 cells expressing FSHR were treated ± 30ng/ml eFSH, FSH21, FSH24 or a FSHR biased agonist; eLHdg. eFSH and FSH21 rapidly dissociated FSHR oligomers into monomers at 2-minute treatment (P < 0.001), and pentamers at 5- and 15-minutes. FSH24 displayed slower kinetics, dissociating FSHR oligomers at 5-minutes (P < 0.01). In contrast, eLHdg enhanced FSHR oligomerisation into predominantly tetramers (P < 0.01) and trimers (P < 0.05) at 5- and 15-minutes, respectively. Analysis of cAMP production by glosensor and Cre-luciferase reporter gene assays showed higher cAMP production by eFSH and FSH21, suggesting monomers/lower-order oligomers favour cAMP production. We next investigated the concentration-dependent effects of FSH glycoforms on FSHR oligomerization. Interestingly, at 5-minutes FSH24 induced rapid FSHR oligomer formation followed by FSH21 at 15-minutes. Only low-level cAMP production was observed with all ligand treatments, with significant increases in cAMP at 15 minutes by eFSH and FSH21. These data suggest functional specificity of FSH glycoforms at different concentrations may be mediated by FSHR oligomer rearrangements. Thus, highlighting potential novel avenues for therapeutic targeting of the FSHR to improve IVF outcomes.