Endocrine Abstracts

The Class A G protein-coupled receptor (GPCR), follicle-stimulating hormone receptor (FSHR), and its associated heterodimeric glycoprotein hormone (FSH) are essential for reproduction. As such, they’re key targets of assisted conception. Post-translational modification of FSH gives rise to two predominant glycosylation variants, which are modulated with ageing: partially glycosylated FSH (FSH21/18), predominates in women’s reproductive prime (20’s), displays faster binding kinetics to the FSHR, and is a more potent activator of cAMP-dependent signalling. Contrasting with fully glycosylated FSH (FSH24) which is more predominant in peri-menopausal women (50’s), and less bioactive. Recent studies have suggested a link between receptor trafficking and signalling, yet how FSH glycoforms modulate FSHR trafficking remains unknown. This study aimed to determine how FSH glycoforms modulate FSHR trafficking and link to signal activation. HEK293 cells transiently expressing the FSHR were pre-treated with a dynamin inhibitor, Dyngo-4a (50μM), to inhibit FSHR internalisation, before treatment with either FSH21/18 and FSH24 and cAMP analysed. When assessing live cAMP, pre-treatment with Dyngo-4a significantly attenuated both FSH21/18 and FSH24 activation, moreover, this was additionally supported by attenuation of FSH glycoform-dependent cre-luciferase activity. Confocal microscopy analysis of FSHR endosomal routing revealed temporal FSH-glycoform-dependent differences in the routing of FSHR to EEA1-positve endosomes, with FSH21/18 displaying increased FSHR-EEA1 co-localisation, contrasting to no changes observed in FSH24-treated cells in comparison to control. siRNA knockdown of the early endosomal adapter protein, APPL1, which has been linked to cAMP production supported distinct differences in FSHR endosomal routing as APPL1 knockdown had no effect on FSH21/18 dependent cAMP-signalling. However, enhanced FSH24-dependent cre-luciferase activity was observed. Together, these data suggest that differential FSH glycosylation may modulate the endosomal routing of FSHR to fine-tune cAMP production. This may have implications for altered FSH/FSHR actions in the ageing ovary, highlighting novel targeting mechanisms for enhancing assisted conception.