Abstract

Follicle-stimulating hormone (FSH) and its target G protein-coupled receptor (FSHR) are essential for reproduction. Recent studies have established that the hypo-glycosylated pituitary FSH glycoform (FSH21/18), is more bioactive in vitro and in vivo than the fully-glycosylated variant (FSH24). FSH21/18 predominates in women of reproductive prime and FSH24 in peri-post-menopausal women, suggesting distinct functional roles of these FSH glycoforms. The aim of this study was to determine if differential FSH glycosylation modulated FSHR oligomerization and resulting impact on cAMP signaling. Using a modified super-resolution imaging technique (PD-PALM) to assess FSHR complexes in HEK293 cells expressing FSHR, we observed time and concentration-dependent modulation of FSHR oligomerization by FSH glycoforms. High eFSH and FSH21/18 concentrations rapidly dissociated FSHR oligomers into monomers, whereas FSH24 displayed slower kinetics. The FSHR β-arrestin biased agonist, truncated eLHβ (Δ121-149) combined with asparagine56-deglycosylated eLHα (dg-eLHt), increased FSHR homomerization. In contrast, low FSH21/18 and FSH24 concentrations promoted FSHR association into oligomers. Dissociation of FSHR oligomers correlated with time points where higher cAMP production was observed. Taken together, these data suggest that FSH glycosylation may modulate the kinetics and amplitude of cAMP production, in part, by forming distinct FSHR complexes, highlighting potential avenues for novel therapeutic targeting of the FSHR to improve IVF outcomes.

Highlights

  • The actions of follicle-stimulating hormone (FSH) and its receptor (FSHR) are essential for reproduction [1,2,3,4,5]

  • Acute 2-minute treatment of HEK293 cells expressing FSHR with either equine FSH (eFSH) or FSH21/18 significantly decreased the overall percentage of associated FSHR, with 20.0 ± 1.3% and 17.5 ± 1.6% associated as dimers and oligomers, respectively (Figure 1Aii)

  • We have shown that pituitary Follicle-stimulating hormone (FSH) glycoforms regulate FSHR homomerization in a time- and concentration-dependent manner

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Summary

Introduction

The actions of follicle-stimulating hormone (FSH) and its receptor (FSHR) are essential for reproduction [1,2,3,4,5]. FSHb differs in amino acid sequence and glycosylation pattern to other glycoprotein hormone beta subunits, conferring biological specificity and selectivity of FSH to FSHR [12]. Functional analysis has shown that this difference in glycosylation pattern results in modulation of binding to the FSHR [14] and the amplitude of the canonical FSHR signaling pathway, the Gas/cAMP/PKA signaling, with FSH21/18 displaying faster binding kinetics and more potent activation of Gas signaling [15,16,17]. Important functional differences have been observed, with differences in ovarian and testicular gene expression in mice injected with either FSH21/18 or FSH24 [19]. How these differences in the signaling properties of FSH21/18 and FSH24 are interpreted by FSHR remain unknown

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