Investigating the molecular regulation of follicle-stimulating hormone-mediated receptor oligomerisation and trafficking, and related signalling.

Abstract

Follicle-stimulating hormone (FSH), and its associated G protein-coupled receptor (GPCR), the FSH receptor (FSHR), play multiple crucial roles in female reproduction, such as folliculogenesis, dominant follicle selection and maintenance of steroidogenesis. Therefore, as key targets of assisted conception, there is therapeutic interest in identifying new modalities for modulating their functions. FSHR primarily mediates its effects via coupling to the Gαs/cAMP/PKA pathway but can also activate multiple signalling pathways via various mechanisms, including mediating additional signalling platforms via internalisation and trafficking to endosomes. Nonetheless, how this signal pleiotropy is initiated remains largely unknown. Post-translational modification of FSH gives rise to two predominant glycoforms; partially glycosylated FSH (FSH21/18), which is more bioactive in vitro and more abundant in women in their reproductive prime, in contrast to fully glycosylated FSH (FSH24), which is less bioactive and more abundant in peri/menopausal women. As well as endogenous ligands, drug discovery programs have resulted in the production of small molecule FSHR modulators that can enhance/inhibit the actions of FSH. This may have important therapeutic benefits for enhancing FSHR activity by providing alternative routes for drug administration and a means to target reproductive pathologies associated with menopausal-related elevation of FSH. Nevertheless, how these modulators propagate their effects also remains unclear. An important modality of how GPCRs can fine-tune receptor signalling is via self-association to form dimer and oligomer complexes. The FSHR has been shown to self-associate, however, the functional role of FSHR oligomerisation, and the impact on signalling and trafficking remains elusive. Therefore, the aim of this thesis was to determine how native ligands and pharmacological FSHR modulators modulate FSHR oligomerisation, downstream signalling, and trafficking. Results showed that FSH glycoforms differentially modulate FSHR monomers, dimers, and oligomers in a temporal- and concentration-dependent manner, correlating with the magnitude and timing of cAMP production and possible β-arrestin/ERK signal pathway selectivity. Furthermore, the differences in FSH glycoform-dependent cAMP-dependent signalling may be further regulated by differences in the routing of the internalised FSHR by different FSH glycoforms. Treatments with the potent FSHR agonist Compound 5 (C5) showed rapid maximal enhancement of FSHR activity that is possibly mediated by FSHR trafficking and recycling to the cell-surface. Moreover, three newly identified FSHR non-competitive antagonists, that enhance FSH binding but reduced FSH activity, may regulate these differences through changes in FSHR oligomerisation. These data suggest that different FSHR ligands and modulators display nuanced mechanisms to modulate FSHR signalling, with the potential impact on physiological outcomes to be determined. Such mechanisms may have the ability to be regulated to modulate FSHR function, with a potential to improve fertility and treat age-related reproductive pathologies.

Date of Award	1 Jul 2023
Original language	English
Awarding Institution	King's College London
Supervisor	Kim Jonas (Supervisor) & Anthony Albert (Supervisor)