GSE184352
Human pluripotent stem cell-derived cells endogenously expressing follicle-stimulating hormone receptors: modeling the function of an inactivating receptor mutation
Follicle-stimulating hormone (FSH) is crucial in the development and regulation of reproductive functions. The actions of human FSH and its receptor (FSHR) and mutations therein have mainly been studied using in vivo models, primary cells, cancer cells and cell lines ectopically expressing the FSHR. To allow studies of endogenous FSHR function in vitro, we differentiated FSHR-expressing cells from human pluripotent stem cells. FSH stimulation of the wild-type (WT), but not the inactivating Finnish founder mutant (A189V) receptor, activated the canonical cAMP-dependent signaling pathway and downstream mediators. Interestingly, the WT and A189V mutant receptors activated distinct pathways in a G-protein-coupled receptor signaling assay. FSH stimulation of WT FSHR-expressing cells induced phosphorylation of PI3Kp85a/g, MAPKAPK2 and proteins involved in calcium-dependent signaling, among others, while in the A189V mutant cells FSH induced phosphorylation of e.g., P90RSK, ERK3, and focal adhesion kinase. Lastly, we expressed FSHR in HEK293 cells followed by affinity purification mass spectrometry analyses, to investigate protein-protein interaction partners of FSHR at resting state and upon FSH stimulation. We found 19 specific high-confidence interacting proteins for WT FSHR and 14 for A189V FSHR, several of which have been linked to infertility. In conclusion, our protocol allows detailed studies of FSH action and disease modeling in human cells endogenously expressing FSHR. Overall design: Two samples (separate experiments) of human cells endogenously expressing FSHR (follicle stimulating hormone receptor) derived by 8-day directed differentiation of human embryonic stem cells (H9).