In vitro impact of distinct FSH glycosylation variants on FSH receptor-stimulated signal transduction and functional selectivity

Follicle-stimulating hormone (FSH) exists as different major glycoforms
that differ in glycosylation of the hormone-specific β-subunit.
Tetra-glycosylated FSH (FSH24) and tri-glycosylated FSH (FSH21) are the
most abundant forms found in humans. Employing distinct readouts in HEK293
cells stably expressing the human FSH receptor, we here compared
intracellular signaling triggered by human pituitary FSH preparations
(FSH21and FSH24) as well as by equine FSH (eFSH), and human recombinant
FSH (recFSH), each exhibiting distinct glycosylation patterns. The
potencies in eliciting cAMP production were
eFSH>FSH21>FSH24>recFSH, whereas in the ERK1/2
activation readout potencies were FSH21≥eFSH>recFSH>FSH24.
In β-arrestin 1/2 CRISPR/Cas9 HEK293 KO cells, FSH21 exhibited a
preference towards β-arrestin-mediated ERK1/2 activation as revealed by a
drastic decrease in pERK during the first 15min exposure to this
glycoform. Further, exposure of β-arrestin1/2 KO cells to H89 additionally
decreased pERK1/2 albeit to a significantly lower extent in response to
FSH21. Concurrent silencing of both β-arrestin and PKA signaling, did not
completely suppress pERK in response to FSH glycoform, suggesting that
pathways other than those dependent on Gs-protein and β-arrestins also
contribute to FSH-stimulated pERK1/2. All four FSH glycoforms stimulated
iCa2+ accumulation through influx from Ca2+ channels as well as release
from intracellular stores; however, iCa2+ accumulation in response to
FSH21 was more dependent on the latter, suggesting differences in the
mechanisms through which FSH glycoforms promote  iCa2+
accumulation.These data indicate that FSH glycosylation plays an important
role in defining both the intensity of signal activation and the balance
among mechanistically different pathways mediating activation of distinct
signaling cascades.