In Vitro Impact of FSH Glycosylation Variants on FSH Receptor-stimulated Signal Transduction and Functional Selectivity.

FSH exists as different glycoforms that differ in glycosylation of the hormone-specific β-subunit. Tetra-glycosylated FSH (FSH) and hypo-glycosylated FSH (FSH) are the most abundant glycoforms found in humans. Employing distinct readouts in HEK293 cells expressing the FSH receptor, we compared signaling triggered by human pituitary FSH preparations (FSH and FSH) as well as by equine FSH (FSH), and human recombinant FSH (FSH), each exhibiting distinct glycosylation patterns. The potency in eliciting cAMP production was greater for FSH than for FSH, FSH, and FSH, whereas in the ERK1/2 activation readout, potency was highest for FSH followed by FSH, FSH, and FSH. In β-arrestin1/2 CRISPR/Cas9 HEK293-KO cells, FSH exhibited a preference toward β-arrestin-mediated ERK1/2 activation as revealed by a drastic decrease in pERK during the first 15-minute exposure to this glycoform. Exposure of β-arrestin1/2 KO cells to H89 additionally decreased pERK1/2, albeit to a significantly lower extent in response to FSH. Concurrent silencing of β-arrestin and PKA signaling, incompletely suppressed pERK response to FSH glycoforms, suggesting that pathways other than those dependent on Gs-protein and β-arrestins also contribute to FSH-stimulated pERK1/2. All FSH glycoforms stimulated intracellular Ca (iCa) accumulation through both influx from Ca channels and release from intracellular stores; however, iCa in response to FSH depended more on the latter, suggesting differences in mechanisms through which glycoforms promote iCa accumulation. These data indicate that FSH glycosylation plays an important role in defining not only the intensity but also the functional selectivity for the mechanisms leading to activation of distinct signaling cascades.