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

Abstract

FSH exists as different glycoforms that differ in glycosylation of the hormone-specific β-subunit. Tetra-glycosylated FSH (FSH²⁴) and hypo-glycosylated FSH (FSH^18/21) 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^18/21 and FSH²⁴) as well as by equine FSH (eFSH), and human recombinant FSH (recFSH), each exhibiting distinct glycosylation patterns. The potency in eliciting cAMP production was greater for eFSH than for FSH^18/21, FSH²⁴, and recFSH, whereas in the ERK1/2 activation readout, potency was highest for FSH^18/21 followed by eFSH, recFSH, and FSH²⁴. In β-arrestin1/2 CRISPR/Cas9 HEK293-KO cells, FSH^18/21 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^18/21. 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 Ca²⁺ (iCa²⁺) accumulation through both influx from Ca²⁺ channels and release from intracellular stores; however, iCa²⁺ in response to FSH^18/21 depended more on the latter, suggesting differences in mechanisms through which glycoforms promote iCa²⁺ 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.