Single-Cell Analyses Reveal That KISS1R-Expressing Cells Undergo Sustained Kisspeptin-Induced Signaling That Is Dependent upon An Influx of Extracellular Ca2+

Abstract

Abstract The kisspeptin receptor (KISS1R) is a Gαq/11-coupled seven-transmembrane receptor activated by a group of peptides referred to as kisspeptins (Kps). The Kp/KISS1R signaling system is a powerful regulator of GnRH secretion, and inactivating mutations in this system are associated with hypogonadotropic hypogonadism. A recent study revealed that Kp triggers prolonged signaling; not from the inability of the receptor to undergo rapid desensitization, but instead from the maintenance of a dynamic and active pool of KISS1R at the cell surface. To investigate this further, we hypothesized that if a dynamic pool of receptor is maintained at the cell surface for a protracted period, chronic Kp-10 treatment would trigger the sustained activation of Gαq/11 as evidenced through the prolonged activation of phospholipase C, protein kinase C, and prolonged mobilization of intracellular Ca2+. Through single-cell analyses, we tested our hypothesis in human embryonic kidney (HEK) 293 cells and found that was indeed the case. We subsequently determined that prolonged KISS1R signaling was not a phenomenon specific to HEK 293 cells but is likely a conserved property of KISS1R-expressing cells because evidence of sustained KISS1R signaling was also observed in the GT1–7 GnRH neuronal and Chinese hamster ovary cell lines. While exploring the regulation of prolonged KISS1R signaling, we identified a critical role for extracellular Ca2+. We found that although free intracellular Ca2+, primarily derived from intracellular stores, was sufficient to trigger the acute activation of a major KISS1R secondary effector, protein kinase C, it was insufficient to sustain chronic KISS1R signaling; instead extracellular Ca2+ was absolutely required for this.