Characterization of KISS1 receptor signaling and the effects of gain-of-function and loss-of-function mutations
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Abstract
Background: Current reports by the Center of Disease Control suggest that 7.3 million women between the age of 15 and 44 struggle with infertility. Although current therapeutics have aided many of these women to reach reproductive competence, many remain diagnosed with idiopathic infertility. Recent identification of a novel mutation in the kisspeptin receptor (KISS1R), producing idiopathic hypogonadotropic hypogonadism, has implicated this receptor and its ligand in the regulation of the hypothalamic-pituitary-gonadal axis at the level of the hypothalamus. To date, the mechanisms by which KISS1R, a G-coupled protein receptor, elicits activation of signaling pathways and culminates in pulsatile release of GnRH are poorly understood.
Aims: The goal of this study is to characterize the mechanisms by which KISS1R activates signaling in cell lines stably expressing WT KISS1R in comparison to two KISS1R mutants, R386P and L148S identified in patients with precocious puberty and hypogonadotropic hypogonadism, respectively.
Methods: Intracellular calcium flux, as detected by a fluorescence microplate reader, was utilized to detect dynamic ligand-dependent activation of KISS1R signaling. Biotinylation of the HEK-WT-KISS1R was used to identify how quickly the receptor undergoes degradation in the absence and presence of ligand. Phosphorylation of ERK, an extracellular regulated kinase of the MAPK signaling pathway, was examined as an indicator of downstream effector activation. Inositol phosphate assays were performed to validate previous data suggesting that KISS1R activation is mediated through IP3- and DAG-dependent signaling.
Results: A ligand-dependent CHO-KISS1R mediated calcium response was identified to be biphasic and unlike that of the G-coupled protein receptor, CHO-TRHR, which produces an acute calcium response to ligand. For HEK-WT-KISS1R and HEK-R386P-KISS1R, the second phase of the calcium response could be eliminated by the removal of exogenous ligand and was dependent on an extracellular calcium source. Biotinylation of HEK-WT-KISS1R revealed that the receptor does not undergo rapid degradation in the absence or presence of ligand, and can be detected for up to 24 h. A calcium dose response curve suggested that the HEK-R386P-KISS1R mutant can produce a more robust response to ligand compared to HEK-WT-KISS1R. The HEK-L 148S-KISS1R was unable to elicit a calcium response to ligand stimulation under the same conditions. Activation of MAPK signaling pathways, as determined by measurement of phosphorylated ERK over a time course, was identified for all three receptors, and was found to be robust for HEK-R386P-KISS1R but greatly reduced for HEK-L 148S-KISS1R in comparison to HEK-WT-KISS1R. Inositol phosphate accumulation in response to ligand activation was detected for HEKWT-KISS1R and HEK-R386P-KISS1R, but with no appreciable difference between the receptors' responses to increasing concentrations of kisspeptin-10 stimulation.
Conclusion: Activation of KISS1R produces a biphasic calcium response that is dependent on the presence of ligand and extracellular calcium. These properties are believed to be intrinsic to the receptor, suggesting that the receptor may regulate its own desensitization and resensitization. The HEK-L 148S-KISS1R mutation was unable to elicit appreciable activation of downstream signaling pathways which is in accord with previous characterization of this mutant to be unable to signal through G-protein-dependent pathways. The mutant HEK-R386P-KISS1R activates a calcium-dependent, MAPK-specific signaling cascade that is more robust than that of HEK-WT-KISS1R and may be responsible for the accelerated activation of the hypothalamic-pituitary-gonadal axis, as observed in the patient identified with this mutation who presented with precocious puberty. Current therapeutics targeting KISS1R are not effective and a better understanding of the mechanisms by which KISS1R promotes signaling is needed in order for them to be successful in the clinical setting.
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Thesis (M.A.)--Boston University
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