Functional regulation of kisspeptin receptor by calmodulin and Ca2+/calmodulin-dependent protein kinase II

Abstract

The kisspeptin receptor (KISS1R), functioning as a metastasis suppressor and gatekeeper of GnRH neurons, is a potent activator of intracellular Ca2+. The surge in cytoplasmic Ca2+ mediates the exocytosis of GnRH from GnRH neurons. However, the regulatory processes which enable KISS1R to sense increasing intracellular Ca2+ and avoid Ca2+ excitotoxicity via a signalling off-switch mechanism remain unclear. This thesis provides evidence for the interaction between KISS1R and the Ca2+ regulated proteins of calmodulin (CaM), and αCa2+/CaM-dependent-protein kinase II (α-CaMKII). Binding of CaM to KISS1R was shown with three independent approaches. Firstly, cell-free spectrofluorimeter assays showed that CaM selectively binds to intracellular loop (IL) 2 and IL3 of the KISS1R. Secondly, KISS1R co-immunoprecipitation experiments identified ligand/Ca2+-dependent binding of KISS1R to HEK-293 endogenous CaM. Thirdly, confocal experiments showed CFPCaM co-localises with YFP-KISS1R. The functional relevance of CaM binding was examined with alanine substitution of critical residues of the CaM binding motifs in IL2 and IL3 of KISS1R. This approach revealed that the receptor activity (relative maximum responsiveness) was increased in the mutated residues of the juxtamembrane regions of IL3 and the N-terminus of IL2 relative to wild-type KISS1R. The Ca2+/CaM regulated αCaMKII was also found to interact with KISS1R by selectively phosphorylating T77 of IL1. Phosphomimetic mutations of T77 into E or D created a receptor that was unable to elicit inositol phosphate production upon ligand stimulation. Finally, in vivo studies using ovariectomised rats that were intracerebroventricularly administered with a cell-permeable αCaMKII inhibitor augmented the effects of kisspeptin ligand stimulation of plasma luteinizing hormone levels. Taken together, this thesis demonstrates that the KISS1R-G protein coupling is regulated by Ca2+-dependent CaM binding and αCaMKII-mediated KISS1R phosphorylation.