Evidence for human kisspeptin receptor homo-oligomerisation and its functional relevance.

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Abstract: The signalling of kisspeptin-1 through the kisspeptin-1 receptor (KISS1R) is central to mammalian reproduction. Naturally occurring heterozygous KISS1R mutations and Kiss1r +/- knockout mice are less affected than their homozygous counterparts, suggesting that the mutant receptors possibly form oligomers with the wild-type (WT) KISS1R, rescuing the receptor function to some extent. To test this hypothesis, the heterozygous KISS1R mutations R38P, P46Q, S125L and R198G, reported in the literature in cases of delayed puberty, were characterised. In silico analysis predicted that all four mutations affected the receptor function to varying extents, which was substantiated by in vitro studies. Determination of cell surface receptor expression and kisspeptin-stimulated signalling response was carried out post transient transfection of the receptor constructs in CHO cells. Results revealed that these mutations (homozygous condition) impaired the cell surface receptor expression, as quantified by flow cytometry, with a concomitant attenuation of inositol phosphate production. Co-transfection of the WT KISS1R with equal amounts of the mutant receptors, to mimic the heterozygous condition of the mutation in the patients, restored the receptor function and, with increasing amounts of mutant receptors, resulted in attenuation of receptor function. As a direct proof of receptor oligomerisation, co-expression of epitope-tagged KISS1R constructs was carried out. Co-immunoprecipitation and imaging FRET studies revealed that KISS1R forms homo-oligomers in a constitutive manner and that the transmembrane domain 7 contributed to the oligomerisation interface, as demonstrated by the impairment in oligomerisation upon deletion of this domain. Thus, characterisation of heterozygous KISS1R mutations corroborated the oligomerisation status of the KISS1 receptor and helped in establishing a genotype-phenotype association.