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Title: Functional studies of kisspeptin analogues and the human kisspeptin receptor
Author: Zhang, X.
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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Kisspeptins and their cognate receptor, the kisspeptin receptor, play pivotal roles in the regulation of the development of puberty and cancer metastasis. However, the signalling pathways activated by kisspeptins and the kisspeptin receptor remain largely to be demonstrated. Aims of the thesis are to investigate the biological functions of kisspeptin analogues and the human kisspeptin receptor. Since kisspeptins show low metabolic stability, it may limit the studies in the long-term signalling of kisspeptins and the kisspeptin receptor. To improve the metabolic stability of kisspeptins, in the thesis, three novel phosphinic peptides were designed and synthesised based on the amino acid sequence of KP-10, which are the last ten amino acids shared by all native and functional kisspeptins. Whether the synthetic peptides can bind to and activate the kisspeptin receptor and their ability to inhibit MMP were tested. The results showed that among synthesized peptides, compound B possesses the kisspeptin receptor-agonistic activity and could function as a selective inhibitor of MMP-2. However, the binding of the synthesized peptides to the kisspeptin receptor was undetectable using the presented methods. In addition, the Pro-rich region within the C-terminal tail of the kisspeptin receptor has been proposed to function as a SH3 binding motif to mediate the interactions of the receptor with non-G protein effectors. Therefore, the interaction of the kisspeptin receptor with two candidates, p85α or c-SRC, was elucidated by using co-IP assays and their potential biological functions were examined. Direct interactions of the human kisspeptin receptor with p85α and c-SRC were observed in human breast cells and the interactions were not affected by short-time KP-10 stimulation. Functionally, the kisspeptin receptor may directly bind to p85α or c-SRC to activate Gq/11- independent PI3K/AKT and c-SRC-dependent ERK1/2 signalling pathways. Importantly, a weak negative crosstalk of the kisspeptin receptor with insulin receptor, which kisspeptins inhibited insulin-induced phosphorylation of AKT, was observed. Furthermore, two Cys residues, Cys338 and Cys340, in the C-terminal tail of the kisspeptin receptor are predicted to be palmitoylation sites and, therefore, be important for the expression and function of the kisspeptin receptor. In the thesis, the roles of Cys338 and Cys340 on the receptor expression and signalling were investigated by using mutagenesis studies. The Ser substitution of Cys338 had little effect on receptor expression, internalization and functions. By contrast, although the mutation of Cys340 to Ser had little effect on expression and the downstream signalling evens measured, it did affect the internalization of the receptor. The Ser substitution of both Cys338 and Cys340 decreased the total expression of the receptor and abolished receptor signalling and internalization. The results may indicate that Cys340 is a primary palmitoylation site and Cys338 acts as an alternative palmitoylation site to rescue the signalling, at least some aspects, of the receptor, when is Cys340 mutated. Alternatively, Cys338 may be normally palmitoylated, but this has effects on signalling functions not measured here, and does not impact on receptor internalization.
Supervisor: Lu, Z. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral