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Title: Molecular studies on the interaction of leptin with its receptor
Author: Mistrik, Pavel
ISNI:       0000 0001 3411 1663
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2000
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Leptin is a hormonal protein involved in energy homeostasis, acting to inhibit food intake, stimulate energy expenditure and influence insulin secretion, lipo-lysis and sugar transport. Its action is mediated by a specific receptor whose activation is highly controversial. As a member of the cytokine receptor super-family, it has been predicted to be activated by ligand-induced dimerization. However, recent evidence has suggested that this receptor exists as a dimer in both ligand-free and ligand-bound states. The aim of this project was to determine the kinetics and stoichiometry of leptin receptor interaction with its ligand, using a variety of biophysical techniques, namely BiaCore and microcalorimetry. To achieve this, it was necessary to express the leptin receptor. Because the receptor cDNA was not available at the start of this project, the initial goal was to obtain the cDNA encoding the extracellular domain of the receptor by RT-PCR. The open reading frame consisting of 839 a.a. encoded by 2517 nucleotides was generated by several molecular approaches, as the mRNA is a rare species. To generate large amounts of the receptor required for microcalorimetry, Baculovirus expression system for the leptin receptor production was devel-oped. At the same time BiaCore analysis of the interaction was performed since it requires small amounts of protein, and commercially available protein could be used. BiaCore was used to measure the thermodynamics of the interaction. Hu-man or mouse receptor chimeras comprising two receptor extracellular domains fused to the Fc region of IgGI were captured on to the sensor via Protein G. The kinetics and stoichiometry of interactions with human, mouse or rat lep-tin were measured. This data demonstrated a high affinity interaction. The KD was 0.2 +/- 0.1 nM, with ka = (1.9 +/- 0.4) x10e6 M-1s-1 and kd = (4.6 +/- 0.9) x10e-4 s-1 for human leptin with its cognate receptor. The observed stoichiometry was 1:1. Little difference was observed for different species of leptin. Thus, leptin forms a very stable 1:1 complex with its receptor. This observation indicates that the leptin receptor oligomerization state is not altered during its interaction with a ligand. This contradicts the common paradigm of cytokine receptor activation. A truncated version of the leptin molecule with deleted glutamine at position 28 was also expressed in E. coli. Its affinity for human and mouse leptin receptor chimeras was analysed by BiaCore, which revealed a 10-fold decrease in affinity, indicating a possible involvement of Q28 in binding.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Hormonal protein; Energy homeostasis; Kinetics