Profiling chemokine signalling bias of CCR4, CCR7 and CCR10

Kelly, Christopher James (2019) Profiling chemokine signalling bias of CCR4, CCR7 and CCR10. PhD thesis, University of Glasgow.

Due to Embargo and/or Third Party Copyright restrictions, this thesis is not available in this service.

Abstract

Inflammatory chemokine signalling is implicated in a broad range of pathologies and has been intensely researched. However, chemokine receptors remain poorly druggable. This may be due to the complexity of chemokine signalling; multiple chemokines can bind a single receptor, and an individual chemokine can bind multiple receptors. Additionally, many receptors demonstrate signalling bias, where different ligands elicit diverse responses. Bias signalling is an area of emerging interest with relevance in drug development, and there is a call to better characterise chemokine signalling bias. CCR4, CCR7 and CCR10 share a similar molecular evolution; each interacts with a ligand pair resultant from gene duplication, with one ligand in each pair being “dominant” in receptor activation, i.e. able to fully activate the receptor triggering recruitment of G-proteins and-arrestin. It may be possible, with a greater understanding of the underlying biology of biased signalling, to generate future antagonists of these receptors that selectively inhibit individual signalling pathways, as has been demonstrated previously with other GPCRs.

Biased signalling is believed to result from slight variation in ligand/receptor binding that lock the receptor in specific conformations for coupling to signalling apparatus. In an effort to visualise these subtle differences, a novel unnatural amino acid (UAA) capture based methodology was attempted. An expression vector for a UAA compatible tRNA/Synthetase pair, as well as mutants of 16 residues of CCR7, were generated. UAA integration was observed with all 16 mutants, however protein level and ligand binding varied significantly between sites of integration. UV crosslinking of ligand and receptor was attempted, but no combination of chemokine and substitution site demonstrated successful chemokine capture.

In an effort to fully profile the bias of CCRs 4, 7 and 10, BRET based methods were employed to assess -arrestin and G-protein recruitment by the receptors in response to their cognate ligands. These data confirmed the reported bias, that CCL22, 19 and 21 trigger -arrestin recruitment to CCR4, 7 and 10 respectively, but CCL17 and CCL28 failed to trigger recruitment, and CCL21 did so at poorer efficiency than CCL19. Interestingly, this pattern remained at the level of G-protein recruitment also, in contrast to the previously reported signalling properties of these chemokines.

Recently, CCR7 was identified as a target of ST8sia4 mediated polysialylation, with significant effects on the biological activity of CCL21. As such, the role of ST8sia4 in chemokine biology was further examined. These data revealed that HEK293T cells, as well as other adherent cell lines, demonstrate little to no expression of ST8sia4. Reintroduction of polysialic acid modification of CCR7 restored the capacity of CCL21 to compete with labelled CCL19 on HEK293T cells, resulting in a partial phenocopy of primary cells in the same assay, and resulted in a shift in potency of CCL21 for arrestin recruitment. This was mirrored at the level of G-protein recruitment, indicating that the previously noted lack of G-protein in response to CCL21 was most likely due to the lack of polysialylation of CCR7. Conversely, inclusion of ST8sia4 expression in the assessment of CCR4 signalling demonstrated no change from previous experiments. CCR10 however was revealed to be a previously undescribed target of ST8sia4 mediated polysialylation, with implications for the signalling potential of both of its cognate ligands.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: chemokines, chemokine signalling, biased agonism, unnatural amino acids, CCR4, CCR7, CCR10, ST8sia4, polysialylation, polysialic acid.
Subjects: Q Science > QR Microbiology > QR180 Immunology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation > Immunology
Funder's Name: GLAZgo Discovery Centre, Wellcome Trust (WELLCOTR)
Supervisor's Name: Graham, Professor Gerard and Milligan, Professor Graeme and Dainty, Doctor Ian
Date of Award: 2019
Embargo Date: 25 August 2019
Depositing User: Mr Christopher J Kelly
Unique ID: glathesis:2019-41049
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 26 Feb 2019 15:00
Last Modified: 02 Apr 2019 08:05
URI: http://theses.gla.ac.uk/id/eprint/41049

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