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Modulation of chemoattractant receptor function by C-terminus interactions

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Title: Modulation of chemoattractant receptor function by C-terminus interactions
Authors: Patel, Pallavi
Item Type: Thesis or dissertation
Abstract: The chemokine receptors CCR3 and CCR4 play key roles in leukocyte recruitment in allergy. Consequently, both receptors are the focus of efforts to block receptor function with potential for the treatment of diseases such as asthma. Responses to chemokines are tightly regulated by the processes of receptor endocytosis and recycling. This thesis set out to examine the mechanisms by which the expression of both receptors are regulated. CCR3 is a key mediator of eosinophil migration and binds the eotaxin family of chemokines. Using a variety of assays with CCR3 transfectants and eosinophils, I observed that CCL11 and CCL24 were more potent ligands than CCL26, pointing to a role for ligand bias in CCR3 activation. Mutation of the C-terminus was found to negatively affect CCR3 expression. This could be rescued by the use of a small molecule antagonist, UCB 35625 as a pharmacoperone, presumed to help the refolding of misfolded CCR3. Given the importance of the CCR3 C-terminus, the identification of novel binding partners for the CCR3 C-terminus was investigated using a yeast-two hybrid (Y2H) approach. This was inconclusive. CCR4 is expressed by Th2 cells and binds the chemokines CCL17 and CCL22. Using a transfectant system, I observed that both ligands induced rapid CCR4 endocytosis, resulting in proteasomal degradation. This was sensitive to C-terminal truncation. Constitutive internalisation of CCR4 and degradation was also observed with cell surface replenishment wholly dependent upon de novo synthesis of CCR4. A Y2H screen identified a novel binding partner, FBXL5, that interacted with the CCR4 C-terminus. This interaction was verified by confocal microscopy, with colocalisation of CCR4 and FBXL5 observed following CCL22 treatment. I conclude that the C-termini of both chemokine receptors plays an important role in modulating receptor trafficking and function. This domain may prove to be a molecular “hot-spot” for receptor blockade by antagonists.
Content Version: Open Access
Issue Date: Apr-2020
Date Awarded: Sep-2020
URI: http://hdl.handle.net/10044/1/83211
DOI: https://doi.org/10.25560/83211
Copyright Statement: Creative Commons Attribution NonCommercial No Derivatives Licence
Supervisor: Pease, James
Solari, Roberto
Sponsor/Funder: Biotechnology and Biological Sciences Research Council (Great Britain)
GlaxoSmithKline
Department: National Heart & Lung Institute
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Doctor of Philosophy (PhD)
Appears in Collections:National Heart and Lung Institute PhD theses



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