Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.715095
Title: Characterisation of the second C5a receptor, C5aR2
Author: Bell, Rachel H.
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2017
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Abstract:
C5a is a small protein produced as part of the complement cascade with inflammatory activity. Elevated levels of C5a have been implicated in a number of inflammatory conditions such as rheumatoid arthritis, inflammatory bowel disease, and asthma. C5a is rapidly metabolised into the less active C5a des-Arg, allowing control of C5a induced inflammation. There are two receptors for C5a in humans, hC5aR1 and hC5aR2, which share around 35% sequence identity but have different affinities for C5a and C5a des-Arg. hC5aR1 is a well characterised G protein-coupled receptor that undergoes ligand-dependent internalisation, whereas hC5aR2 does not couple to G-proteins and undergoes constitutive recycling. The structural basis for these differences is currently unknown. The main aim of this work was to test the hypothesis that hC5aR2 had a different ligand binding mechanism to hC5aR1 and in addition that C5a and C5a des-Arg would be bound differently by hC5aR2. A series of hC5aR2 receptor mutants was designed by modelling the structure of the receptor on hC5aR1 and CXCR4. After production, purification and labelling of recombinant human C5a and C5a des-Arg, ligand binding by mutant and wild type receptors stably transfected into Chinese Hamster Ovary cells was investigated by flow cytometry. Internalisation of the receptors from the cell surface in response to ligand was used as a measure of function. Mutations in hC5aR2 were found that could affect either C5a or C5a des-Arg binding, in contrast to hC5aR1, where analogous mutations affected the binding of both ligands. Through comparisons with hC5aR1 binding data in the literature, it is apparent hC5aR2 does seem to have a different ligand binding mechanism. Some hC5aR2 mutants showed increased levels of constitutive internalisation, suggesting that the binding mechanism is also coupled to endocytic pathways, which is also not seen with hC5aR1. In addition, ligand dependent internalisation of hC5aR2 transfected into CHO cells was observed in this work.
Supervisor: Monk, Peter N. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.715095  DOI: Not available
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