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Title: Dimerization of complement C5a receptors in inflammatory responses
Author: Abd, Ahmed
ISNI:       0000 0004 6422 1531
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2017
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Inflammation is a complex pathophysiologic process that occurs in response to tissue injury induced though various stimuli. It involves cellular and humoral responses in which various cell types and inflammatory mediators are engaged. The inflammatory response must be tightly controlled, otherwise it results in chronic inflammation and perhaps continuous tissue damage. During the activation of the complement cascade, several small fragments, known as anaphylatoxins, are released. One of these anaphylatoxins is produced from the complement protein C5 and is known as C5a. C5a is a multifunctional polypeptide that is involved in cellular immune responses. The receptors for C5a, C5a1 and C5a2, are among the large family known as G protein-coupled receptors. Unlike C5a1, C5a2 is incapable of signalling through G proteins but can induce β-arrestin translocation and recruitment. C5a2 function is still enigmatic and it has been suggested as a decoy receptor for its ligands, as a signalling receptor or as a signalling modifier of C5a1, possibly through the formation of heterodimers. In the current study, we aimed to study possible interactions between the C5a1 and C5a2 receptors. To achieve these goals, heterologous expression of different C5a receptors was studied in clearly defined settings using transfected RBL cells. The possibility of direct physical interaction between the two receptors was explored using fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) using tagged C5a1 and C5a2 receptors. Possible dimerization was further examined using untagged receptors through cointernalization of the C5a1 and C5a2 receptors. Various signalling assays were used to analyse the possible effect of C5a2 on C5a1 such as degranulation, intracellular Ca2+ mobilization, MAPK (ERK1/2, p38 and JNK) signalling and internalization assays. FRET results showed possible homodimerization of C5a1 receptors but not heterodimerization with C5a2. BRET and receptor co-internalization studies also could not detect clear heterodimerization between C5a1 and C5a2. The possible indirect effect of C5a2 on C5a1 was assessed by comparing the C5a1 signalling upon coexpression with C5a2. Intracellular Ca2+ mobilization was similar when C5a1 expressed with or without C5a2. However, the RBL degranulatory response to C5a was lower when C5a2 was co-expressed with C5a1. Phosphorylation of ERK1/2 was also decreased when C5a2 was coexpressed with C5a1. In addition, the presence of C5a2 decreased C5a1 internalization. Taken together, when co-expressed in RBL cells, possible interaction between C5a receptors was observed. This interaction is not necessarily due to direct physical interaction but could be through scavenging effect on the ligand or sequestration of intracellular partners.
Supervisor: Monk, Peter ; Partridge, Lynda Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available