Mapping the signalling pathways and interactions for the complement C5a receptors using novel methodologies

Croker, Daniel E. (2014). Mapping the signalling pathways and interactions for the complement C5a receptors using novel methodologies PhD Thesis, Institute for Molecular Bioscience, The University of Queensland. doi:10.14264/uql.2014.200

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Author Croker, Daniel E.
Thesis Title Mapping the signalling pathways and interactions for the complement C5a receptors using novel methodologies
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
DOI 10.14264/uql.2014.200
Publication date 2014-01-01
Thesis type PhD Thesis
Supervisor Matthew A. Cooper
Reena Halai
Total pages 105
Total colour pages 15
Total black and white pages 90
Language eng
Subjects 1107 Immunology
0601 Biochemistry and Cell Biology
Formatted abstract
The complement system is a major component of our innate immune system that acts as our first line of defence against infectious organisms and foreign bodies. Following activation of the complement cascade the anaphylatoxins C3a, C4a and C5a are generated prior to the formation of the membrane attack complex (C5b-C9). Carboxypeptidases rapidly digest C5a to C5a-des Arg, which becomes the dominant, and purportedly ‘inactive’ form present in the body. C5a and C5a-des Arg bind to two GPCRs, the C5a receptor (C5aR) and C5a receptor like-2 receptor (C5L2). G-protein coupled receptors (GPCR) are of major importance in drug development, with ~30% of currently marketed drugs targeting these receptors. C5aR is a classical GPCR that couples to and signals through Gα proteins, whilst C5L2 is thought to be a non-signalling GPCR due to amino acid changes in the Gα protein binding motif. The role of C5L2 is still not well understood, and was postulated to be a simple non-signalling recycling decoy receptor. Both the C5a receptors have been shown to have important roles in many inflammatory conditions. To better understand the role of C5a receptor signalling, pathways activated by C5a and C5a-des Arg were mapped using traditional secondary messenger and label-free assay techniques in transfected and native cells (human monocyte derived macrophages; HMDM). C5a-des Arg was found to possess a potency similar to C5a on many of the assay technologies. The label-free assay data evidenced the same rank order of potency as the secondary messenger assays, but with a reduction in potency of all the ligands tested compared to the secondary messenger assays.

As GPCRs are known to form oligomers, we hypothesised that activation of C5aR could lead to heteromer formation with C5L2 as a mechanism to modulate C5aR signalling possibly through β-arrestin recruitment. Notably, analysis of C5a and C5a-des Arg signalling showed little or no difference between the two ligands. Only when looking at receptor heteromer formation was a clear difference observed with C5a-des Arg being unable to up-regulate heteromer formation. This led concomitantly to an up-regulation of the release of the anti-inflammatory cytokine IL-10 and G-CSF by C5a, but not the pro-inflammatory cytokines IL-6 and TNFα. Activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was a key component of this up-regulation and was mediated by the increased recruitment of β-arrestin by C5a through C5aR. The increased recruitment of the β-arrestin-C5L2 complex had a down regulatory role on C5aR signalling through ERK1/2. This improved understanding of the roles the C5a receptors play in acute inflammatory conditions may lead to the development of ligands that could be of use to treat severe acute inflammation associated with conditions such as sepsis.
Keyword Complement C5a receptors
Receptor dimerisation
Receptor signalling

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Created: Thu, 17 Jul 2014, 17:59:39 EST by Daniel Croker on behalf of Scholarly Communication and Digitisation Service