Function, structure and therapeutic potential of complement C5a receptors

Abstract

Complement fragment (C)5a is a 74 residue pro-inflammatory polypeptide produced during activation of the complement cascade of serum proteins in response to foreign surfaces such as microorganisms and tissue damaged by physical or chemical injury. C5a binds to at least two seven-transmembrane domain receptors, C5aR (C5R1, CD88) and C5L2 (gpr77), expressed ubiquitously on a wide variety of cells but particularly on the surface of immune cells like macrophages, neutrophils and T cells. C5aR is a classical G protein-coupled receptor that signals through G alpha i and G alpha 16, whereas C5L2 does not appear to couple to G proteins and has no known signalling activity. Although C5a was first described as an anaphylatoxin and later as a leukocyte chemoattractant, the widespread expression of C5aR suggested more general functionality. Our understanding of the physiology of C5a has improved significantly in recent years through exploitation of receptor knockout and knocking mice, C5 and C5a antibodies, soluble recombinant C5a and C5a analogues and newly developed receptor antagonists. C5a is now also implicated in non-immunological functions associated with developmental biology, CNS development and neurodegeneration, tissue regeneration, and haematopoiesis. Combined receptor mutagenesis, molecular modelling, structure-activity relationship studies and species dependence for ligand potency on C5aR have been helpful for identifying ligand binding sites on the receptor and for defining mechanisms of receptor activation and inactivation. This review will highlight major developments in C5a receptor research that support C5aR as an important therapeutic target. The intriguing possibilities raised by the existence of a non-signalling C5a receptor are also discussed.

Figure 1

Three pathways for complement activation.

Figure 2

Solution structure of human C5a determined from 1H NMR spectroscopy in H₂0/D₂O (Zhang et al., 1997), showing (left) : four helices MLQK₄KIEEIAAK₁₂YKH (blue), SVV₁₈KKCCYDGA₂₆CVNN (orange), DE₃₂TCEQRAA₃₉RISLGP (black), R₄₆CIKAFTQCCVVA₆₃SQ (violet) joined by loops (green) and a C terminal D₆₉GLGR₇₄ (red), which adopts a 1.5 turn helix joined to the four helix bundle by a short loop (green); (right): electrostatic map showing residues with charged acidic (red) or basic (blue) side chains.

Figure 3

Sequences of the C5a receptors, C5aR and C5L2, with potential glycosylation sites asterisked. Both receptors (42–45 kDa) also have characteristic arrays of Asp and Tyr residues at the N-termini; overall sequence identity is 35%. The degree of conservation for individual residues is shown by the depth of shading on the C5L2. Residues identified in site-directed or random saturation mutagenesis studies as having an important role in ligand binding, and/or signalling by C5aR are shown in blue. Sequences critical for G-protein coupling in C5aR, which are changed in C5L2, are shown in red.

Figure 4

Structures of small molecule ligands for C5aR.

Figure 5

Modelled interaction between 3D53 (green) and human C5aR (orange) showing ligand-binding pocket (left: side view, right: top view) with Arg, Trp, dCha and AcPhe components of 3D53 fitting between helices of C5aR with key receptor residues labelled according to Ballesteros and Palczewski (2001). Figure updated since Higginbottom et al. (2005).

Figure 6

The ‘interactome' of C5aR. C5aR interacts directly or indirectly with kinases (purple), GTP binding/regulatory proteins (red), transcription factors (pink), other signalling enzymes (blue) or structural proteins (grey). Internalization of C5aR is mediated by clathrin, which associates with receptor-bound β-arrestin (Ar) and the actin cytoskeleton. Proteins, such as hsp27, phosphorylated by MAP kinase-activated protein kinase 2 (MAPKAP-K2), may regulate the actin cytoskeleton. MAPKAP-K2 is itself activated by the mitogen-activated kinase (MAPK/ERK/JNK) cascade, in turn activated by kinase Akt (also known as PK-B) or by p21-associated protein kinase (PAK) complexed with Rac/Cdc42 guanine nucleotide exchange factor PIXα, cdc42 and G-protein-coupled receptor kinase-interactor 2 (GIT2). G-protein α-subunits are deactivated by regulator of G-protein signalling 1 (RGS1) that stimulates GTP conversion to GDP; in the GDP-bound state, α-subunits can bind to and modulate the activity of the NADPH-oxidase component p67^phox. βγ-subunits directly activate PAK and indirectly activate PK-Cβ by increasing diacylglycerol and intracellular Ca²⁺ ([Ca²⁺]_i) through phospholipase Cβ (PLCβ). βγ may be sequestered by G-protein-coupled receptor kinase (GRK), which also phosphorylates C5aR along with PK-Cβ. Transcription factors signal transducer and activator of transcription 3 (STAT3), cAMP responsive element binding protein (CREB) and nuclear factor (NF)-κB are activated at the convergence of the kinase pathways, and apoptosis inhibited by phosphorylation of Bcl-associated death promoter (BAD) and upregulation of caspase degradation. JNK, c-Jun N-terminal kinase; NADPH, nicotinamide adenine dinucleotide phosphate.