We have demonstrated that an alternative C5a receptor (C5aR) ligand, the homodimer of ribosomal protein S19 (RP S19), contains a unique C-terminus (I(134)-H(145)) that is distinct from the moieties involved in the C5a-C5aR interaction. To examine the role of I(134)-H(145) in the ligand-C5aR interaction, we connected this peptide to the C-terminus of C5a (C5a/RP S19) and found that it endowed the second binding moiety of RP S19 (L(131)DR) with a relatively higher binding affinity to the C5aR on a human mast cell line, HMC-1. In contrast to the C5aR, the second C5aR C5L2 worked as a decoy receptor. As a result, the mitogen-activated protein kinase (MAPK) downstream of the Gi protein exchanged extracellular-signal regulated kinase for p38MAPK. This alternative p38MAPK activation could be pharmacologically suppressed not only by the downregulation of phosphoinositide 3-kinase (PI3K) by LY294002, but also by the over-activation of protein kinase C by phorbol 12-myristate 13-acetate. The activation was reproduced upon C5a-C5aR interaction by a simultaneous suppression of PI3K and phospholipase C with LY294002 and U73122 at low concentrations. Moreover, p38MAPK phosphorylation upstream of the pertussis toxin-dependent extracellular Ca(2+) entry was also suppressed by high concentrations of MgCl(2), which blocks melastatin-type transient receptor potential Ca(2+) channels (TRPMs). The active conformation of C5aR upon the ligation by C5a, at least on HMC-1 cells, is changed by the additional interaction of the I(134)-H(145) peptide, which seems to guide the alternative activation of p38MAPK. This activation is then amplified by a novel positive feedback loop between p38MAPK and TRPM.

C5a receptor has been identified as a leukocyte chemotactic receptor to two intrinsic chemical mediators, C5a and the S19 ribosomal protein dimer, so far. We found an Escherichia coli protein that also induced the chemotactic responses of monocytes and polymorphonuclear leukocytes via the C5a receptor. We identified the E. coli-derived chemoattractant to be Skp by the molecular size and the N-terminal amino acid sequence. Skp is a periplasmic chaperone protein widely present in gram-negative bacterial species. Immunoabsorption experiments indicated that Skp was the major leukocyte chemotactic factor in the E. coli extract. Receptor-antagonizing experiments with analogue peptides of S19 ribosomal protein and C5a receptor is one of the most important leukocyte receptors involving in the inflammatory reaction. C5a receptor was initially identified as the receptor of C5a, which is a 74 amino acid peptide liberated from the ␣-chain of complement C5 by C5 convertases during the complement activation. C5a attracts polymorphonuclear leukocytes (PMNs), monocytes, and many other leukocytes via the C5a receptor. In addition to this, C5a stimulates PMNs via the C5a receptor to release granule contents and to produce radical oxygen species.1 C5a also promotes monocytes/macrophages via the C5a receptor to synthesize interleukin (IL)-1, IL-6, and several other cytokines.2 The C5a receptor gene, which is located on chromosome 19 q13.3-13.4 (from the Genome Database), is also expressed in nonmyeloid cells such as astrocytes and hepatocytes at least under inflamed conditions. Hepatocyte C5a receptor would participate in the acute phase protein synthesis. Besides, we have realized that the S19 ribosomal protein (RP S19) dimer attracts monocytes via the C5a receptor as in the case of C5a.3 RP S19 is a component of the small subunit of ribosome, being composed of 145 amino acid residues. RP S19 is intermolecularly crosslinked by a transglutaminase-catalyzed reaction 4,5 during apoptotic process and the dimer is liberated from the apoptotic cells. 6,7 The RP S19 dimer attracts monocytes/ macrophages and promotes them to phagocytically clear the apoptotic cells from which the chemoattractant molecule has been originated. In contrast to this, the RP S19 dimer antagonizes the C5a receptor-mediated chemotaxis of PMNs. 3Whereas a calculated homology in the amino acid sequence between C5a and RP S19 is only 4%, C5a and the RP S19 dimer activate monocyte C5a receptor by the same interaction mechanism. The C5a receptor, composed of 350 amino acid residues, is a member of the G-protein-coupled 7 transmembrane protein receptor family. The ligand-receptor interaction between C5a or the RP S19 dimer and C5a receptor is a two-step binding process. 8 In the first binding, a basic cluster of the ligand molecules; a cluster three-dimensionally formed by His15, Arg46, and Lys49 of C5a, or a cluster with the Lys41-His42-Lys43 tandem of RP S19, seems to bind to the amino-terminal acidic moiety of C5a receptor, which is composed of several Asp residues ...

C-terminus of S19 ribosomal protein (RP S19) endows the cross-linked homodimer with a dual effect on the C5a receptor in leucocyte chemoattraction; agonistic effect on the monocyte receptor, and antagonistic effect on the neutrophil receptor. C5a exhibits the uniform agonistic effect on this receptor of both cell types. We have currently prepared a recombinant C5a-chimeric protein bearing the C-terminus of RP S19 (C5a/RP S19 chimera) to be used as a substitute of the RP S19 dimer. In vitro, this chimera similarly inhibited the intracellular Ca(2+) mobilization of neutrophils induced by C5a to the RP S19 dimer did. In the guinea pig skin, 10(-7) M C5a/RP S19 chimera exhibited an inhibitory capacity to the neutrophil infiltration induced by 3 x 10(-7) M C5a without enhancing monocyte infiltration. In reverse passive Arthus reaction, the neutrophil infiltration associated with plasma extravasation was significantly reduced by the simultaneous administration of 10(-7) M C5a/RP S19 chimera with antibodies. The C5a/RP S19 chimera is a useful tool not only to examine the molecular mechanism that underlies the functional difference of the C5a receptor between monocytes and neutrophils, but also to prevent C5a-mediated hyper-response of neutrophils in acute inflammation.