Characterization of a C3a Receptor in Rainbow Trout and Xenopus: The First Identification of C3a Receptors in Nonmammalian Species

Abstract

Virtually nothing is known about the structure, function, and evolutionary origins of the C3aR in nonmammalian species. Because C3aR and C5aR are thought to have arisen from the same common ancestor, the recent characterization of a C5aR in teleost fish implied the presence of a C3aR in this animal group. In this study we report the cloning of a trout cDNA encoding a 364-aa molecule (TC3aR) that shows a high degree of sequence homology and a strong phylogenetic relationship with mammalian C3aRs. Northern blotting demonstrated that TC3aR was expressed primarily in blood leukocytes. Flow cytometric analysis and immunofluorescence microscopy showed that Abs raised against TC3aR stained to a high degree all blood B lymphocytes and, to a lesser extent, all granulocytes. More importantly, these Abs inhibited trout C3a-mediated intracellular calcium mobilization in trout leukocytes. A fascinating structural feature of TC3aR is the lack of a significant portion of the second extracellular loop (ECL2). In all C3aR molecules characterized to date, the ECL2 is exceptionally large when compared with the same region of C5aR. However, the exact function of the extra portion of ECL2 is unknown. The lack of this segment in TC3aR suggests that the extra piece of ECL2 was not necessary for the interaction of the ancestral C3aR with its ligand. Our findings represent the first C3aR characterized in nonmammalian species and support the hypothesis that if C3aR and C5aR diverged from a common ancestor, this event occurred before the emergence of teleost fish.

Histamine is stored in mast cells of most evolutionarily advanced fish and regulates the fish inflammatory response

“…More recently, heterotypic aggregation with activated T cells (39) and the direct recognition of soluble mediators derived from infectious agents, such us lipopolysaccharide, have also been found to induce MC activation and mediator release (40). All these ''alternative'' mechanisms may operate in the activation of fish MCs, because the presence of receptors for IgM (41), anaphylatoxins (42) and soluble mediators derived from infectious agents (43) are all functional in these animals.…”

C4a: An Anaphylatoxin in Name Only

“…Expression of the receptors for C3a and C5a on nearly all cells types provides broad support for these newly described biological activities [reviewed in [4]]. From an evolutionary point of view, the C3a and C5a receptors have integrated anaphylatoxin-mediated control of adaptive immune responses at the level of the bony fishes since these receptors have been conserved in teleost fish for over 300 million years [79,80]. The control of other biological functions by C3a and C5a in organisms phylogenetically simpler than the teleost fish remains to be established.…”

Conservation of Structural and Functional Features in a Primordial CD80/86 Molecule from Rainbow Trout (Oncorhynchus mykiss), a Primitive Teleost Fish

“…Collection of trout leukocytes from blood and head kidney was done as previously reported by us (24,25). Briefly, fish blood was obtained from the caudal vein and immediately diluted (1/5) with DMEM (Invitrogen) supplemented with 100 U/ml penicillin, 100 g/ml streptomycin, and 25 U/ml heparin and then placed on ice.…”