Molecular cloning and characterization of rainbow trout (Oncorhynchus mykiss) C5a anaphylatoxin receptor

Abstract

In the course of suppression subtractive hybridization between the cDNA of phytohemagglutinin-stimulated and non-stimulated head kidney cells in rainbow trout (Oncorhynchus mykiss), a cDNA clone was obtained that showed most similarity to mammalian receptor for the anaphylatoxin of the fifth complement component (C5aR). The rainbow trout C5aR cDNA contains a 1,679-bp nucleotide sequence that encodes a 350-amino-acid putative protein with 29.0-31.5% identity to mammalian C5aR. Rainbow trout C5aR has seven putative transmembrane domains that are common to mammalian C5aR. A cysteine residue in the second cytoplasmic domain in mammalian C5aR, required for formation of disulfide-linked dimers, is seen in trout C5aR but not in other similar rhodopsin-like receptors. An arginine residue in the fifth transmembrane domain, which is critical for activation of the C5aR by C5a, is also conserved in rainbow trout C5aR. The rainbow trout C5aR gene has a 1.9-kb intron in the position 12 bp upstream of the start codon, which is similar to that seen right after the start codon in human C5aR gene. These results indicate that the clone is a rainbow trout homologue of mammalian C5aR. Southern blot hybridization suggested that C5aR is a single-copy gene. Northern blotting and RT-PCR analyses detected higher amounts of the transcript in head kidney and posterior kidney, but much lower levels in peripheral blood leukocytes and spleen, faint expression in brain and gills, heart, intestine and very faint expression in liver and muscle.