Characterization of a C3a receptor in rainbow trout and Xenopus: the first identification of C3a receptors in nonmammalian species.
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.
Boshra
Hani
H
Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104, USA.
Wang
Tiehui
T
Hove-Madsen
Leif
L
Hansen
John
J
Li
Jun
J
Matlapudi
Anjun
A
Secombes
Christopher J
CJ
Tort
Lluis
L
Sunyer
J Oriol
JO
D000595:Amino Acid Sequence; D000818:Animals; D019138:Antibodies, Blocking / Q000737:chemistry; D001666:Binding Sites, Antibody; D015152:Blotting, Northern; D015139:Blotting, Southern; D002118:Calcium / Q000037:antagonists & inhibitors / Q000378:metabolism; D015926:Complement C3a / Q000037:antagonists & inhibitors / Q000502:physiology; D003169:Complement Inactivator Proteins / Q000502:physiology; D018076:DNA, Complementary / Q000302:isolation & purification; D019084:Fluorescent Antibody Technique, Indirect; D006168:Guinea Pigs; D006801:Humans; D007424:Intracellular Fluid / Q000276:immunology / Q000378:metabolism; D007962:Leukocytes / Q000276:immunology / Q000378:metabolism; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss; D051381:Rats; D011951:Receptors, Complement / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014981:Xenopus
D019138:Antibodies, Blocking; D003169:Complement Inactivator Proteins; D018076:DNA, Complementary; D008565:Membrane Proteins; D011951:Receptors, Complement; C085567:complement C3a receptor; D015926:Complement C3a; D002118:Calcium