Cloning and functional characterization of the mouse C3a anaphylatoxin receptor gene.
The mouse anaphylatoxin C3a receptor (mC3aR) gene was isolated using a human C3aR cDNA probe. The genomic fragment contains an open reading frame of 1431 base pairs that encodes a peptide of 477 amino acids. A cDNA with identical sequence was subsequently obtained from the mouse pre-B cell line 70Z/3 by reverse transcriptase polymerase chain reaction based on sequence of the mC3aR gene. Northern blot analysis suggested expression of the mC3aR in lung and heart, and to a lesser extent, in brain, liver, muscle, kidney, and testis. The deduced amino acid sequence of the mouse C3aR is 65% identical to that of the human C3aR. Like the human receptor, mouse C3aR contains a predicted large extracellular loop of approximately 165 amino acids (residues 161-325) between the fourth and fifth transmembrane domains. This loop, however, is the least conserved structure (45% identical sequence) of all the extracellular and intracellular domains between the mouse and human C3aRs. The mouse gene product bound 125I-labeled human C3a with a Kd of 2.54 nM when expressed in the stably transfected rat basophilic leukemic cell line RBL-2H3. Bound C3a could be effectively displaced by excess quantities of unlabeled C3a, but not by C4a or C5a. C3a induced dose-dependent calcium mobilization in the transfected cells, which could be blocked by pertussis toxin treatment. These results confirm that the cloned gene encodes a functional C3aR capable of coupling to a pertussis toxin-sensitive G protein. The sequence divergence of the large extracellular loop does not appear to affect C3a binding and transmembrane signaling.
Hsu
M H
MH
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Ember
J A
JA
Wang
M
M
Prossnitz
E R
ER
Hugli
T E
TE
Ye
R D
RD
D000595:Amino Acid Sequence; D000706:Anaphylatoxins / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D011951:Receptors, Complement / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014162:Transfection
D000706:Anaphylatoxins; D008565:Membrane Proteins; D011951:Receptors, Complement; C085567:complement C3a receptor