The mouse anaphylatoxin C3a receptor: molecular cloning, genomic organization, and functional expression.

Abstract

The anaphylatoxin C3a receptor (C3aR) is unique among the family of G protein-coupled receptors in possessing an unusually large predicted second extracellular loop. To isolate the mouse C3aR, a probe derived from this extracellular loop was used to screen a mouse brain cDNA library. A 3.3-kb cDNA encoding an open reading frame of 477 amino acids was identified. The predicted amino acid contained four predicted N-linked glycosylation sites and was 65% identical to the 482 amino acids comprising the coding region of the human C3aR. Northern blot analysis revealed that this gene was expressed in a variety of mouse tissue and was especially abundant in heart and lung tissues. The mouse C3aR cDNA was used as a probe to isolate a mouse C3aR genomic clone. The nucleotide sequence of the mouse C3aR genomic clone was identical to the cDNA throughout the coding region, indicating that the receptor is encoded on a single exon. The C3aR cDNA was subcloned into a mammalian expression vector and transiently expressed in HEK-293 cells. Binding of radiolabeled C3a to the transfected cells was competed in a dose-dependent manner by increasing concentrations of unlabeled C3a, with a 50% inhibiting concentration of 10 nM. Similar to the human C3aR, RBL-2H3 rat basophilic cells stably expressing this receptor responded in a dose-dependent manner to C3a, a synthetic C3a peptide agonist, but not C4a or C5a, with a vigorous calcium mobilization.