THE MOUSE ANAPHYLATOXIN C3A RECEPTOR - MOLECULAR-CLONING, GENOMIC ORGANIZATION, AND FUNCTIONAL EXPRESSION

The anaphylatoxin C3a receptor (C3aR) is unique among the family of G protein-coupled receptors in possessing an unusually large predicted s econd extracellular loop. To isolate the mouse C3aR, a probe derived f rom this extracellular loop was used to screen a mouse brain cDNA libr ary. A 3.3-kb cDNA encoding an open reading frame of 477 amino acids w as identified. The predicted amino acid contained four predicted N-lin ked glycosylation sites and was 65% identical to the 482 amino acids c omprising 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 cDN A was used as a probe to isolate a mouse C3aR genomic clone. The nucle otide sequence of the mouse C3aR genomic clone was identical to the cD NA throughout the coding region, indicating that the receptor is encod ed on a single exon. The C3aR cDNA was subcloned into a mammalian expr ession vector and transiently expressed in HEK-293 cells. Binding of r adiolabeled C3a to the transfected cells was competed in a dose-depend ent manner by increasing concentrations of unlabeled C3a, with a 50% i nhibiting concentration of 10 nM. Similar to the human C3aR, RBL-2H3 r at basophilic cells stably expressing this receptor responded in a dos e-dependent manner to C3a, a synthetic C3a peptide agonist, but not C4 a or C5a, with a vigorous calcium mobilization.