Structural diversity in the extracellular faces of peptidergic G-protein-coupled receptors. Molecular cloning of the mouse C5a anaphylatoxin receptor.

The mouse C5a receptor gene was isolated using the human C5a receptor cDNA probe recently described (Gerard, N. P., and C. Gerard. 1991. Nature 349:614). By analogy with the human gene, the mouse homolog contains two exons with the 5' untranslated region and initiating methionine codon present in exon 1 and the remainder of the molecule in exon 2. Generation of an expressible cDNA for the mouse C5a receptor was accomplished using the polymerase chain reaction and a sense oligodeoxynucleotide primer which included an initiation codon just 5' to the sequence encoding the N-linked glycosylation site. When transfected into human 293 kidney epithelial cells the cloned cDNA directs expression of a binding site for human C5a anaphylatoxin with a binding constant of 2.5 +/- 0.3 nM; the human C5a receptor expressed under identical conditions has a Kd of 1.7 +/- 0.2 nM. Overall, the deduced amino acid sequences of the receptors are 65% identical given the analogous gene structures. Alignment of the sequences as seven transmembrane segment receptors reveals that the greatest structural diversity (approximately 70%) exists in the putative extracellular domains. In contrast, species differences among other members of this family of seven membrane-spanning receptors is generally only 10 to 20%, even for receptors whose ligands are relatively small and not expected to interact with sites on the extracellular surfaces. A high degree of structural identify is observed for the C5a receptors in the transmembrane segments and in all but one of the loops predicted to exist in the cytoplasm. Inasmuch as critical structures responsible for high affinity binding of the 74 amino acid polypeptide to both C5a receptors involve features conserved between species, these data provide the starting point for mutagenesis studies to determine the nature of the binding and activation sites for the chemotactic receptors. Additionally, these data provide a reagent for immunologic and molecular genetic studies on the role of C5a receptors in inflammatory models.