Abstract
Anaphylatoxin C5a is a potent inflammatory mediator associated with pathogenesis and progression of several inflammation-associated disorders. Small molecule C5a receptor (C5aR) antagonist development is hampered by species-specific receptor biology and the associated inability to use standard rat and mouse in vivo models. Gerbil is one rodent species reportedly responsive to small molecule C5aR antagonists with human C5aR affinity. We report the identification of the gerbil C5aR cDNA using a degenerate primer PCR cloning strategy. The nucleotide sequence revealed an open reading frame encoding a 347-amino acid protein. The cloned receptor (expressed in Sf9 cells) bound recombinant human C5a with nanomolar affinity. Alignment of the gerbil C5aR sequence with those from other species showed that a Trp residue in transmembrane domain V is the only transmembrane domain amino acid unique to small molecule C5aR antagonist-responsive species (i.e. gerbil, human, and non-human primate). Site-directed mutagenesis was used to generate human and mouse C5aRs with a residue exchange of this Trp residue. Mutation of Trp to Leu in human C5aR completely eliminated small molecule antagonist-receptor interaction. In contrast, mutation of Leu to Trp in mouse C5aR enabled small molecule antagonist-receptor interaction. This crucial Trp residue is located deeper within transmembrane domain V than residues reportedly involved in C5a- and cyclic peptide C5a antagonist-receptor interaction, suggesting a novel interaction site(s) for small molecule antagonists. These data provide insight into the basis for small molecule antagonist species selectivity and further define sites critical for C5aR activation and function.
Highlights
Mediated through binding to its G-protein-coupled receptor (C5aR)2 and the subsequent activation of pertussis toxin-sensitive G-proteins [14], mitogen-activated protein kinase [15, 16], and phospholipase C [17] and calcium mobilization
We have identified the gerbil C5a receptor (C5aR) sequence and a key Trp residue in transmembrane domain (TM) V associated with small molecule antagonist binding and function
Cloning and Sequence Analysis of Mongolian Gerbil C5aR—Gerbil C5aR was cloned based on the interspecies sequence homology of C5aR transmembrane domains
Summary
Mediated through binding to its G-protein-coupled receptor (C5aR) and the subsequent activation of pertussis toxin-sensitive G-proteins [14], mitogen-activated protein kinase [15, 16], and phospholipase C [17] and calcium mobilization. Cyclic peptide [32] and small molecule [24] C5aR antagonists demonstrate a greater degree of species selectivity This suggests different C5aR binding and activation determinants for C5a peptide and small molecule antagonists. A small molecule C5aR antagonist (W-54011) inhibits C5a-mediated responses in human, cynomolgus monkey, and gerbil neutrophils, but not in mouse, rat, guinea pig, rabbit, or dog neutrophils [24]. Because of this observed small molecule antagonist species selectivity, we sought to clone gerbil C5aR and to identify amino acid residues potentially responsible for the observed species-selective pharmacology. The location of this amino acid, in relation to those previously identified as important for C5a and cyclic peptide antagonist binding, suggests that the small molecule antagonists tested interact deeper within the transmembrane domains to affect C5aR signaling and function
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