title: Molecular characterization of the gerbil C5a receptor and identification of a transmembrane domain V amino acid that is crucial for small molecule antagonist interaction.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Neurogen Corporation, Branford, Connecticut 06405, USA. swaters@nrgn.com
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Waters
authors.forename: Robbin M
authors.initials: RM
authors.lastname: Brodbeck
authors.forename: Jeremy
authors.initials: J
authors.lastname: Steflik
authors.forename: Jianying
authors.initials: J
authors.lastname: Yu
authors.forename: Carolyn
authors.initials: C
authors.lastname: Baltazar
authors.forename: Amy E
authors.initials: AE
authors.lastname: Peck
authors.forename: Daniel
authors.initials: D
authors.lastname: Severance
authors.forename: Lu Yan
authors.initials: LY
authors.lastname: Zhang
authors.forename: Kevin
authors.initials: K
authors.lastname: Currie
authors.forename: Bertrand L
authors.initials: BL
authors.lastname: Chenard
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Hutchison
authors.forename: George
authors.initials: G
authors.lastname: Maynard
authors.forename: James E
authors.initials: JE
authors.lastname: Krause
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry*; D003001:Cloning, Molecular; D015870:Gene Expression; D005849:Gerbillinae; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D044087:Receptor, Anaphylatoxin C5a / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D013045:Species Specificity; D013329:Structure-Activity Relationship; D014364:Tryptophan
chemical_list: D044087:Receptor, Anaphylatoxin C5a; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014364:Tryptophan