Site‐Directed Mutagenesis of Conserved Charged Residues in the Helical Region of the Human C5a Receptor: Arg206 Determines High‐Affinity Binding Sites of C5a Receptor

The human C5a receptor (C5aR) belongs to the family of G‐protein‐coupled receptors with seven transmembrane helices. This part of the molecule is thought to contain part of the ligand‐binding pocket, specifically to bind the C‐terminal Arg of human C5a. Guided by sequence similarity and molecular modelling studies, several residues including polar (Asn119, Thr168, Gln259) as well as all conserved charged amino acids in the upper transmembrane region of the C5aR (Asp37, Asp82, Arg175, Arg206, Asp282) were exchanged by site‐directed mutagenesis. Receptor mutants were transiently expressed in COS cells and analyzed for altered binding behaviour and/or localization at the cell surface by immunofluorescence. For all residues, suitable mutants could be found that exhibited wild‐type affinity towards the ligand, providing evidence against a major contribution of these residues to high‐affinity ligand binding. Some mutants, however, exhibited a complete (Asp282→Ala) or partial loss of ligand‐binding capacity (Arg175→Ala, Arg206→Gln) despite adequate expression levels on the cell surface. This phenotype was further analyzed in the [Gln206]C5aR mutant: quantitative flow cytometric analysis of epitope‐tagged receptor derivatives in 293 cells confirmed an equal level of wild‐type and mutant C5aR on the cell surface. Competitive binding curves revealed the presence of only a small population (