Open AccessSite‐Directed Mutagenesis of Conserved Charged Residues in the Helical Region of the Human C5a Receptor
Author(s) -
Raffetseder Ute,
Röper Detlef,
Mery Laurence,
Gietz Claudia,
Klos Andreas,
Grötzinger Joachim,
Wollmer Axel,
Boulay François,
Köhl Jörg,
Bautsch Wilfried
Publication year - 1996
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1996.00082.x
Subject(s) - c5a receptor , mutagenesis , mutant , biology , transmembrane domain , xenopus , receptor , binding site , ligand (biochemistry) , population , site directed mutagenesis , amino acid , wild type , biochemistry , microbiology and biotechnology , genetics , gene , antibody , demography , complement system , sociology
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 (<10%) of high‐affinity sites ( K d ≈2nM), which was functionally active at 20 nM in the heterologous Xenopus oocyte expression system after coexpression of Gα‐16. The number of high‐affinity sites of wild‐type and [Gln206]C5aR in 293 cells could be up‐regulated by coexpression of Giα‐2 and down‐regulated by GTP[γS]‐mediated uncoupling of the G‐protein receptor interaction in membrane preparations. These findings are compatible with a model in which the Arg206 residue located in the upper third of transmembrane helix V determines high‐affinity binding in the human C5aR by affecting the intracellular G‐protein coupling.