Mutational analysis of the human complement 5a receptor: Identification of a potential role of asp 37 and asp 82 in ligand binding

The receptor for the inflammatory and chemotactic agent complement 5a (C5a) is a member of the G‐protein coupled receptor (GPCR) superfamily. Site‐directed mutagenesis of the human C5a receptor was performed to determine which amino acids were important for ligand binding. Specific regions of the C5a receptor were mutated based on their similarities to the ligand binding domain of other GPCRs. These mutated receptors were then transiently expressed in COS‐7 cells in order to test their ability to bind [125|]C5a. Because of the basic nature of the ligand, we concentrated on mutating acidic amino acid residues located at the N‐terminal and transmembrane regions of the receptor. Mutation of Asp 37, located near the first transmembrane domain, or Asp 82, located within the second transmembrane domain, to valine resulted in a total loss of specific [125l]C5a binding to membrane preparations of transfected cells. Furthermore, mutation of Asp 82 to alanine, leucine, or glutamate also resulted in an absence of specific binding. However, mutation of Asp 82 to asparagine did not eliminate the ability of the receptor to bind [125l]C5a. Mutation of each of the N‐terminal extracellular domain aspartate residues, Asp 282 (located within the seventh transmembrane domain), or Glu 179 or Glu 180 (located within the second extracellular loop) to valine also did not significantly affect [125l]C5a binding. These studies thus identified two acidic amino acid residues of the C5a receptor which are important for binding [125l]C5a. © 1995 Wiley‐Liss, Inc.