title: Identification of an N-formyl peptide receptor ligand binding domain by a gain-of-function approach.
abstract: Replacement of N-formyl peptide receptor (FPR) domains with those from a homologous receptor, FPR2, resulted in chimeric receptors displaying low binding affinity to fMet-Leu-Phe (fMLF). To characterize fMLF binding domain, we adopted a "gain-of-function" approach by selective replacement of non-conserved residues in the FPR2 portion of the chimeric receptors with those from the FPR. This led to the identification of 3 clusters of residues required for high-affinity fMLF binding. Introduction of 2 positively charged amino acids, Arg84 and Lys85, dramatically improved binding affinity of one chimeric receptor (Kd from 105 nM to 1.6 nM). Similarly, restoration of either Gly89/His90 or Phe102/Thr103 improved the binding affinity of another chimeric receptor from a Kd of 275 nM to a 2.3 Kd and 3.3 nM, respectively. Increased ligand binding affinity was accompanied by a gain in calcium mobilization capability, suggesting functional coupling to G proteins. These results demonstrate the presence of structural determinants in the first extracellular loop and its adjacent transmembrane domains that are essential for high affinity fMLF binding.
authors.affiliation: Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: O
authors.initials: O
authors.lastname: Quehenberger
authors.forename: Z K
authors.initials: ZK
authors.lastname: Pan
authors.forename: E R
authors.initials: ER
authors.lastname: Prossnitz
authors.forename: S L
authors.initials: SL
authors.lastname: Cavanagh
authors.forename: C G
authors.initials: CG
authors.lastname: Cochrane
authors.forename: R D
authors.initials: RD
authors.lastname: Ye
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D008024:Ligands; D008969:Molecular Sequence Data; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018000:Receptors, Peptide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D008024:Ligands; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins