title: High-affinity binding of peptide agonists to the human B1 bradykinin receptor depends on interaction between the peptide N-terminal L-lysine and the fourth extracellular domain of the receptor.
abstract: The aim of this study was to identify the location of the N terminus of peptide agonist ligands when bound to the human B1 bradykinin (BK) receptor. To reach this aim, we exploited the fact that high-affinity binding of kinin peptides to the human B1 receptor subtype requires a peptide N-terminal L-Lys, whereas high-affinity binding to the B2 receptor subtype does not require this residue. This was done by comparing the affinities of BK, a B2 receptor-selective peptide, and kallidin or Lys-BK, a less receptor-selective peptide, for chimeric proteins in which each B1 receptor domain had been substituted in the human B2 receptor and expressed in HEK293 cells. Individual substitution of transmembrane domains 1-7 (TM-I-VII) and extracellular domains 1-4 (EC-I-IV) of the B1 receptor in the B2 receptor influenced the affinities of BK and Lys-BK approximately equally. In contrast, substitution of B1 EC-IV dramatically reduced the affinity and potency of BK, whereas these parameters for Lys-BK were essentially unaltered. Substitution of either the N- or C-terminal half of B1 EC-IV in the B2 receptor only had a limited effect on the peptide binding constants, indicating the involvement of multiple residues throughout this domain. Complementary mutations of the N-terminal residue in Lys-BK revealed that both the positive charge and the proper spatial orientation of this residue were required for interaction with B1 EC-IV. Thus, the N-terminal residue of peptide agonists when bound to the human B1 receptor is positioned extracellularly and interacts with EC-IV.
authors.affiliation: Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas, USA.
authors.forename: D B
authors.initials: DB
authors.lastname: Fathy
authors.forename: D J
authors.initials: DJ
authors.lastname: Kyle
authors.forename: L M
authors.initials: LM
authors.lastname: Leeb-Lundberg
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D001920:Bradykinin / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011487:Protein Conformation; D043783:Receptor, Bradykinin B1; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D010455:Peptides; D043783:Receptor, Bradykinin B1; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D011993:Recombinant Fusion Proteins; D008239:Lysine; D001920:Bradykinin