title: Binding affinities and activation of Asp712Ala and Cys100Ser mutated kinin B1 receptor forms suggest a bimodal scheme for the molecule of bound-DABK.
abstract: Mutant forms of kinin B(1) receptor (B(1)R) and analogs of the full agonist des-Arg(9)-bradykinin (DABK) were investigated aiming to verify the importance of selected receptor residues and of each agonist-peptide residue in the specific binding and activation. Linked by a specific disulfide bond (Cys(100)-Cys(650)), the N-terminal (N(t)) and the EC3 loop C-terminal (C(t)) segments of angiotensin II (AngII) receptor 1 (AT(1)R) have been identified to form an extracellular site for binding the agonist N(t) segment (Asp(1) and Arg(2) residues). Asp(712) residue at the receptor EC3 loop binds the peptide Arg(2) residue. By homology, a similar site might be considered for DABK binding to B(1)R since this receptor contains the same structural elements for composing the site in AT(1)R, namely the disulfide bond and the EC3 loop Asp(712) residue. DABK, Ala(n)-DABK analogs (n=Ala(1)-, Ala(2)-, Ala(3)-, Ala(4)-, Ala(5)-, Ala(6)-, Ala(7)-, Ala(8)-DABK), and other analogs were selected to binding wild-type, Asp712Ala and Cys100Ser mutated B(1)R receptors. The results obtained suggested that the same bimodal scheme adopted for AngII-AT(1)R system may be applied to DABK binding to B(1)R. The most crucial similarity in the two cases is that the N(t) segments of peptides equally bind to the homologous Asp(712) residue of both AT(1)R and B(1)R extracellular sites. Confirming this preliminary supposition, mutation of residues located at the B(1)R extracellular site as EC3 loop Asp(712) and Cys(100) caused the same modifications in biological assays observed in AT(1)R submitted to homologous mutations, such as significant weakening of agonist binding and reduction of post-receptor-activation processes. These findings provided enough support for defining a site that determines the specific binding of DABK to B(1)R receptors.
authors: Biophysics Department, Universidade Federal de São Paulo, Rua Botucatu, 862 7th floor, São Paulo, SP, Brazil.
authors: E S
authors: ES
authors: Rodrigues
authors: R P
authors: RP
authors: Martin
authors: R F
authors: RF
authors: Silva
authors: C R
authors: CR
authors: Nakaie
authors: L
authors: L
authors: Oliveira
authors: S I
authors: SI
authors: Shimuta
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D005753:Gastric Mucosa / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D043783:Receptor, Bradykinin B1 / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013270:Stomach / Q000187:drug effects*; D013329:Structure-Activity Relationship; D046509:Tissue Culture Techniques
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C119712:Agtrap protein, mouse; D010455:Peptides; D043783:Receptor, Bradykinin B1