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.