Essential role of TM V and VI for binding the C-terminal sequences of Des-Arg-kinins

Abstract In the kallikrein–kinin and renin–angiotensin systems the main receptors, B 1 and B 2 (kinin receptors) and AT 1 and AT 2 (angiotensin receptors) respectively, are seven-transmembrane domain G-protein-coupled receptors. Considering that the B 1 agonists Des-Arg 9 -BK (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe), Lys-desArg 9 -BK or Des-Arg 10 -KD (Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe) and the AT 1 agonist (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) have the same two residues at the C-terminal region (i.e. Pro-Phe), we hypothesized that TM V and TM VI of the B 1 receptor could play an essential role in agonist binding and activity, being these regions receptor sites for binding the C-terminal sequences of Des-Arg-kinins similarly to that observed to AT 1 receptor. To investigate this hypothesis, we replaced Arg 212 for Ala at the top of the TM V and the sequence 274–282 (CPYHFFAFL) in TM VI of the rat kinin B 1 receptor by the B 2 receptor homologous sequence, 289–297 (FPFQISTFL) and subsequently analyzed the consequences of these mutations by competition binding and functional assays. Despite correct expression, observed at the mRNA and protein level by RT-PCR and confocal microscopy, respectively, no agonist binding and function was verified for the mutated receptors. Therefore, our results suggest an important role for Arg 212 in the TM V and a region of TM VI of rat B 1 receptor in the interaction with the C-terminal residues of Des-Arg-kinins, similar to that observed with AngII.