title: 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, B1 and B2 (kinin receptors) and AT1 and AT2 (angiotensin receptors) respectively, are seven-transmembrane domain G-protein-coupled receptors. Considering that the B1 agonists Des-Arg9-BK (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe), Lys-desArg9-BK or Des-Arg10-KD (Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe) and the AT1 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 B1 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 AT1 receptor. To investigate this hypothesis, we replaced Arg212 for Ala at the top of the TM V and the sequence 274-282 (CPYHFFAFL) in TM VI of the rat kinin B1 receptor by the B2 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 Arg212 in the TM V and a region of TM VI of rat B1 receptor in the interaction with the C-terminal residues of Des-Arg-kinins, similar to that observed with AngII.
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, UNIFESP, São Paulo 04023-062, Brazil. edson@biofis.epm.br <edson@biofis.epm.br>
authors.forename: Edson L
authors.initials: EL
authors.lastname: Santos
authors.forename: Kely
authors.initials: K
authors.lastname: de Picoli Souza
authors.forename: Elena
authors.initials: E
authors.lastname: Cibrián-Uhalte
authors.forename: Suzana M
authors.initials: SM
authors.lastname: Oliveira
authors.forename: Michael
authors.initials: M
authors.lastname: Bader
authors.forename: Claudio M
authors.initials: CM
authors.lastname: Costa-Neto
authors.forename: Laerte
authors.initials: L
authors.lastname: Oliveira
authors.forename: João B
authors.initials: JB
authors.lastname: Pesquero
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001920:Bradykinin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006367:HeLa Cells; D006801:Humans; D007609:Kallidin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D051381:Rats; D043783:Receptor, Bradykinin B1 / Q000819:agonists / Q000737:chemistry* / Q000378:metabolism
chemical_list: D043783:Receptor, Bradykinin B1; C024626:bradykinin, des-Arg(9)-; D007609:Kallidin; C052787:kallidin, des-Arg(10)-; D001920:Bradykinin