Prolonged signalling and trafficking of the bradykinin B2 receptor stimulated with the amphibian peptide maximakinin: Insight into the endosomal inactivation of kinins

Maximakinin, a 19-residue peptide from the amphibian Bombina maxima, incorporates the full sequence of bradykinin (BK) at its C-terminus with a hydrophilic 10-residue N-terminal extension. As a putative venom component, it may stimulate BK B2 receptors (B2Rs) in a distinct manner relative to the fragile mammalian agonist BK. Maximakinin affinity for B2Rs and angiotensin converting enzyme (ACE) and its pharmacological profile have been compared to those of BK. Maximakinin is an agonist of the human and rabbit B2R with a 8-12 fold lesser potency, but a prolonged duration of action relative to BK (ERK MAP kinase activation, c-Fos induction in HEK 293 cells). Maximakinin had a moderately inferior affinity (∼6-fold vs. BK) for recombinant ACE based on [3H]enalaprilat binding displacement. Unlike BK, maximakinin induced the internalization of the fusion protein B2R-green fluorescent protein (GFP) and the downregulation of this construction over a 12-h stimulation period, reproducing the effect of inactivation-resistant B2R agonists. Alternate homologues of BK extended at the N-terminus showed intermediate behaviours between BK and maximakinin in the B2R-GFP downregulation assay. The recycling of B2R-GFP at the cell surface after a 3-h BK treatment was notably inhibited by cotreatment with E-64 or bafilomycin A1, supporting that an endosomal cysteine protease degrades kinins in a process that determines the cycling and fate of the B2R. Maximakinin is the first known natural kinin sequence that elicits a prolonged cellular signalling, thus suggesting a possible basis for a venomous action and a naturally selected one for the design of B2R-transported biotechnological cargoes.