Effects of inactivation-resistant agonists on the signalling, desensitization and down-regulation of bradykinin B2 receptors

Background and purpose: A peptide bradykinin (BK) B-2 receptor agonist partially resistant to degradation, B-9972, down-regulates this receptor subtype. We have used another recently described non-peptide agonist, compound 47a, as a tool to study further the effects of metabolically more stable and thus persistent, agonists of the BK B-2 receptor on signalling, desensitization and down-regulation of this receptor. Experimental approach and key results: Compound 47a was a partial agonist at the B-2 receptor in the human umbilical vein, where it shared with B-9972 a very slow relaxation on washout, and in HEK 293 cell lines expressing tagged forms [myc, green fluorescent protein (GFP)] of the rabbit B-2 receptor. Compound 47a desensitized the umbilical vein to BK. In the cellular systems, the inactivation-resistant agonists induced [Ca2+](i) transients as brief as those of BK but affected other functions with a longer duration than BK [12 h; receptor endocytosis, endosomal beta-arrestin(1/2) translocation, protein kinase C-dependent extracellular signal-regulated kinases (ERK)1/2 phosphorylation and c-Fos expression]. The B-2 receptor-GFP was degraded in cells exposed to B-9972 or compound 47a for 12 h. The non-peptide B-2 receptor antagonist LF 16-0687 prevented all effects of compound 47a, which were also absent in cells lacking recombinant B-2 receptors. Conclusion and implications: Inactivation-resistant agonists revealed a long-lasting assembly of the agonist-B-2 receptor-beta-arrestin complexes in endosomal structures and induce 'biased signalling' (in terms of activation of ERK and c-Fos) as a function of time. Further, B-9972 and compound 47a, unlike BK, efficiently down-regulated BK B-2 receptors.