B-9972 (D-Arg-[Hyp3,Igl5,Oic7,Igl8]-bradykinin) Is an Inactivation-Resistant Agonist of the Bradykinin B2 Receptor Derived from the Peptide Antagonist B-9430 (D-Arg-[Hyp3,Igl5,D-Igl7,Oic8]-bradykinin): Pharmacologic Profile and Effective Induction of Receptor Degradation

Authors:
  • LGLajos Gera
    Lajos Gera
  • GMGuillaume Morissette
    Guillaume Morissette
  • JSJohn M. Stewart
    John M. Stewart
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Abstract
The bradykinin B2 receptor is a heptahelical receptor regulated by a cycle of phosphorylation, endocytosis, and extensive recycling at the cell surface following agonist stimulation. B-9430 (d-Arg-[Hyp3,Igl5,d-Igl7,Oic8]-bradykinin) is a second generation peptide antagonist found to be competitive at the human B2 receptor and insurmountable at the rabbit B2 receptor (contractility assays, isolated human umbilical and rabbit jugular veins). Two isomers of this peptide were prepared: B-10344 (d-Arg-[Hyp3,Igl5,Oic7,d-Igl8]-bradykinin; inverted sequence Oic7, d-Igl8) and B-9972 (d-Arg-[Hyp3,Igl5,Oic7,Igl8]-bradykinin); they are low- and high-potency agonists, respectively, in vascular preparations. The potency gap between bradykinin and B-9972 is narrow in contractility assays, despite the fact that B-9972 affinity is 7-fold inferior at the rabbit B2 receptor (radioligand binding competition assay). The effects of agonists on receptors were compared using two chimerical constructions based on rabbit B2 receptors: conjugate of the B2 receptor with green fluorescent protein (B2R-GFP) and the N-terminally tagged conjugate of the myc epitope with the B2 receptor. Imaging and immunoblotting showed that B-9972 induced a persistent endocytosis of cell surface B2 receptors in human embryonic kidney 293 cells with slow receptor degradation (weak after 3 h of treatment, important at 12 h) and B2R-GFP desensitization ([3H]bradykinin endocytosis and extracellular signal-regulated kinase 1/2 phosphorylation assays). Bradykinin was not active in this respect but when combined with captopril, induced some degradation. B-9430 reduced the endocytosis and degradation of B2 receptors by the agonists. The results illustrate the agonist-antagonist transition in B2 receptor peptide ligands with a constrained C-terminal structure, the importance of species in their pharmacological profile, and the possibility of selectively degrading receptors using a peptidase-resistant agonist.

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