Design of Fluorescent Bradykinin Analogs: Application to Imaging of B2 Receptor-Mediated Agonist Endocytosis and Trafficking and Angiotensin-Converting Enzyme

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Abstract
The known structure-activity relationship and docking models for peptide ligands of the bradykinin B2 receptor indicate a certain tolerance to N-terminal extension. We took advantage of this by generating two fluorescent bradykinin analogs containing 5(6)-carboxyfluorescein (CF) optionally used with the ε-aminocaproyl spacer condensed at the N terminus of the agonist. Pharmacological studies indicated that CF-bradykinin was virtually inactive as a B2 receptor ligand and agonist, whereas CF-ε-aminocaproyl-bradykinin (CF-εACA-BK) was 400- to 1000-fold less potent than bradykinin (competition of [3H]bradykinin binding to B2 receptors, contractility of the human isolated umbilical vein). Nevertheless, CF-εACA-BK (5 μM) was taken up by human embryonic kidney 293a cells expressing recombinant B2 receptors, but not by those cotreated with an antagonist or expressing a truncated receptor that is pharmacologically intact but not phosphorylable. A higher-affinity CF-conjugated peptide, the antagonist CF-εACA-d-Arg-[Hyp3,Igl5,d-Igl7,Oic8]-bradykinin (B-10380), labeled both intact and truncated receptor forms at the cell surface. The fluorescent agonist CF-εACA-BK was found in vesicles positive for β-arrestin1, Rab5, and Rab7, then apparently degraded as a function of time because the fluorescence was transferred from the vesicles to the cytosol in a vesicular-ATPase-dependent process (3 h). The ectopeptidase angiotensin-converting enzyme (ACE) is a major kininase. The binding affinity of CF-εACA-BK for this carboxydipeptidase is identical to that of bradykinin ([3H]enalaprilat displacement assay). Recombinant ACE is essentially a plasma membrane protein in CF-εACA-BK imaging of intact cells. Micromolar CF-εACA-BK is a probe for the two major physiological targets of bradykinin, the B2 receptor and ACE. As an agonist, it is subjected to β-arrestin-mediated endocytosis, trafficking, and subsequent ligand degradation.

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