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[摘要]：The known structure-activity relationship and docking models for peptide ligands of the bradykinin B-2 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 epsilon-aminocaproyl spacer condensed at the N terminus of the agonist. Pharmacological studies indicated that CF-bradykinin was virtually inactive as a B 2 receptor ligand and agonist, whereas CF-epsilon-aminocaproyl-bradykinin (CF-epsilon ACA-BK) was 400- to 1000-fold less potent than bradykinin (competition of [H-3] bradykinin binding to B 2 receptors, contractility of the human isolated umbilical vein). Nevertheless, CF-epsilon ACA-BK (5 mu M) was taken up by human embryonic kidney 293a cells expressing recombinant B-2 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-epsilon ACA-D-Arg-[Hyp(3), Igl(5), D-Igl(7), Oic(8)]-bradykinin (B-10380), labeled both intact and truncated receptor forms at the cell surface. The fluorescent agonist CF-epsilon ACA-BK was found in vesicles positive for beta-arrestin(1), 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-epsilon ACA-BK for this carboxydipeptidase is identical to that of bradykinin ([H-3]enalaprilat displacement assay). Recombinant ACE is essentially a plasma membrane protein in CF-epsilon ACA-BK imaging of intact cells. Micromolar CF-epsilon ACA-BK is a probe for the two major physiological targets of bradykinin, the B-2 receptor and ACE. As an agonist, it is subjected to beta-arrestin-mediated endocytosis, trafficking, and subsequent ligand degradation. |
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