Peptide agonists and antagonists of both
bradykinin (BK) B(1) and
B(2) receptors (B(1)R,
B(2)R) are known to tolerate to a certain level N-terminal sequence extensions. Using this strategy, we produced and characterized the full set of fluorescent
ligands by extending both agonists and antagonist
peptides at both receptor subtypes with
5(6)-carboxyfluorescein (CF) and the ε-aminocaproyl (ε-ACA) optional spacer. Alternatively,
kinin receptor
ligands were extended with another
carboxylic acid cargo (
chlorambucil, biotinyl, pentafluorocinnamoyl, AlexaFluor-350 (
AF350), ferrocenoyl,
cetirizine) or with
fluorescein isothiocyanate. N-terminal extension always reduced receptor affinity, more importantly for bulkier substituents and more so for the agonist version compared to the antagonist. This loss was generally alleviated by the presence of the spacer and modulated by the species of origin for the receptor. We report and review the pharmacological properties of these N-terminally extended
peptides and the use of fluorophore-conjugated
ligands in imaging of cell receptors and of
angiotensin converting enzyme (ACE) in intact cells. Antagonists (B(1)R: B-10376: CF-ε-ACA-
Lys-Lys-[Hyp(3), CpG(5), D-
Tic(7), CpG(8)]des-Arg(9)-BK;
B(2)R:
B-10380: CF-ε-ACA-D-Arg-[Hyp(3), Igl(5), D-Igl(7), Oic(8)]-BK and
fluorescein-5-thiocarbamoyl (
FTC)-
B-9430) label the plasma membrane of cells expressing the cognate receptors. The
B(2)R agonists CF-ε-ACA-BK, AF350-ε-ACA-BK and FTC-B-9972 are found in endosomes and model the endosomal degradation of BK in a complementary manner. The uneven surface fluorescence associated to the B(1)R agonist B-10378 (CF-ε-ACA-Lys-des-Arg(9)-BK) is compatible with a particular form of agonist-induced receptor translocation. CF-ε-ACA-BK binds to the carboxydipeptidase ACE with an affinity identical to that of BK.
Metal- or
drug-containing cargoes further show the prospect of
ligands that confer special signaling to
kinin receptors.