Characterization of B‐9972, a peptidase‐resistant agonist of the bradykinin B2 receptors: effects on the endocytosis and recycling of the receptors

The bradykinin (BK) B 2 receptor (B 2 R) is a good model of a G protein coupled receptor regulated by a cycle of phosphorylation, endocytosis and extensive recycling at the cell surface following agonist stimulation. B‐9430 (D‐Arg‐[Hyp 3 , Igl 5 , D‐Igl 7 , Oic 8 ]‐BK) is a second generation peptide antagonist found to be competitive at the human B 2 R and insurmountable at the rabbit B 2 R (contractility assays, isolated human umbilical and rabbit jugular veins). Two isomers of this peptide were prepared: B‐10344 (inverted sequence Oic 7 , D‐Igl 8 ) and B‐9972 (Oic 7 ‐Igl 8 ); respectively low and high affinity agonists of the B 2 R. Two functional chimerical constructions based on the rabbit B 2 R were exploited to compare the effects of different agonists on receptor cycling: a green fluorescent protein conjugate (B 2 R‐GFP) and the novel N‐terminal tagged myc‐B 2 R. Imaging and immunoblotting showed that B‐9972 induced a persistent endocytosis of cell surface B 2 Rs in HEK 293 cells with a slow receptor degradation (weak after 3 hours of treatment, important at 12 hours). BK combined with captopril reproduced a part of the effects of B‐9972 on B 2 R downregulation. B‐9430 reduces the morphological effects and downregulation of B 2 Rs by the agonists, although B‐9430 exerted some partial agonist effects on recombinant receptors expressed at high densities (calcium transients, etc.). The results illustrate the agonist‐antagonist transition in B 2 R peptide ligands with constrained C‐terminal structures, the importance of species of origin for their pharmacological profile and the possibility of downregulating receptors selectively using a peptidase‐resistant agonist.