Biotechnological Fluorescent Ligands of the Bradykinin B1 Receptor: Protein Ligands for a Peptide Receptor

The bradykinin (BK) B 1 receptor (B 1 R) is a peculiar G protein coupled receptor that is strongly regulated to the point of being inducible in immunopathology. Limited clinical evidence suggests that its expression in peripheral blood mononuclear cells is a biomarker of active inflammatory states. In an effort to develop a novel imaging/diagnostic tool, we report the rational design and testing of a fusion protein that is a ligand of the human B 1 R but not likely to label peptidases. This ligand is composed of a fluorescent protein (FP) (enhanced green FP [EGFP] or mCherry) prolonged at its N-terminus by a spacer peptide and a classical peptide agonist or antagonist (des-Arg 9 -BK, [Leu 8 ]des-Arg 9 -BK, respectively). The design of the spacer-ligand joint peptide was validated by a competition assay for [ 3 H]Lys-des-Arg 9 -BK binding to the human B 1 R applied to 4 synthetic peptides of 18 or 19 residues. The labeling of B 1 R-expressing cells with EGFP or mCherry fused with 7 of such peptides was performed in parallel (microscopy). Both assays indicated that the best design was FP-(Asn-Gly) n -Lys-des-Arg 9 -BK; n = 15 was superior to n = 5, suggesting benefits from minimizing steric hindrance between the FP and the receptor. Cell labeling concerned mostly plasma membranes and was inhibited by a B 1 R antagonist. EGFP-(Asn-Gly) 15 -Lys-des-Arg 9 -BK competed for the binding of [ 3 H]Lys-des-Arg 9 -BK to human recombinant B 1 R, being only 10-fold less potent than the unlabeled form of Lys-des-Arg 9 -BK to do so. The fusion protein did not label HEK 293a cells expressing recombinant human BK B 2 receptors or angiotensin converting enzyme. This study identifies a modular C-terminal sequence that can be adapted to protein cargoes, conferring high affinity for the BK B 1 R, with possible applications in diagnostic cytofluorometry, histology and drug delivery (e.g., in oncology).