Biotechnological Fluorescent Ligands of the Bradykinin B(1) 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).