Bifunctional fusion proteins containing the sequence of the bradykinin homologue maximakinin: activities at the rat bradykinin B receptor.

To support bradykinin (BK) B receptor (BR) detection and therapeutic stimulation, we developed and characterized fusion proteins consisting of the BK homolog maximakinin (MK), or variants, positioned at the C-terminus of functional proteins (enhanced green fluorescent protein (EGFP), the peroxidase APEX2, or human serum albumin (HSA)). EGFP-MK loses its reactivity with anti-BK antibodies and molecular mass as it progresses in the endosomal tract of cells expressing rat BRs (immunoblots, epifluorescence microscopy). APEX2-(NG)-MK is a bona fide agonist of the rat, but not of the human BR (calcium and c-Fos signaling) and is compatible with the cytochemistry reagent TrueBlue (microscopy), a luminol-based reagent, or 3,3',5,5'-tetramethylbenzidine (luminescence or colourimetric BR detection, cell well plate format). APEX2-(NG)-MK is a non-isotopic ligand suitable for drug discovery via binding competition. Affinity-purified secreted forms of HSA fused with peptides possessing the C-terminal MK or BK sequence failed to stimulate the rat BR in the concentration range of 50-600 nmol/L. However, the non-secreted construction myc-HSA-MK is a BR agonist, indicating that protein denaturation made the C-terminal sequence available for receptor binding. Fusion protein ligands of the BR are stable but subjected to slow intracellular inactivation, strong species specificity, and possible steric hindrance between the receptor and large proteins.