Development and Characterization of a Fluorescent Ligand for Leukotriene B4 Receptor 2 in Cells and Tissues

The leukotriene B4
receptor 2 (BLT2) is a G-protein coupled receptor
activated by 12­(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT),
which has been proposed as a promising therapeutic target for diabetic
wound healing and gastrointestinal lesions. In this study, the rational
design of a fluorescent probe based on the synthetic BLT2 agonist
CAY10583 is described. The synthesis of several derivatives of CAY10583
coupled to fluorescein resulted in a traceable ligand suitable for
different fluorescence-based techniques. An HTRF-based displacement
assay (Tag-lite) on stably transfected CHO-K1 cells was developed
to characterize binding properties of diverse BLT2 ligands. Highly
specific binding to the BLT2 receptor was demonstrated in staining
experiments on mouse skin tissue, and specific modulation of BLT2-induced
cAMP signaling provided further evidence for receptor binding and
ligand functionality. In conclusion, the fluorescent ligands developed
in this study are suitable to investigate the pharmacology of BLT2
receptor ligands in a variety of assay systems.