Received for publication January 17, 2008.
Revised May 10, 2008.
Accepted for publication May 12, 2008.

Derivatized 2-furoyl-LIGRLO-amide, a versatile and selective probe for Proteinase-Activated Receptor 2 (PAR₂): binding and visualization

Abstract

The Proteinase-Activated Receptor-2 (PAR₂) activating peptide with an N-terminal furoyl group modification, 2-furoyl-LIGRLO-NH₂ (2fLI), was derivatized via its free ornithine amino group to yield ³H-propionyl-2fLI and Alexa 594-2fLI that were used as receptor probes for ligand binding assays and receptor visualization both for cultured cells in vitro and for colonic epithelial cells in vivo. The binding of the radiolabeled and fluorescent PAR₂ probes was shown to be present in PAR₂-transfected KNRK cells but not in vector-alone-transfected cells and was abolished by pretreatment of cells with saturating concentrations of receptor-selective PAR₂ peptide agonists like SLIGRL-NH₂ and the parent agonist, 2fLI but not by reverse-sequence peptides like 2-furoyl-OLRGIL-NH₂ that cannot activate PAR₂. The relative orders of potencies for a series of PAR₂ peptide agonists to compete for the binding of ³H-propionyl-2fLI (2fLI >> SLIGRL-NH₂ trans-cinnamoyl-LIGRLO-NH₂>SLIGKV-NH₂>SLIGKT-NH₂) mirrored qualitatively their relative potencies for PAR₂-mediated calcium signaling in the same cells or for vasorelaxation in a rat aorta vascular assay. In the vascular assay, the potency of Alexa 594-2fLI was the same as 2fLI. We conclude that ornithine-derivatized 2fLI peptides are conveniently-synthesized PAR₂ probes that will be of value for future studies of receptor binding and visualization studies.

Key words: PAR-2, Protease activated receptor, calcium, colon, fluorescent receptor labeling, ligand binding