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 Fluor 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 Kirsten normal rat kidney cells, but not in vector-alone-transfected cells, and was abolished by pretreatment of cells with saturating concentrations of receptor-selective PAR2 peptide agonists such as SLIGRL-NH2 and the parent agonist 2fLI but not by reverse-sequence peptides such as 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 Fluor 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.
Footnotes
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These studies were supported in large part by term grants from the Canadian Institutes of Health Research (CIRH) (to M.D.H. and N.V.) and by a grant from the Crohn's and Colitis Foundation of Canada (to N.V.), with supplementary support from the National Institutes of Health Grant 1R01MN07568301A1 (to M.D.H.) and the Foundation Bettencourt-Schueller through an INSERM-Avenir program (to N.V.). S.H. was the recipient of an Alberta Heritage Foundation for Medical Research scholarship and a Canadian Association of Gastroenterology scholarship. R.R. is currently supported by a Canadian Association of Gastroenterology/CIHR/Ortho-Jensen Postdoctoral fellowship.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.136432.
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ABBREVIATIONS: PAR, proteinase-activated receptor; Alexa Fluor 594, pyranol[3,4-g:5,6-g′]diquinolin-13-ium, 6-[2-carboxy-4(or 5)[[(2,5-dioxo-1-pyrrolidinyl)oxycarbonyl]phenyl]-1,2,2,10,10,11-hexamethyl-4,8-bis(sulfomethyl)-succinimidyl ester; Alexa Fluor 594-2fLI, 2-furoyl-LIGRL(N-Alexa Fluor 594)-O-NH2; Alexa Fluor 594-2fOL, reverse-sequence receptor-inactive 2-furoyl-(N-Alexa Fluor 594)OLRGIL-NH2; 2fLI, 2-furoyl-LIGRLO-NH2; 2fO or 2fOL, 2-furoyl-OLRGIL-NH2; [3H]propionyl-2fLI, 2-furoyl-LIGRL(N-[3H]propionyl)-O-NH2; PAR2-AP, PAR2-activating peptide; KNRK, Kirsten normal rat kidney; IC50, concentration of binding competitor for which specific ligand binding is inhibited by 50%; RrIC50, RaIC50, and RbIC50, relative IC50s for radioligand binding, Alexa-594 2fLI binding, and vascular bioassay, respectively, normalized to the value for SLIGRL-NH2 = 1.0; HPLC, high-performance liquid chromatography; OCT, ornithine carbamyl transferase; HEK, human embryonic kidney; A23187, calcimycin; tc, trans-cinnamoyl.
- Received January 16, 2008.
- Accepted May 12, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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