PROBING THE BINDING DOMAIN OF THE NK2 RECEPTOR WITH FLUORESCENT LIGANDS - EVIDENCE THAT HEPTAPEPTIDE AGONISTS AND ANTAGONISTS BIND DIFFERENTLY

We have investigated the interaction of fluorescent peptide ligands wi th the G protein-coupled receptor NK2 using novel spectrofluorometric approaches. Several heptapeptide antagonists of structure PhCO-Xaa-Ala -D-Trp-Phe-D-Pro-Pro-Nle-NH2 were labelled on position 1 (Xaa) with th e environment-sensitive nitrobenzoxadiazole (NBD) probe, differing onl y in the length of the spacer between the NBD group and the peptide. U pon binding of the labelled antagonist to NK2 receptors stably express ed in Chinese hamster ovary (CHO) cells, an increase in NBD fluorescen ce was observed when the spacer length was less than 10 Angstrom. Coll isional quenching experiments using iodide and Co2+ ions were performe d to define the accessibility of the NBD group on bound ligands to the solvent. By comparing ligands with spacer arms of varying lengths, we found that the binding pocket is buried at a depth of 5-10 Angstrom. In contrast, N-terminally NBD-labelled agonists, decapeptide neurokini n A (NKA) or heptapeptide Nle(10) NKA[4-10], bound to the NK2 receptor were accessible td the solvent. Binding of fluorescent Ligands to the NK2 receptor was accompanied by an enhancement in the fluorescence an isotropy. The changes in fluorescence properties were used to determin e the kinetic parameters of antagonist binding and dissociation. These results indicate that the binding site on the NK2 receptor for the am ino-terminal end of the heptapeptide antagonists is buried in the hydr ophobic pocket of the receptor protein and clearly distinct from the b inding site for the amino-terminal end of agonists, which is accessibl e to the solvent. Heptapeptide agonist and antagonist may therefore ha ve distinct but probably overlapping binding sites. These results supp ort recent observations suggesting that peptide agonists dock in the e xtracellular regions of seven-transmembrane receptors. The methodology described here should be of broad applicability for investigating lig and-receptor recognition.