The authors have investigated the interaction of fluorescent peptide ligands with 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 labeled on position 1 (Xaa) with the environment-sensitive nitrobenzoxadiazole (NBD) probe, differing only in the length of the spacer between the NBD group and the peptide. Upon binding of the labeled antagonist to NK2 receptors stably expressed in Chinese hamster ovary (CHO) cells, an increase in NBD fluorescence was obsd. when the spacer length was less than 10 .ANG.. Collisional quenching expts. using iodide and Co2+ ions were performed to define the accessibility of the NBD group on bound ligands to the solvent. By comparing ligands with spacer arms of varying lengths, the authors found that the binding pocket is buried at a depth of 5-10 .ANG.. In contrast, N-terminally NBD-labeled agonists, decapeptide neurokinin A (NKA) or heptapeptide Nle10-NKA[4-10], bound to the NK2 receptor were accessible to the solvent. Binding of fluorescent ligands to the NK2 receptor was accompanied by an enhancement in the fluorescence anisotropy. The changes in fluorescence properties were used to det. the kinetic parameters of antagonist binding and dissocn. These results indicate that the binding site on the NK2 receptor for the amino-terminal end of the heptapeptide antagonists is buried in the hydrophobic pocket of the receptor protein and clearly distinct from the binding site for the amino-terminal end of agonists, which is accessible to the solvent. Heptapeptide agonist and antagonist may therefore have distinct but probably overlapping binding sites. These results support recent observations suggesting that peptide agonists dock in the extracellular regions of seven-transmembrane receptors. The methodol. described here should be of broad applicability for investigating ligand-receptor recognition. [on SciFinder (R)]