CHARACTERIZATION OF NONPEPTIDE ANTAGONIST AND PEPTIDE AGONIST BINDING-SITES OF THE NK1 RECEPTOR WITH FLUORESCENT LIGANDS

Ligand recognition of the NK1 receptor (substance P receptor) by pepti de agonist and non-peptide antagonist has been investigated and compar ed by the use of fluorescent ligands and spectrofluorometric methods, Analogues of substance P (SP) labeled with the environment-sensitive f luorescent group 5-dimethylaminonaphthalene-1 sulfonyl (dansyl) at eit her position 3, 8, or 11 or with fluorescein at the N-alpha position w ere synthesized and characterized. Peptides modified at the alpha-amin o group or at positions 3 or 11 conserved a relatively good affinity f or NK1 and agonistic properties, Modification at position 8 resulted i n an 18,000-fold decrease in affinity, A fluorescent dansyl analogue o f the non-peptide antagonist CP96,345 was prepared and characterized. The quantum yield of fluorescence for dansyl-CP96,345 was much higher than for any of the dansyl-labeled peptides indicating that the micro- environment of the binding site is more hydrophobic for the nonpeptide antagonist than for the peptide agonists, Comparison of collisional q uenching of fluorescence by the water-soluble hydroxy-Tempo compound s howed that dansyl-CP96,345 is buried and virtually inaccessible to aqu eous quenchers, whereas dansyl-or fluoresceinyl-labeled peptides were exposed to the solvent, Anisotropy of all fluorescent ligands increase d upon binding to NK1 indicating a restricted motional freedom, Howeve r, this increase in anisotropy was more pronounced for the dansyl atta ched to the non-peptide antagonist CP96,345 than for the fluorescent p robes attached to different positions of SP, In conclusion, our data i ndicate that the environment surrounding non-peptide antagonist and pe ptide agonists are vastly different when bound to the NK1 receptor. Th ese results support recent observations by mutagenesis and cross-linki ng work suggesting that peptide agonists have their major interaction points in the N-terminal extension and the loops forming the extracell ular face of the NK1 receptor, Our data also suggest that neither the C terminus nor the N terminus of SP appears to penetrate deeply below the extracellular surface in the transmembrane domain of the receptor.