Characterization of non-peptide antagonist and peptide agonist binding sites of the NK1 receptor with fluorescent ligands.
Ligand recognition of the NK1 receptor (substance P receptor) by peptide agonist and non-peptide antagonist has been investigated and compared by the use of fluorescent ligands and spectrofluorometric methods. Analogues of substance P (SP) labeled with the environment-sensitive fluorescent group 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) at either position 3, 8, or 11 or with fluorescein at the Nalpha position were synthesized and characterized. Peptides modified at the alpha-amino group or at positions 3 or 11 conserved a relatively good affinity for NK1 and agonistic properties. Modification at position 8 resulted in an 18, 000-fold decrease in affinity. A fluorescent dansyl analogue of 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 non-peptide antagonist than for the peptide agonists. Comparison of collisional quenching of fluorescence by the water-soluble hydroxy-Tempo compound showed that dansyl-CP96,345 is buried and virtually inaccessible to aqueous quenchers, whereas dansyl- or fluoresceinyl-labeled peptides were exposed to the solvent. Anisotropy of all fluorescent ligands increased upon binding to NK1 indicating a restricted motional freedom. However, this increase in anisotropy was more pronounced for the dansyl attached to the non-peptide antagonist CP96,345 than for the fluorescent probes attached to different positions of SP. In conclusion, our data indicate that the environment surrounding non-peptide antagonist and peptide agonists are vastly different when bound to the NK1 receptor. These results support recent observations by mutagenesis and cross-linking work suggesting that peptide agonists have their major interaction points in the N-terminal extension and the loops forming the extracellular 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.
Turcatti
G
G
Geneva Biomedical Research Institute, Glaxo Wellcome, CH-1228 Geneva, Switzerland.
Zoffmann
S
S
Lowe
J A
JA
Drozda
S E
SE
Chassaing
G
G
Schwartz
T W
TW
Chollet
A
A
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001713:Biphenyl Compounds / Q000737:chemistry*; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D003619:Dansyl Compounds / Q000737:chemistry; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D006801:Humans; D008024:Ligands; D008562:Membrane Glycoproteins / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D064729:Neurokinin-1 Receptor Antagonists; D017433:Protein Structure, Secondary; D018040:Receptors, Neurokinin-1 / Q000819:agonists*; D011994:Recombinant Proteins / Q000737:chemistry; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013373:Substance P / Q000031:analogs & derivatives*
D001713:Biphenyl Compounds; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D008024:Ligands; D008562:Membrane Glycoproteins; D064729:Neurokinin-1 Receptor Antagonists; D018040:Receptors, Neurokinin-1; D011994:Recombinant Proteins; D013373:Substance P; C066892:CP 96345