Turcatti G, Zoffmann S, Lowe J A, Drozda S E, Chassaing G, Schwartz T W, Chollet A
Geneva Biomedical Research Institute, Glaxo Wellcome, CH-1228 Geneva, Switzerland.
J Biol Chem. 1997 Aug 22;272(34):21167-75. doi: 10.1074/jbc.272.34.21167.
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.
通过使用荧光配体和荧光光谱法，对肽激动剂和非肽拮抗剂与NK1受体（P物质受体）的配体识别进行了研究和比较。合成并表征了在3、8或11位用对环境敏感的荧光基团5-二甲基氨基萘-1-磺酰基（丹磺酰基）标记的P物质（SP）类似物，或在Nα位用荧光素标记的类似物。在α-氨基或3或11位修饰的肽对NK1保留了相对较好的亲和力和激动特性。在8位修饰导致亲和力降低18000倍。制备并表征了非肽拮抗剂CP96,345的荧光丹磺酰类似物。丹磺酰-CP96,345的荧光量子产率远高于任何丹磺酰标记的肽，这表明与肽激动剂相比，非肽拮抗剂结合位点的微环境更疏水。水溶性羟基-Tempo化合物对荧光的碰撞猝灭比较表明，丹磺酰-CP96,345被掩埋，水相猝灭剂几乎无法接近，而丹磺酰或荧光素标记的肽则暴露于溶剂中。所有荧光配体与NK1结合后各向异性增加，表明运动自由度受限。然而，与连接到SP不同位置的荧光探针相比，连接到非肽拮抗剂CP96,345上的丹磺酰基的各向异性增加更为明显。总之，我们的数据表明，与NK1受体结合时，非肽拮抗剂和肽激动剂周围的环境有很大不同。这些结果支持了最近通过诱变和交联研究得出的观察结果，即肽激动剂在NK1受体N端延伸区和形成细胞外表面的环中有主要相互作用点。我们的数据还表明，SP的C端和N端似乎都没有深入到受体跨膜结构域的细胞外表面以下。
