Holst B, Hastrup H, Raffetseder U, Martini L, Schwartz T W
Laboratory for Molecular Pharmacology, Department of Pharmacology, The Panum Institute, University of Copenhagen DK-2200 and the 7TM Pharma A/S, DK-2100 Copenhagen, Denmark.
J Biol Chem. 2001 Jun 8;276(23):19793-9. doi: 10.1074/jbc.M100621200. Epub 2001 Feb 22.
The NK1 neurokinin receptor presents two non-ideal binding phenomena, two-component binding curves for all agonists and significant differences between agonist affinity determined by homologous versus heterologous competition binding. Whole cell binding with fusion proteins constructed between either Galpha(s) or Galpha(q) and the NK1 receptor with a truncated tail, which secured non-promiscuous G-protein interaction, demonstrated monocomponent agonist binding closely corresponding to either of the two affinity states found in the wild-type receptor. High affinity binding of both substance P and neurokinin A was observed in the tail-truncated Galpha(s) fusion construct, whereas the lower affinity component was displayed by the tail-truncated Galpha(q) fusion. The elusive difference between the affinity determined in heterologous versus homologous binding assays for substance P and especially for neurokinin A was eliminated in the G-protein fusions. An NK1 receptor mutant with a single substitution at the extracellular end of TM-III-(F111S), which totally uncoupled the receptor from Galpha(s) signaling, showed binding properties that were monocomponent and otherwise very similar to those observed in the tail-truncated Galpha(q) fusion construct. Thus, the heterogenous pharmacological phenotype displayed by the NK1 receptor is a reflection of the occurrence of two active conformations or molecular phenotypes representing complexes with the Galpha(s) and Galpha(q) species, respectively. We propose that these molecular forms do not interchange readily, conceivably because of the occurrence of microdomains or "signal-transductosomes" within the cell membrane.
NK1神经激肽受体呈现出两种非理想结合现象，所有激动剂的双组分结合曲线以及同源竞争结合与异源竞争结合所测定的激动剂亲和力之间存在显著差异。使用在Gα(s)或Gα(q)与截短尾部的NK1受体之间构建的融合蛋白进行全细胞结合，这种结合确保了非混杂的G蛋白相互作用，结果表明单组分激动剂结合与野生型受体中发现的两种亲和力状态中的任何一种紧密对应。在截短尾部的Gα(s)融合构建体中观察到P物质和神经激肽A的高亲和力结合，而截短尾部的Gα(q)融合体则显示出较低亲和力组分。在G蛋白融合体中消除了P物质尤其是神经激肽A在异源结合试验与同源结合试验中所测定亲和力之间难以捉摸的差异。在跨膜区III细胞外端有一个单取代（F111S）的NK1受体突变体完全使受体与Gα(s)信号传导解偶联，其结合特性为单组分，并且在其他方面与截短尾部的Gα(q)融合构建体中观察到的非常相似。因此，NK1受体所显示的异质药理表型反映了分别代表与Gα(s)和Gα(q)物种形成复合物的两种活性构象或分子表型的存在。我们提出这些分子形式不容易相互转换，这可能是由于细胞膜内存在微结构域或“信号转导体”。
