The two NK-1 binding sites correspond to distinct, independent, and non-interconvertible receptor conformational states as confirmed by plasmon-waveguide resonance spectroscopy
The two NK-1 binding sites correspond to distinct, independent, and non-interconvertible receptor conformational states as confirmed by plasmon-waveguide resonance spectroscopy
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DOI：
10.1021/bi052586d
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发表时间：
2006-04-25
期刊：
影响因子：
2.9
通讯作者：
Sagan, S
中科院分区：
文献类型：
--
作者：
Alves, ID;Delaroche, D;Sagan, S
两个非化学计量的配体结合位点已被报道的NK-1受体，使用经典的方法（放射性配体结合和第二信使测定）。最常见的（主要，NK-顶）结合位点结合P物质（SP），并与腺苷酸环化酶途径有关。较少的（次要的，NK-1 m）结合位点结合P物质、SP的C末端六肽和七肽类似物以及NK-2内源性配体神经激肽A，并与磷脂酶C途径偶联。在这里，我们已经研究了这两个结合位点与等离子体波导共振（PWR）光谱，允许的热力学和动力学的配体-受体结合过程和伴随的受体的结构变化进行监测，通过测量的各向异性光学性质的脂质双层的受体被纳入。用这种方法分析了P物质、神经激肽A和丙酰[Met（O-2）（11）]SP（7-11）这三种肽与部分纯化的NK-1受体的结合。P物质和神经激肽A以两种亲和力以双相方式与重构受体结合（分别为K-dl = 0.14 +/-0.02nM和K-d2 = 1.4 +/-0.18nM和K-dl = 5.5 +/-0.7nM和K-d2 = 620 +/-117nM），而对于丙酰基[Met（O-2）（II）]SP（7-11）仅观察到一种结合亲和力（Kd = 5.5 +/- 0.4 nM）。此外，在一个配体与受体结合后再加入另一个配体的结合实验表明，这些配体与每个结合位点的结合不受另一个位点已经被占据的事实的影响。这些数据有力地证明了这两个结合位点在这些实验的时间尺度上（1-2 h）是独立的和不可相互转换的。
Two nonstoichiometric ligand binding sites have been previously reported for the NK-1 receptor, with the use of classical methods (radioligand binding and second messenger assays). The most populated (major, NK-IM) binding site binds substance P (SP) and is related to the adenylyl cyclase pathway. The less populated (minor, NK-1m) binding site binds substance P, C-terminal hexa- and heptapeptide analogues of SP, and the NK-2 endogenous ligand, neurokinin A, and is coupled to the phospholipase C pathway. Here, we have examined these two binding sites with plasmon-waveguide resonance (PWR) spectroscopy that allows the thermodynamics and kinetics of ligand-receptor binding processes and the accompanying structural changes of the receptor to be monitored, through measurements of the anisotropic optical properties of lipid bilayers into which the receptor is incorporated. The binding of the three peptides, substance P, neurokinin A, and propionyl [Met(O-2)(11)]SP(7-11), to the partially purified NK-1 receptor has been analyzed by this method. Substance P and neurokinin A bind to the reconstituted receptor in a biphasic manner with two affinities (K-dl = 0.14 +/- 0.02 nM and K-d2 = 1.4 +/- 0.18 nM, and K-dl = 5.5 +/- 0.7 nM and K-d2 = 620 +/- 117 nM, respectively), whereas only one binding affinity (K-d = 5.5 +/- 0.4 nM) could be observed for propionyl[Met(O-2)(II)]SP(7-11). Moreover, binding experiments in which one ligand was added after another one has been bound to the receptor have shown that the binding of these ligands to each binding site was unaffected by the fact that the other site was already occupied. These data strongly suggestthat these two binding sites are independent and non-interconvertible on the time scale of these experiments (1-2 h).