Alves Isabel D, Delaroche Diane, Mouillac Bernard, Salamon Zdzislaw, Tollin Gordon, Hruby Victor J, Lavielle Solange, Sagan Sandrine
Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA.
Biochemistry. 2006 Apr 25;45(16):5309-18. doi: 10.1021/bi052586d.
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-1M) 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(d1) = 0.14 +/- 0.02 nM and K(d2) = 1.4 +/- 0.18 nM, and K(d1) = 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))(11)]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 suggest that these two binding sites are independent and non-interconvertible on the time scale of these experiments (1-2 h).
先前使用经典方法（放射性配体结合和第二信使测定）已报道NK-1受体存在两个非化学计量的配体结合位点。占据最多的（主要的，NK-1M）结合位点结合P物质（SP），并与腺苷酸环化酶途径相关。占据较少的（次要的，NK-1m）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。在此，我们使用表面等离子体波导共振（PWR）光谱法研究了这两个结合位点，该方法通过测量掺入受体的脂质双层的各向异性光学性质，能够监测配体-受体结合过程的热力学和动力学以及受体伴随的结构变化。通过该方法分析了三种肽P物质、神经激肽A和丙酰基[Met（O(2)）(11)]SP(7-11)与部分纯化的NK-1受体的结合。P物质和神经激肽A以双相方式与重组受体结合，具有两种亲和力（K(d1) = 0.14 +/- 0.02 nM和K(d2) = 1.4 +/- 0.18 nM，以及K(d1) = 5.5 +/- 0.7 nM和K(d2) = 620 +/- 117 nM），而对于丙酰基[Met（O(2)）(11)]SP(7-11)只能观察到一种结合亲和力（K(d) = 5.5 +/- 0.4 nM）。此外，在一个配体已与受体结合后再添加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被占据这一事实的影响。这些数据强烈表明，在这些实验的时间尺度（1-2小时）上，这两个结合位点是独立且不可相互转换的。
