Alves Isabel D, Ciano Kathy A, Boguslavski Valentina, Varga Eva, Salamon Zdzislaw, Yamamura Henry I, Hruby Victor J, Tollin Gordon
Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson 85721, USA.
J Biol Chem. 2004 Oct 22;279(43):44673-82. doi: 10.1074/jbc.M404713200. Epub 2004 Aug 17.
A better understanding of signal transduction mechanisms is of critical importance. Methodologies that allow studies to be done while receptors are incorporated into lipid bilayers are advantageous. One such technique is plasmon-waveguide resonance (PWR) spectroscopy, which can follow changes in conformation accompanying protein-ligand, protein-protein, and protein-lipid interactions occurring in G-protein-coupled receptors in real time with high sensitivity and without the need for molecular labeling. Here we investigated several aspects of human delta-opioid receptor (hDOR)-G-protein interactions: 1) the effect of different types of agonists on the interaction with individual G-protein subtypes; 2) the affinities of the separate G-protein alpha and betagamma subunits to different ligand-occupied states of the receptor; and 3) the effect of the presence of the G-protein on the interactions of the ligand with the receptor. To accomplish this we have incorporated the receptor into a solid supported lipid bilayer in the presence of ligand or G-protein and monitored the PWR spectral changes induced by the reciprocal G-protein or ligand interactions. We found a high degree of selectivity in the interactions of different agonist-bound states of the receptor with the different G-protein subtypes. This has important implications for agonist-directed trafficking and selective drug design. Studies with the separated alpha and betagamma subunits show that cooperativity exists in these interactions. The high affinities of the separated subunits to the receptor point to the possibility of independent promotion of specific signaling events. The presence of G-proteins increased the affinity of agonists to the hDOR, and caused faster binding kinetics and different ligand-induced conformational changes. Because ligand also influences G-protein binding, reciprocity exists between these two binding processes.
更好地理解信号转导机制至关重要。能够在受体整合到脂质双层中时进行研究的方法具有优势。一种这样的技术是表面等离子体波导共振（PWR）光谱学，它可以实时、高灵敏度地跟踪G蛋白偶联受体中发生的蛋白质-配体、蛋白质-蛋白质和蛋白质-脂质相互作用所伴随的构象变化，且无需分子标记。在这里，我们研究了人类δ-阿片受体（hDOR）与G蛋白相互作用的几个方面：1）不同类型激动剂对与单个G蛋白亚型相互作用的影响；2）单独的G蛋白α和βγ亚基对受体不同配体占据状态的亲和力；3）G蛋白的存在对配体与受体相互作用的影响。为了实现这一点，我们在存在配体或G蛋白的情况下将受体整合到固体支持的脂质双层中，并监测由G蛋白或配体相互作用引起的PWR光谱变化。我们发现受体不同激动剂结合状态与不同G蛋白亚型之间的相互作用具有高度选择性。这对激动剂导向的转运和选择性药物设计具有重要意义。对分离的α和βγ亚基的研究表明这些相互作用中存在协同作用。分离的亚基对受体的高亲和力表明有可能独立促进特定的信号事件。G蛋白的存在增加了激动剂对hDOR的亲和力，并导致更快的结合动力学和不同的配体诱导的构象变化。由于配体也影响G蛋白结合，这两个结合过程之间存在相互作用。
