Ligand Regulation of the Quaternary Organization of Cell Surface M3 Muscarinic Acetylcholine Receptors Analyzed by Fluorescence Resonance Energy Transfer (FRET) Imaging and Homogeneous Time-resolved FRET
Ligand Regulation of the Quaternary Organization of Cell Surface M3 Muscarinic Acetylcholine Receptors Analyzed by Fluorescence Resonance Energy Transfer (FRET) Imaging and Homogeneous Time-resolved FRET
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DOI：
10.1074/jbc.m110.122184
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发表时间：
2010-07-23
影响因子：
4.8
通讯作者：
Milligan, Graeme
中科院分区：
文献类型：
--
作者：
Alvarez-Curto, Elisa;Ward, Richard J.;Milligan, Graeme
Flp-In-TM T-REx（TM）293细胞组成性表达野生型人M-3毒蕈碱乙酰胆碱受体构建体，并且能够表达仅由该受体的合成配体（RASSL）形式按需激活的受体，维持对毒蕈碱激动剂卡巴胆碱的反应，但仅在诱导RASSL后才对氯氮平N-氧化物产生反应。这两种结构共定位在质膜上，并产生与直接物理相互作用一致的强比率荧光共振能量转移（FRET）信号。增加FRET供体RASSL的诱导水平基本上不改变野生型受体FRET-受体水平。然而，比率FRET调制在一个钟形的方式与最大水平的供体导致减少FRET。卡巴胆碱，而不是拮抗剂阿托品，显着降低FRET信号。基于SNAP-标签技术并采用在Flp-In-TMTREx（TM）293细胞中稳定表达的人M-3受体的野生型和RASSL形式的细胞表面均质时间分辨FRET也鉴定了细胞表面二聚体/寡聚体复合物。然而，现在，信号被适当的选择性激动剂增强。在野生型受体，大量增加的FRET信号卡巴胆碱和乙酰胆碱的浓度依赖性与EC 50值一致的两个配体的相对亲和力。这些研究证实了人M-3毒蕈碱乙酰胆碱受体作为二聚体/寡聚体复合物存在于细胞表面的能力，并证明了这种复合物的组织可以通过配体结合来修饰。然而，配体对这种复合物的影响的结论可能取决于所使用的方法。
Flp-In-TM T-REx(TM) 293 cells expressing a wild type human M-3 muscarinic acetylcholine receptor construct constitutively and able to express a receptor activated solely by synthetic ligand (RASSL) form of this receptor on demand maintained response to the muscarinic agonist carbachol but developed response to clozapine N-oxide only upon induction of the RASSL. The two constructs co-localized at the plasma membrane and generated strong ratiometric fluorescence resonance energy transfer (FRET) signals consistent with direct physical interactions. Increasing levels of induction of the FRET donor RASSL did not alter wild type receptor FRET-acceptor levels substantially. However, ratiometric FRET was modulated in a bell-shaped fashion with maximal levels of the donor resulting in decreased FRET. Carbachol, but not the antagonist atropine, significantly reduced the FRET signal. Cell surface homogeneous time-resolved FRET, based on SNAP-tag technology and employing wild type and RASSL forms of the human M-3 receptor expressed stably in Flp-In-TM TREx(TM) 293 cells, also identified cell surface dimeric/oligomeric complexes. Now, however, signals were enhanced by appropriate selective agonists. At the wild type receptor, large increases in FRET signal to carbachol and acetylcholine were concentration-dependent with EC50 values consistent with the relative affinities of the two ligands. These studies confirm the capacity of the human M-3 muscarinic acetylcholine receptor to exist as dimeric/oligomeric complexes at the surface of cells and demonstrate that the organization of such complexes can be modified by ligand binding. However, conclusions as to the effect of ligands on such complexes may depend on the approach used.