Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules
Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules
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DOI：
10.1073/pnas.0907915107
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发表时间：
2010-02-09
影响因子：
11.1
通讯作者：
Birdsall, Nigel J. M.
中科院分区：
文献类型：
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作者：
Hern, Jonathan A.;Baig, Asma H.;Birdsall, Nigel J. M.
G蛋白偶联受体（GPCR）是人类基因组中最大的跨膜信号蛋白家族。GPCR信号级联反应中的事件已得到很好的表征，但受体组成及其膜分布通常仍是未知的。虽然有证据表明GPCR超家族的一些成员以组成型二聚体或更高的寡聚体存在，但对结果的解释一直存在争议，最近的研究表明单体GPCR也可能具有功能。由于在该领域内存在争议，为了解决这个问题，我们在活细胞中使用全内反射荧光显微镜（TIRFM）来可视化模型GPCR（M-1毒蕈碱乙酰胆碱受体）的数千个单个分子。通过跟踪单个受体随时间的位置，可以直接确定它们的迁移率、聚集和二聚化动力学，分辨率类似于30 ms和类似于20 nm。在分离的CHO细胞中，受体随机分布在质膜上。在任何给定的时间，大约30%的受体分子以二聚体的形式存在，我们没有发现更高的寡聚体的证据。双色TIRFM建立了二聚体形成的动态性质，M-1受体在几秒钟的时间尺度上经历单体和二聚体之间的相互转化。
G-protein-coupled receptors (GPCRs) are the largest family of transmembrane signaling proteins in the human genome. Events in the GPCR signaling cascade have been well characterized, but the receptor composition and its membrane distribution are still generally unknown. Although there is evidence that some members of the GPCR superfamily exist as constitutive dimers or higher oligomers, interpretation of the results has been disputed, and recent studies indicate that monomeric GPCRs may also be functional. Because there is controversy within the field, to address the issue we have used total internal reflection fluorescence microscopy (TIRFM) in living cells to visualize thousands of individual molecules of a model GPCR, the M-1 muscarinic acetylcholine receptor. By tracking the position of individual receptors over time, their mobility, clustering, and dimerization kinetics could be directly determined with a resolution of similar to 30 ms and similar to 20 nm. In isolated CHO cells, receptors are randomly distributed over the plasma membrane. At any given time, similar to 30% of the receptor molecules exist as dimers, and we found no evidence for higher oligomers. Two-color TIRFM established the dynamic nature of dimer formation with M-1 receptors undergoing interconversion between monomers and dimers on the timescale of seconds.