McDonald Neil A, Henstridge Christopher M, Connolly Christopher N, Irving Andrew J
Neurosciences Institute, Division of Pathology and Neuroscience, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, UK.
Mol Cell Neurosci. 2007 Jun;35(2):237-48. doi: 10.1016/j.mcn.2007.02.016. Epub 2007 Mar 3.
N-terminally tagged CB1 receptor fusion proteins, incorporating enhanced green fluorescent protein (GFP) or super-ecliptic pHluorin (SEP), were generated to study CB1 receptor trafficking and cell surface receptor expression in live COS7 and HEK293 cells and hippocampal neurons. An artificial signal sequence (SS) was required for efficient surface expression of CB1 receptor chimeras, which behaved like wild-type CB1 receptors in functional assays. Treatment with cannabinoid ligands led to a rapid down-regulation of SS-GFP-CB1 from the plasma membrane in COS7 and HEK293 cells, associated with trafficking into cytosolic vesicles. Activation of CB1 receptors was also linked with a time-dependent reduction in cell surface SEP-CB1 fluorescence and incorporation of the construct into acidic endosomes, revealed following exposure to NH4Cl. In live hippocampal neurons, SEP-CB1 fluorescence was largely restricted to the axon, consistent with its polarised surface expression. Thus, these new molecular tools are well suited for studying CB1 receptor trafficking and a new generation of GPCR chimeras incorporating SEP at the N-terminus will be especially useful for monitoring dynamic changes in cell surface receptor expression in living cells.
生成了包含增强型绿色荧光蛋白（GFP）或超嗜碱pH荧光蛋白（SEP）的N端标记CB1受体融合蛋白，以研究CB1受体在活的COS7细胞、HEK293细胞和海马神经元中的转运及细胞表面受体表达。CB1受体嵌合体的有效表面表达需要一个人工信号序列（SS），其在功能测定中表现得像野生型CB1受体。用大麻素配体处理导致COS7细胞和HEK293细胞中SS-GFP-CB1从质膜快速下调，这与转运到胞质囊泡有关。CB1受体的激活还与细胞表面SEP-CB1荧光的时间依赖性降低以及构建体掺入酸性内体有关，在暴露于NH4Cl后可观察到这一现象。在活的海马神经元中，SEP-CB1荧光主要局限于轴突，这与其极化的表面表达一致。因此，这些新的分子工具非常适合用于研究CB1受体转运，并且新一代在N端掺入SEP的GPCR嵌合体对于监测活细胞中细胞表面受体表达的动态变化将特别有用。
