Gorrie G H, Vallis Y, Stephenson A, Whitfield J, Browning B, Smart T G, Moss S J
Medical Research Council Laboratory of Molecular Cell Biology and Department of Pharmacology, University College, London WC1E 6BT, United Kingdom.
J Neurosci. 1997 Sep 1;17(17):6587-96. doi: 10.1523/JNEUROSCI.17-17-06587.1997.
GABAA receptors are believed to be pentameric hetero-oligomers, which can be constructed from six subunits (alpha, beta, gamma, delta, epsilon, and rho) with multiple members, generating a large potential for receptor heterogeneity. The mechanisms used by neurons to control the assembly of these receptors, however, remain unresolved. Using Semliki Forest virus expression we have analyzed the assembly of 9E10 epitope-tagged receptors comprising alpha1 and beta2 subunits in baby hamster kidney cells and cultured superior cervical ganglia neurons. Homomeric subunits were retained within the endoplasmic reticulum, whereas heteromeric receptors were able to access the cell surface in both cell types. Sucrose density gradient fractionation demonstrated that the homomeric subunits were incapable of oligomerization, exhibiting 5 S sedimentation coefficients. Pulse-chase analysis revealed that homomers were degraded, with half-lives of approximately 2 hr for both the alpha1((9E10)) and beta2((9E10)) subunits. Oligomerization of the alpha1((9E10)) and beta2((9E10)) subunits was evident, as demonstrated by the formation of a stable 9 S complex, but this process seemed inefficient. Interestingly the appearance of cell surface receptors was slow, lagging up to 6 hr after the formation of the 9 S receptor complex. Using metabolic labeling a ratio of alpha1((9E10)):beta2((9E10)) of 1:1 was found in this 9 S fraction. Together the results suggest that GABAA receptor assembly occurs by similar mechanisms in both cell types, with retention in the endoplasmic reticulum featuring as a major control mechanism to prevent unassembled receptor subunits accessing the cell surface.
γ-氨基丁酸A型（GABAA）受体被认为是五聚体异源寡聚体，它可由六个亚基（α、β、γ、δ、ε和ρ）构建而成，每个亚基都有多个成员，这使得受体具有极大的异质性潜力。然而，神经元控制这些受体组装的机制仍未得到解决。利用辛德毕斯病毒表达系统，我们分析了在幼仓鼠肾细胞和培养中的颈上神经节神经元中，包含α1和β2亚基的9E10表位标记受体的组装情况。同型亚基保留在内质网中，而异源受体在这两种细胞类型中都能够到达细胞表面。蔗糖密度梯度分级分离表明，同型亚基无法进行寡聚化，沉降系数为5S。脉冲追踪分析显示，同型亚基会被降解，α1（（9E10））和β2（（9E10））亚基的半衰期约为2小时。α1（（9E10））和β2（（9E10））亚基的寡聚化很明显，表现为形成了稳定的9S复合物，但这个过程似乎效率不高。有趣的是，细胞表面受体的出现很缓慢，在9S受体复合物形成后延迟长达6小时。通过代谢标记发现在这个9S组分中α1（（9E10））:β2（（9E10））的比例为1:1。这些结果共同表明，GABAA受体在两种细胞类型中的组装机制相似，以内质网滞留作为主要的控制机制，以防止未组装的受体亚基到达细胞表面。
