Transmembrane topography of nicotinic acetylcholine receptor: immunochemical tests contradict theoretical predictions based on hydrophobicity profiles.
Transmembrane topography of nicotinic acetylcholine receptor: immunochemical tests contradict theoretical predictions based on hydrophobicity profiles.
复制标题
烟碱乙酰胆碱受体的跨膜形貌：免疫化学测试与基于疏水性特征的理论预测相矛盾。
DOI：
10.1021/bi00357a052
复制
发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Lindstrom,J
中科院分区：
文献类型：
--
作者：
Ratnam,M;Nguyen,DL;Rivier,J;Sargent,PB;Lindstrom,J
摘要：在我们之前的论文[Ratnam， M., Sargent， P. B., Sarin， V., Fox， J. L., Le Nguyen， D., Rivier， J., Criado， M., and Lindstrom， J.(1986))中，我们展示了对鱼雷乙酰胆碱受体纯化亚基的肽映射研究结果，表明序列/3429-441位于受体的细胞质表面。由于这一发现与早期的受体跨膜结构理论模型相矛盾，该理论模型将0亚基的这一序列置于细胞外表面，因此我们通过更直接的方法研究了相应序列（389-408）和a亚基邻近序列的位置。我们合成了包括序列330-346，a349-364, 360-378， a379-385和389-408的肽和这些肽的较短部分。这些肽对应于一个高度免疫原性的区域，通过使用125i标记的肽作为抗原，我们能够在我们的单克隆抗体库中检测到2到6个亚基，这些亚基特异性地与这些肽结合，除了a389-408。我们从针对变性a亚基的抗血清和针对该肽的抗血清中获得了a389-408的特异性抗体。这些抗体对a389-396具有特异性。在结合实验中，针对所有这五种肽的抗体只有在囊泡渗透后才能与富含受体的膜囊泡结合，从而允许抗体进入受体的细胞质表面，这表明结合这些抗体的受体序列位于膜的胞内侧。电子显微镜使用胶体金可视化结合抗体被用来最终证明，所有这些序列暴露在受体的细胞质表面。这些结果，连同我们之前的证明，每个亚基的c端10个氨基酸暴露在细胞质表面，表明疏水结构域M4 (a409-426)，以前从亲水谱预测是跨膜的，实际上并没有跨膜。此外，这些结果表明，假定的两性跨膜结构域M5 （a364-399）也不会跨膜。因此，我们的结果表明，膜蛋白的跨膜拓扑结构不能严格地从其初级氨基酸序列的路径谱中推断出来。我们提出了一个与现有数据一致的受体亚基多肽链跨膜取向模型。针对合成肽的抗体是研究蛋白质结构的可靠探针，因为它们经常被发现与含有这些序列的天然蛋白质发生特异性交叉反应（Niman et al., 1983）。针对烟碱乙酰胆碱受体初级序列各部分对应的合成肽的抗体已被用于可视化这些抗体在富含受体的膜中的结合域（Lindstrom等人，1984;Ratnam & Lindstrom, 1984； Criado等人，1985a, b； Young等人，1985；La Rochelle等人，1985）。在这些研究中选择合成肽的理由是基于受体结构的合理理论模型（Noda et al., 1983; Claudio et al., 1983）。这项工作得到了美国国立卫生研究院（NS11323）、肌肉萎缩症协会、亚历山大·奥assis公益基金会和洛杉矶基金会对JL的资助。
Revised Manuscript Received December 10, 1985 abstract: In our preceding paper [Ratnam, M., Sargent, P. B., Sarin, V., Fox, J. L., Le Nguyen, D., Rivier, J., Criado, M., & Lindstrom, J.(1986) Biochemistry (preceding paper in this issue)], we presented results from peptide mapping studies of purified subunits of the Torpedo acetylcholine receptor which suggested that the sequence/3429-441 is on the cytoplasmic surface of the receptor. Since thisfinding contradicts earlier theoretical models of the transmembrane structure of the receptor, which placed this sequence of the 0 subunit on the extracellular surface, we investigated the location of the corresponding sequence (389-408) and adjacent sequences of the a subunit by a more direct approach. We synthesizedpeptides including the sequences «330-346, a349-364,«360-378, a379-385, and «389-408 and shorter parts of these peptides. These peptides corresponded to a highly immunogenic region, and by using 125I-labeled peptides as antigens, we were able to detect in our library of monoclonal antibodies to a subunits between two and six which bound specifically to each of these peptides, except a389-408. We obtained antibodies specific for a389-408 both from antisera against the denatured a subunit and from antisera made against the peptide. These antibodies were specific to a389-396. In binding assays, antibodies specific for all of these five peptides bound to receptor-rich membrane vesicles only after permeabilization of the vesicles to permit access of the antibodies to the cytoplasmic surface of the receptors, suggesting that the receptor sequences which bound these antibodies were located on the intracellular side of the membrane. Electron microscopy using colloidal gold to visualize the bound antibodies was used to conclusivelydemonstrate that all of these sequences are exposed on the cytoplasmic surface of the receptor. These results, along with our previous demonstration that the C-terminal 10 amino acids of each subunit are exposed on the cytoplasmic surface, show that the hydrophobic domain M4 (a409-426), previously predicted from hydropathy profiles to be transmembranous, does not, in fact, cross the membrane. Further, these results show that the putative amphipathic trans-membrane domain M5 (a364-399) also does not cross the membrane. Our results thus indicate that the transmembrane topology of a membrane protein cannot be deduced strictly from the hydropathyprofile of its primary amino acid sequence. We present a model for the transmembrane orientation of receptor subunit polypeptide chains which is consistent with current data..^^. ntibodies against synthetic peptides are reliable probes for studying protein structure as they have frequently been found to cross-react specifically with the native proteins containing these sequences (Niman et al., 1983). Antibodies against synthetic peptides corresponding to various parts of the primary sequence of the nicotinic acetylcholine receptor have been used to visualize the binding domains of these antibodiesin re-ceptor-rich membranes (Lindstrom et al., 1984; Ratnam & Lindstrom, 1984; Criado et al., 1985a, b; Young et al., 1985; La Rochelle et al., 1985). Therationale for choosingthe synthetic peptides in these studies was based on plausible theoretical models of the receptor structurededuced from the hydrophobicity profiles of the primary sequences of the sub-units of the receptor (Noda et al., 1983; Claudio et al., 1983; fThis work was supported by grants to JL from the National Institutes of Health (NS11323), the Muscular Dystrophy Association, the Alexander Onassis PublicBenefit Foundation, and the Los Angeles …