BINDING OF MONOCLONAL-ANTIBODIES AGAINST THE CARBOXYL-TERMINAL SEGMENT OF THE NICOTINIC RECEPTOR DELTA-SUBUNIT SUGGESTS AN UNUSUAL TRANSMEMBRANE DISPOSITION OF THIS SEQUENCE REGION
BINDING OF MONOCLONAL-ANTIBODIES AGAINST THE CARBOXYL-TERMINAL SEGMENT OF THE NICOTINIC RECEPTOR DELTA-SUBUNIT SUGGESTS AN UNUSUAL TRANSMEMBRANE DISPOSITION OF THIS SEQUENCE REGION
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DOI：
10.1021/bi00020a013
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发表时间：
1995-05-23
期刊：
影响因子：
2.9
通讯作者：
CONTIFINE, BM
中科院分区：
文献类型：
--
作者：
LEI, SJ;OKITA, DK;CONTIFINE, BM
用电鳐烟碱乙酰胆碱受体（AChR）δ亚基羧基端特异性单克隆抗体（mAb）或合成的δ亚基羧基端序列（C delta-mAb）免疫小鼠，通过免疫电镜观察电鳐富含AChR的突触后膜片段，确定其表位的跨膜分布。一些C delta-mAbs仅识别膜的细胞质侧，一些在类似程度上识别两侧，而其他的大多数但不完全地结合到细胞质侧。C δ-单克隆抗体与膜的结合被含有δ亚基羧基末端区域的合成肽特异性阻断。控制抗乙酰胆碱受体单克隆抗体的α或δ亚基，其表位具有已知的跨膜拓扑结构，独特地认识到预期的突触后膜的一面。使用序列delta 481 - 501的单残基取代的肽类似物鉴定参与C delta-mAb结合的残基。所有C δ-单克隆抗体识别AChR δ亚基羧基末端相同序列片段δ 485 - 493内的表位。这些结果表明，δ亚基的AChR可能有替代的构象，导致暴露的细胞外或细胞质表面上的相同的序列区域。几个Pro残基存在于该区域中。它们中的一个或多个的可选顺式或反式构象可能导致δ亚基的羧基末端序列的不同折叠模式，如对病毒蛋白所述[Liddington，R. C.的方法，Yan，Y.，Moulai，J.，Sahli河，Benjamin，T. L.，和Harrison，S. C.（1991）Nature 354，278 - 284]。
Monoclonal antibodies (mAbs) specific for the carboxyl terminal region of the delta subunit of Torpedo nicotinic acetylcholine receptor (AChR), derived from mice immunized with AChR or a synthetic carboxyl terminal sequence of the delta subunit (C delta-mAbs), were used to determine the transmembrane disposition of their epitope(s) by immunoelectron microscopy, using AChR-rich postsynaptic membrane fragments from Torpedo electroplax. Some C delta-mAbs recognized only the cytoplasmic side of the membranes, some both sides to a similar extent, and others bound mostly, but not exclusively, to the cytoplasmic side. Binding of C delta-mAbs to the membranes was specifically blocked by synthetic peptides containing the carboxyl terminal region of the delta subunit. Control anti-AChR mAbs specific for the alpha or the delta subunits, whose epitopes have known transmembrane topology, uniquely recognized the expected side of the postsynaptic membrane. Residues involved in C delta-mAb binding were identified using single residue substituted peptide analogues of the sequence delta 481-501. All C delta-mAbs recognized epitopes within the same sequence segment, delta 485-493, at the carboxyl terminal of the AChR delta subunit. These results suggest that the delta subunit of the AChR might have alternative conformations, leading to exposure of the same sequence region on the extracellular or the cytoplasmic surface. Several Pro residues are present in this region. The alternative cis or trans conformation of one or more of them might result in different folding patterns of the carboxyl terminal sequence of the delta subunit, as described for a viral protein [Liddington, R. C., Yan, Y., Moulai, J., Sahli, R., Benjamin, T. L., and Harrison, S. C. (1991) Nature 354, 278-284].