Truncated MHC class II cytoplasmic and transmembrane domains: effect on plasma membrane expression.
Truncated MHC class II cytoplasmic and transmembrane domains: effect on plasma membrane expression.
复制标题
截短的 MHC II 类细胞质和跨膜结构域：对质膜表达的影响。
DOI：
10.1016/0161-5890(94)00165-w
复制
发表时间：
1995
影响因子：
3.6
通讯作者：
Wade,TK
中科院分区：
文献类型：
--
作者：
Wade,WF;Khrebtukova,I;Schreiber,KL;McKean,DJ;Wade,TK
主要组织相容性复合体(MHC)II类分子的质膜表达是抗原提呈细胞与T淋巴细胞相互作用所必需的。由α链和β链组成的II类分子是高度多态的，这有利于它们与抗原和银特异性T细胞的相互作用。最近，我们重点研究了II类分子的跨膜(TM)和细胞质(Cy)结构域，试图了解它们可能的功能。使用定点突变在IAK的α或β链的TM和Cy结构域中创建截断，或两者都截断，我们已经确定了I-Ak分子高效表面表达的一些序列要求。未在PM表达的AKβTM突变体不会运输通过内侧高尔基体，内切糖苷酶-H处理的免疫沉淀物的原位染色和蛋白质印迹分析表明。TM第二类突变体缺乏转运并不是由于缺乏与不变链(II)的联系。在两条链上都有Cy结构域截断的II类分子不能有效地转运到PM，并且也有一定比例的分子对内糖苷酶-H敏感。在表达Cy结构域截短的II类分子的细胞中进行的II类原位染色显示，与表达野生型II类分子的转染体的染色相比，显示了一种离散的囊泡模式。免疫荧光数据和内切糖苷酶-H数据表明，Cy结构域是有效运输所必需的。用一组I-AK构象特异性抗体进行的免疫沉淀研究表明，截断两个链的Cy结构域并不影响第II类蛋白的构象。然而，进一步截断AKβ链进入TM域，导致缺乏通过内质网/内侧高尔基体的运输，并降低了突变的第II类蛋白在细胞内的表达(稳定性)。确实相关的TM突变体的α/β链结合了一组构象敏感抗体，除了一个，3F12。我们的结论是，MHC II类分子的α和β链的Cy结构域以及AKβ链的TM结构域序列是有效表达II类PM所必需的。TM突变体缺乏PM表达的原因可能是无法获得3F12特异性表位所定义的具有运输功能的构象，而截断AKαCy结构域是为了防止II链的ER保留序列的完全掩蔽。
Plasma membrane (PM) expression of major histocompatibility complex (MHC) class II molecule is required for the interaction of antigen (Ag) presenting cells and T lymphocytes. Class II molecules composed of an alpha and a beta chain are highly polymorphic which facilitates their interaction with Ag and Ag-specific T cells. Recently, we have focused on the less polymorphic sequences of class II molecules, the transmembrane (TM) and cytoplasmic (Cy) domains, in an attempt to understand what their function might be. Using site-directed mutagenesis to create truncations in the TM and Cy domains of IAk's alpha or beta chain, or both, we have identified some of the sequence requirements for efficient surface expression of I-Akmolecules. AkβTM mutants that are not expressed at the PM are not transported past the medial-Golgi as indicated by in situ staining and Western blot analysis of endoglycosidase-H-treated immunoprecipitates. The lack of transport of TM class II mutants is not due to lack of association with the invariant chain (Ii). Class II molecules with Cy domain truncations in both chains are not efficiently transported to the PM and also have a percentage of molecules that are endoglycosidase-H sensitive. In situ staining of class II in cells expressing Cy domain truncated class II molecules revealed a discrete vesicular pattern compared to the staining of transfectants that expressed wildtype class II molecules. The immunofluorescence data along with the endoglycosidase-H data indicate the Cy domains are required for efficient transport. Immunoprecipitation studies using a panel of I-Akconformation-specific antibodies revealed that the truncation of the Cy domains of both chains did not effect the conformation of class II. However, further truncation of the Akβ chain into the TM domain resulted in lack of transport past the ER/medial-Golgi and diminished expression (stability) of mutant class II proteins within the cells. The alpha/beta chains of the TM mutants that did associate bound a panel of conformation sensitive antibodies except for one, 3F12. We conclude that the Cy domain of the alpha and beta chains of MHC class II, as well as sequences in the TM domains of the Akβ chain are required for efficient class II PM expression. The reason for the lack of PM expression of TM mutants may be the inability to access a transport competent conformation as defined by the 3F12-specific epitope, while truncation of the AkαCy domains is proposed to prevent complete masking of the ER retention sequence of the Ii chain.