Cell surface localization of α3β4 nicotinic acetylcholine receptors is regulated by N-cadherin homotypic binding and actomyosin contractility.
Cell surface localization of α3β4 nicotinic acetylcholine receptors is regulated by N-cadherin homotypic binding and actomyosin contractility.
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DOI：
10.1371/journal.pone.0062435
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Brusés JL
中科院分区：
文献类型：
--
作者：
Brusés JL
神经元烟碱乙酰胆碱受体（nAChR）在整个中枢和外周神经系统中广泛表达，并且定位于细胞膜的突触和突触外位点。然而，调控烟碱受体在细胞膜不同区域定位的机制还不清楚。N-钙粘蛋白是一种细胞粘附分子，介导并置细胞膜之间的同型结合，并通过与胞质结构域的蛋白质相互作用调节肌动蛋白细胞骨架。在突触接触，N-钙粘蛋白通常是本地化邻近的活动区和突触后密度，这表明N-钙粘蛋白有助于组装的突触复合体。为了检测N-钙粘蛋白同型结合是否调节烟碱受体的细胞表面定位，本研究使用了在中国仓鼠卵巢细胞中异源表达的N-钙粘蛋白和α 3 β 4 nAChR亚基C-末端融合至myc标签表位。通过共聚焦显微镜分析α 3 β 4 nAChR在细胞-细胞接触处和在无接触细胞膜处的表达水平。发现α 3 β 4 nAChR分布于缺乏N-钙粘蛋白的接触细胞的整个表面。相比之下，N-钙粘蛋白介导的细胞-细胞接触缺乏α 3 β 4 nAChR。由缺乏β-连环蛋白结合区或整个胞质结构域的N-钙粘蛋白缺失蛋白介导的细胞-细胞接触显示α 3 β 4 nAChRs表达的控制水平。用latrunculin A和细胞松弛素D抑制肌动蛋白聚合并不影响N-钙粘蛋白介导的细胞-细胞接触中α 3 β 4 nAChRs的定位。然而，用Rho相关激酶抑制剂Y27632处理导致N-钙粘蛋白介导的细胞-细胞接触中α 3 β 4 nAChR水平显著增加。极化Caco-2细胞中α 3 β 4 nAChRs定位的分析显示，在顶端细胞膜上特异性表达，并与顶端F-肌动蛋白和肌动蛋白成核剂Arp3共定位。这些结果表明，N-钙粘蛋白同型结合下游的肌动球蛋白收缩性调节α 3 β 4 nAChR的细胞表面定位，可能是通过与特定的F-肌动蛋白库相互作用。
Neuronal nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout the central and peripheral nervous system and are localized at synaptic and extrasynaptic sites of the cell membrane. However, the mechanisms regulating the localization of nicotinic receptors in distinct domains of the cell membrane are not well understood. N-cadherin is a cell adhesion molecule that mediates homotypic binding between apposed cell membranes and regulates the actin cytoskeleton through protein interactions with the cytoplasmic domain. At synaptic contacts, N-cadherin is commonly localized adjacent to the active zone and the postsynaptic density, suggesting that N-cadherin contributes to the assembly of the synaptic complex. To examine whether N-cadherin homotypic binding regulates the cell surface localization of nicotinic receptors, this study used heterologous expression of N-cadherin and α3β4 nAChR subunits C-terminally fused to a myc-tag epitope in Chinese hamster ovary cells. Expression levels of α3β4 nAChRs at cell-cell contacts and at contact-free cell membrane were analyzed by confocal microscopy. α3β4 nAChRs were found distributed over the entire surface of contacting cells lacking N-cadherin. In contrast, N-cadherin-mediated cell-cell contacts were devoid of α3β4 nAChRs. Cell-cell contacts mediated by N-cadherin-deleted proteins lacking the β-catenin binding region or the entire cytoplasmic domain showed control levels of α3β4 nAChRs expression. Inhibition of actin polymerization with latrunculin A and cytochalasin D did not affect α3β4 nAChRs localization within N-cadherin-mediated cell-cell contacts. However, treatment with the Rho associated kinase inhibitor Y27632 resulted in a significant increase in α3β4 nAChR levels within N-cadherin-mediated cell-cell contacts. Analysis of α3β4 nAChRs localization in polarized Caco-2 cells showed specific expression on the apical cell membrane and colocalization with apical F-actin and the actin nucleator Arp3. These results indicate that actomyosin contractility downstream of N-cadherin homotypic binding regulates the cell surface localization of α3β4 nAChRs presumably through interactions with a particular pool of F-actin.
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