依赖的PDZ-CFTR的大分子信号复合物的体外分析
Summary
囊性纤维化跨膜电导调节器（CFTR），上皮细胞的氯离子通道，已与多种蛋白质相互作用和调节重要的细胞过程的CFTR序序介导的相互作用，其中已经有据可查。这个协议描述我们开发组装的PDZ-依赖的CFTR大分子信号复合物的方法<em>在体外</em>。
Abstract
囊性纤维化跨膜电导调节器（CFTR），主要位于上皮细胞的顶膜的氯离子通道，起着至关重要的作用，在跨上流体稳态1-3。 CFTR基因有牵连的两个主要疾病：囊性纤维化（CF）4和第5分泌性腹泻。在CF的CFTR氯离子通道的功能活动，合成或降低。这种疾病会影响美国的约1 6 2500白种人。过度CFTR的活动也被牵连毒素诱导的分泌性腹泻（如霍乱毒素和热稳定大肠杆菌肠毒素）刺激cAMP或cGMP生产在肠道内的7例。
越来越多的证据表明CFTR和其他蛋白质的数目越来越多，包括运输，离子通道，受体，激酶，磷酸酶，信号之间的物理和功能相互作用的存在ING分子和细胞骨架的元素，CFTR和其结合蛋白之间相互作用已被证明要严格调节在体外和体内8-19 CFTR介导的跨膜离子转运。在这个协议中，我们只注重方法，援助在CFTR的羧基末端，它拥有一个蛋白质结合基序之间的相互作用的研究[简称为PSD95/Dlg1/ZO-1（序）序]，和PDZ结构域所指的脚手架蛋白，其中包含一个特定的绑定模块组。到目前为止，几个不同的PDZ骨架蛋白已绑定到CFTR的羧基末端与各种亲缘关系，如NHERF1，NHERF2，PDZK1，PDZK2，CAL（CFTR的相关配体），Shank2，把握20-27。内CFTR的PDZ结构图案是公认的PDZ骨架蛋白是在过去四年在C端（即1477-DTRL-1480在人类的CFTR）20个氨基酸。有趣的是，CFTR的，可以绑定多个PDZ结构域都NHERFs和PDZK1，尽管有不同的亲和力22。这方面的CFTR的具有约束力的多价已被证明是功能性的意义，这表明，PDZ结构骨架蛋白可能促进CFTR的形成大分子信号为特定/选择性和高效率的信号复合物在细胞16-18。
已开发多个生化分析研究，CFTR涉及蛋白质的相互作用，如免疫共沉淀，下拉法，成对结合法，比色法成对结合实验，和复杂的大分子组装检测16-19,28,29 。在这里，我们重点对1的PDZ基序依赖CFTR含大分子体外 ，这是我们的实验室研究蛋白质或域的域的相互作用，涉及的CFTR 16-19,28,29广泛用于复杂装配的详细程序。
Protocol
1。标记细菌中的重组融合蛋白表达和纯化放大定义CFTR的一览表2，MRP2的，MRP4，β2受体，和NHERFs（全长或PDZ1过或PDZ2域地区的C-尾（最后50-100个氨基酸，包含在C-末端的PDZ结构图案） ）通过PCR方法。 PCR产物克隆到pGEX4T-1载体（如GST-NHERFs的，GST-MRP4 CT），GST融合蛋白的MBP融合蛋白的质粒pMAL-C2载体（如的MBP-β2受体的CT，MBP-CFTR的CT ），pET30-S融合蛋白（如他 – S-CFT…
Discussion
在这个协议中，我们展示了一个方法， 在体外装配，检测包含使用纯化蛋白（或蛋白质碎片）和/或细胞裂解大分子信号复合物的CFTR报告以前16-19,29,30。为了达到最佳效果临界点以下，在筹备过程中，需要特别注意：
- 重要的是，洗脱缓冲液的pH值调整到8.0后加入还原型谷胱甘肽的纯化GST融合蛋白，如步骤1）中所述时。此外还原型谷胱甘肽后，pH值可低到3.0。如果PH值未调整?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们的工作一直支持由美国心脏协会（大中华区东南亚联盟）开始赠款援助0765185B，艾尔莎美国帕迪基金会的研究经费，韦恩州立大学校内的启动基金和心血管研究所伊希斯倡议的奖助学金。这种方法在体外 CFTR的大分子复杂的装配最初率先博士AP那仁（田纳西大学健康科学中心）。
Materials
| Name of the reagent | Company | Catalog number | Comments |
| pGEX4T-1 vector | GE Healthcare | 28-9545-49 | formerly Amersham Biosciences |
| pMAL-C2 vector | New England BioLabs | ||
| pET30 vector | EMD Chemicals | 69077-3 | formerly Novagen |
| Glutathione agarose beads | BD Biosciences | 554780 | |
| Amylose resin | New England BioLabs | E8021S | |
| Talon beads | Clontech | 635501 | |
| reduced glutathione | BD Biosciences | 554782 | |
| imidazole | Fisher | BP305-50 | |
| maltose | Fisher | BP684-500 | |
| S-protein agarose | EMD Chemicals | 69704-3 | formerly Novagen |
| Anti-Flag HRP | Sigma | A8592 | |
| Anti-CFTR IgG | Custom-made | R1104 | mAb recognizing CFTR epitope at a.a. 722-734 |
| Anti-MRP2 IgG | Chemicon International | MAB4148 | Now a part of Millipore |
Table 2. Specific reagents and equipment.
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Tags
In Vitro AnalysisPDZ-dependent CFTR Macromolecular Signaling ComplexesCFTRChloride ChannelTransepithelial Fluid HomeostasisCystic FibrosisSecretory DiarrheaCFTR Cl- ChannelPhysical InteractionsFunctional InteractionsTransportersIon ChannelsReceptorsKinasesPhosphatasesSignaling MoleculesCytoskeletal ElementsCFTR Carboxyl Terminal TailPSD95/Dlg1/ZO-1 (PDZ) MotifScaffold Proteins
Cite This Article
Wu, Y., Wang, S., Li, C. In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes. J. Vis. Exp. (66), e4091, doi:10.3791/4091 (2012).