Development of a membrane-anchored ligand and receptor yeast two-hybrid system for ligand-receptor interaction identification.
Development of a membrane-anchored ligand and receptor yeast two-hybrid system for ligand-receptor interaction identification.
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DOI：
10.1038/srep35631
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发表时间：
2016-10-20
影响因子：
4.6
通讯作者：
Han W
中科院分区：
文献类型：
--
作者：
Li J;Gao J;Han L;Zhang Y;Guan W;Zhou L;Yu Y;Han W
识别配体和跨膜受体之间的相互作用对于理解内分泌系统至关重要。然而，目前用于此目的的方法仍然不能进行高通量筛选。本研究建立了膜锚定配体和受体酵母双杂交系统（MALAR-Y2 H）。在该方法中，细胞外配体通过嵌合跨膜结构与细胞内分裂泛素报告系统连接。同时，跨膜受体的猎物蛋白融合到分裂泛素报告系统的另一半。配体和受体的胞外相互作用可以导致酵母中泛素报告系统的功能恢复，并最终导致报告基因的表达。因此，该系统可用于检测细胞外配体与其跨膜受体之间的相互作用。为了验证该方法的有效性和普适性，我们分析了几对小鼠配体和受体之间的相互作用。结果表明，该方法的检测结果与现有的研究结果完全一致，表明MALAR-Y2 H可用于该领域，具有高精度、高效率和通用性强的特点。MALAR-Y2 H具有操作简单、高通量的特点，是研究分泌蛋白和跨膜蛋白相互作用网络的有力平台。
Identifying interactions between ligands and transmembrane receptors is crucial for understanding the endocrine system. However, the present approaches for this purpose are still not capable of high-throughput screening. In this report, a membrane-anchored ligand and receptor yeast two-hybrid (MALAR-Y2H) system was established. In the method, an extracellular ligand is linked with an intracellular split-ubiquitin reporter system via a chimeric transmembrane structure. Meanwhile, the prey proteins of transmembrane receptors are fused to the other half of the split-ubiquitin reporter system. The extracellular interaction of ligands and receptors can lead to the functional recovery of the ubiquitin reporter system in yeast, and eventually lead to the expression of report genes. Consequently, the system can be used to detect the interactions between extracellular ligands and their transmembrane receptors. To test the efficiency and universality of the method, interactions between several pairs of ligands and receptors of mouse were analyzed. The detecting results were shown to be thoroughly consistent with the present knowledge, indicating MALAR-Y2H can be utilized for such purpose with high precision, high efficiency and strong universality. The characteristics of the simple procedure and high-throughput potential make MALAR-Y2H a powerful platform to study protein-protein interaction networks between secreted proteins and transmembrane proteins.
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