Purification and identification of G protein-coupled receptor protein complexes under native conditions
Purification and identification of G protein-coupled receptor protein complexes under native conditions
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DOI：
10.1074/mcp.m600298-mcp200
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发表时间：
2007-05-01
影响因子：
7
通讯作者：
Jockers, Ralf
中科院分区：
文献类型：
--
作者：
Daulat, Avais M.;Maurice, Pascal;Jockers, Ralf
G蛋白偶联受体（gpcr）是最大的膜受体家族，具有重要的治疗意义。gpcr相关蛋白的鉴定是更好地了解这些受体的重要一步。然而，目前的方法并不令人满意，因为只有分离的受体结构域（细胞内环或羧基末端尾部）才能用作“诱饵”。我们在此报告了一种基于串联亲和纯化与质谱相结合的方法，该方法克服了这些限制，因为整个受体用于鉴定活哺乳动物细胞中形成的蛋白质复合物。选择人MT1和MT2褪黑激素受体作为模型gpcr。这两种受体在其羧基末端用串联亲和纯化标签进行标记，并在人胚胎肾293细胞中表达。优化了受体的增溶和纯化条件。该方法通过Gi蛋白的共同纯化得到了验证，Gi蛋白是众所周知的GPCR相互作用伙伴，但很难用目前的蛋白质相互作用测定方法进行鉴定。发现了几个新的和功能相关的MT1-和mt2相关蛋白；其中一些是两种受体共有的，而另一些则是每种亚型所特有的。综上所述，我们的方案首次允许在自然条件下纯化gpcr相关蛋白，其数量适合质谱分析。
G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. The identification of GPCR-associated proteins is an important step toward a better understanding of these receptors. However, current methods are not satisfying as only isolated receptor domains (intracellular loops or carboxyl-terminal tails) can be used as "bait." We report here a method based on tandem affinity purification coupled to mass spectrometry that overcomes these limitations as the entire receptor is used to identify protein complexes formed in living mammalian cells. The human MT1 and MT2 melatonin receptors were chosen as model GPCRs. Both receptors were tagged with the tandem affinity purification tag at their carboxyl-terminal tails and expressed in human embryonic kidney 293 cells. Receptor solubilization and purification conditions were optimized. The method was validated by the co-purification of Gi proteins, which are well known GPCR interaction partners but which are difficult to identify with current protein-protein interaction assays. Several new and functionally relevant MT1- and MT2-associated proteins were identified; some of them were common to both receptors, and others were specific for each subtype. Taken together, our protocol allowed for the first time the purification of GPCR-associated proteins under native conditions in quantities suitable for mass spectrometry analysis.