McAllister Sean D, Hurst Dow P, Barnett-Norris Judy, Lynch Diane, Reggio Patricia H, Abood Mary E
California Pacific Medical Center Research Institute, San Francisco, California 94115, USA.
J Biol Chem. 2004 Nov 12;279(46):48024-37. doi: 10.1074/jbc.M406648200. Epub 2004 Aug 23.
In this study, we tested the hypothesis that a CB(1) TMH3-4-5-6 aromatic microdomain, which includes F3.25(190), F3.36(201), W5.43(280), and W6.48(357), is centrally involved in CB(1) receptor activation, with the F3.36(201)/W6.48(357) interaction key to the maintenance of the CB(1)-inactive state. We have shown previously that when F3.36(201), W5.43(280), and W6.48(357) are individually mutated to alanine, a significant reduction in ligand binding affinity is observed in the presence of WIN 55,212-2 and SR141716A but not CP55,940 and anandamide. In the work presented here, we report a detailed functional analysis of the F3.36(201)A, F3.25(190)A, W5.43(280)A, and W6.48(357)A mutant receptors in stable cell lines created in HEK cells for agonist-stimulated guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding and GIRK1/4 channel current effects in Xenopus oocytes where the mutant proteins were expressed transiently. The F3.36(201)A mutation showed statistically significant increases in ligand-independent stimulation of GTPgammaS binding versus wild type CB(1), although basal levels for the W6.48(357)A mutant were not statistically different from wild type CB(1). F3.36(201)A demonstrated a limited activation profile in the presence of multiple agonists. In contrast, enhanced agonist activation was produced by W6.48(357)A. These results suggest that a F3.36(201)/W6.48(357)-specific contact is an important constraint for the CB(1)-inactive state that may need to break during activation. Modeling studies suggest that the F3.36(201)/W6.48(357) contact can exist in the inactive state of CB(1) and be broken in the activated state via a chi(1) rotamer switch (F3.36(201) trans, W6.48(357) g+) --> (F3.36(201) g+, W6.48(357) trans). The F3.36(201)/W6.48(357) interaction therefore may represent a "toggle switch" for activation of CB(1).
在本研究中，我们验证了以下假设：包含F3.25(190)、F3.36(201)、W5.43(280)和W6.48(357)的CB(1)跨膜螺旋3-4-5-6芳香微区在CB(1)受体激活过程中起核心作用，其中F3.36(201)/W6.48(357)相互作用是维持CB(1)非活性状态的关键。我们之前已经表明，当F3.36(201)、W5.43(280)和W6.48(357)分别突变为丙氨酸时，在存在WIN 55,212-2和SR141716A的情况下，配体结合亲和力显著降低，但在存在CP55,940和花生四烯乙醇胺时未出现这种情况。在本文所呈现的工作中，我们报告了对在HEK细胞中构建的稳定细胞系中的F3.36(201)A、F3.25(190)A、W5.43(280)A和W6.48(357)A突变受体进行的详细功能分析，分析内容包括激动剂刺激的鸟苷5'-3-O-(硫代)三磷酸(GTPγS)结合以及在非洲爪蟾卵母细胞中瞬时表达突变蛋白时对GIRK1/4通道电流的影响。与野生型CB(1)相比，F3.36(201)A突变导致GTPγS结合的非配体依赖性刺激有统计学意义的增加，尽管W6.48(357)A突变体的基础水平与野生型CB(1)无统计学差异。F3.36(201)A在存在多种激动剂时表现出有限的激活模式。相比之下，W6.48(357)A产生了增强的激动剂激活作用。这些结果表明，F3.36(201)/W6.48(357)特异性接触是CB(1)非活性状态的一个重要限制因素，在激活过程中可能需要打破。模型研究表明，F3.36(201)/W6.48(357)接触可以存在于CB(1)的非活性状态，并在激活状态下通过一个χ(1)旋转异构体开关(F3.36(201)反式，W6.48(357) g+) --> (F3.36(201) g+，W6.48(357)反式)而断裂。因此，F3.36(201)/W6.48(357)相互作用可能代表了CB(1)激活的一个“拨动开关”。
