Identification of the intracellular region of the leukotriene B4 receptor type 1 that is specifically involved in Gi activation
Identification of the intracellular region of the leukotriene B4 receptor type 1 that is specifically involved in Gi activation
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DOI：
10.1074/jbc.m610540200
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发表时间：
2007-02-09
影响因子：
4.8
通讯作者：
Shimizu, Takao
中科院分区：
文献类型：
--
作者：
Kuniyeda, Kanako;Okuno, Toshiaki;Shimizu, Takao
许多G蛋白偶联受体可以激活多个G蛋白亚家族成员。白三烯B-4受体1（BLT 1）是一种与白三烯B-4具有高亲和力的G蛋白偶联受体，在机体防御、炎症和免疫中发挥作用。先前的研究已经表明，BLT 1利用不同的G蛋白（G（i）家族和G（16）G蛋白）介导不同的细胞事件，并且BLT 1的胞质尾区的截短不会损害Gi和G16蛋白的活化。为了确定BLT 1对G蛋白偶联的响应区域，我们对其细胞内环进行了广泛的诱变研究。通过G（i）-依赖性鸟苷5 '-（γ-硫代）三磷酸（GTP γ S）结合和G（16）-依赖性磷酸肌醇积累试验判断，发现BLT 1的三个细胞内环（i1、i2和i3）对Gi和G16偶联都很重要。在i3氨基末端具有突变的i3-1突变体表现出极大降低的GTP γ S结合，但由白三烯B刺激触发的完整的肌醇磷酸积累。这些结果表明，i3-1区是必需的，只有Gi激活。此外，在i3-1突变体中，Gi激活的缺陷伴随着野生型受体所见的高亲和力白三烯B4结合状态的丧失。基于牛视紫红质的结构构建的BLT 1的三维模型表明，i3-1区域可能由跨膜螺旋V的胞质末端组成，该跨膜螺旋V将螺旋突出到胞质中。从突变的研究和三维建模，我们提出，延伸的细胞质螺旋连接到跨膜螺旋V的BLT 1可能是一个关键区域选择性激活Gi蛋白。
Many G-protein-coupled receptors can activate more than one G-protein subfamily member. Leukotriene B-4 receptor type 1 (BLT1) is a high affinity G-protein-coupled receptors for leukotriene B-4 functioning in host defense, inflammation, and immunity. Previous studies have shown that BLT1 utilizes different G-proteins (the G(i) family and G(16) G-proteins) in mediating diverse cellular events and that truncation of the cytoplasmic tail of BLT1 does not impair activation of Gi and G16 proteins. To determine responsive regions of BLT1 for G-protein coupling, we performed an extensive mutagenesis study of its intracellular loops. Three intracellular loops (i1, i2, and i3) of BLT1 were found to be important for both Gi and G16 coupling, as judged by G(i)-dependent guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) binding and G(16)-dependent inositol phosphate accumulation assays. The i3-1 mutant, with a mutation at the i3 amino terminus, exhibited greatly reduced GTP gamma S binding but intact inositol phosphate accumulation triggered by leukotriene B, stimulation. These results suggest that the i3-1 region is required only for Gi activation. Moreover, in the i3-1 mutant, the deficiency in Gi activation was accompanied by a loss of the high affinity leukotriene B4 binding state seen with the wild type receptor. A three-dimensional model of BLT1 constructed based on the structure of bovine rhodopsin suggests that the i3-1 region may consist of the cytoplasmic end of the transmembrane helix V, which protrudes the helix into the cytoplasm. From mutational studies and three-dimensional modeling, we propose that the extended cytoplasmic helix connected to the transmembrane helix V of BLT1 might be a key region for selective activation of Gi proteins.