Structure-based analysis of GPCR function:: Evidence for a novel pentameric assembly between the dimeric leukotriene B4 receptor BLT1 and the G-protein
Structure-based analysis of GPCR function:: Evidence for a novel pentameric assembly between the dimeric leukotriene B4 receptor BLT1 and the G-protein
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DOI：
10.1016/s0022-2836(03)00439-x
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发表时间：
2003-06-13
影响因子：
5.6
通讯作者：
Parello, J
中科院分区：
文献类型：
--
作者：
Banères, JL;Parello, J
我们在大肠杆菌中生产了人白三烯 B-4 (LTB4) 受体 BLT1 作为重组蛋白。这种去污剂溶解的受体在与 LTB4 的亲和力方面表现出两种状态：(i) 涉及受体同型二聚体 (BLT1.LTB4)(2) 的低亲和力状态 (k(a) = 7.8 x 10(8) M-1)；我们报告了第六跨膜螺旋在调节该同二聚体稳定性中发挥核心作用的证据； (ii) 受体与负载 GDP 的异源三聚体 G 蛋白 Galpha(i2)beta(1)gamma(2) 相互作用时的高亲和力状态 (k(a) = 1.3 x 10(10) M-1）。 G 蛋白与重组 BLT1 的结合诱导 Galpha 亚基进行 GDP-GTP 交换。这些结果表明，就 LTB4 的高亲和力识别、与 G 蛋白的关联以及 Galpha 的激活而言，分离的 BLT1 完全代表了体内受体。在天然复合物溶液中化学交联和中子散射后结合质谱分析，我们明确地确定只有一个 G 蛋白三聚体与受体二聚体结合形成化学计量定义的 (BLT1.LTB4)(2):Galpha(i2)beta(1)gamma(2) 五聚体组装体。这表明受体二聚化对于 LTB4 诱导信号的转导可能至关重要。 (C) 2003 Elsevier Science Ltd. 保留所有权利。
We produced human leukotriene B-4 (LTB4) receptor BLT1 as a recombinant protein in Escherichia coli. This detergent-solubilized receptor displays two states with regard to its affinity for LTB4: (i) a low-affinity state (k(a) = 7.8 x 10(8) M-1) that involves a receptor homodimer (BLT1.LTB4)(2); we report evidence for a central role of the sixth transmembrane helix in regulating the stability of this homodimer; (ii) a high-affinity state (k(a) = 1.3 x 10(10) M-1) upon interaction of the receptor with the hetero-trimeric GDP-loaded G-protein, Galpha(i2)beta(1)gamma(2). Association of the G-protein with recombinant BLT1 induces GDP-GTP exchange by the Galpha subunit. These results indicate that isolated BLT1 is fully representative of the in vivo receptor with regard to high-affinity recognition of LTB4, association with a G-protein and activation of Galpha. Using a combination of mass spectrometry after chemical cross-linking and neutron-scattering in solution with the native complex, we establish unambiguously that only one G-protein trimer binds to a receptor dimer to form the stoichiometrically defined (BLT1.LTB4)(2):Galpha(i2)beta(1)gamma(2) pentameric assembly. This suggests that receptor dimerization could be crucial to transduction of the LTB4-induced signal. (C) 2003 Elsevier Science Ltd. All rights reserved.