The intracellular loops of the GB2 subunit are crucial for G-protein coupling of the heteromeric γ-aminobutyrate B receptor
The intracellular loops of the GB2 subunit are crucial for G-protein coupling of the heteromeric γ-aminobutyrate B receptor
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DOI：
10.1124/mol.62.2.343
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发表时间：
2002-08-01
影响因子：
3.6
通讯作者：
Blahos, J
中科院分区：
文献类型：
--
作者：
Havlickova, M;Prezeau, L;Blahos, J
γ-氨基丁酸酯B(GABA(B))受体是第一个被发现的G蛋白偶联受体，需要GB1和GB2两个亚基才能形成一个功能性受体。GB1胞外区(ECD)与GABA结合，而GB2含有足够的G蛋白激活的分子决定因素。研究了这两个亚基在G蛋白偶联中的确切作用。GB1和GB2在结构上与代谢性谷氨酸、钙敏感等3个GPCRs家族相关，其中第二环(I2)和第三环(I3)在G蛋白偶联中起重要作用。在这里，我们研究了GB1和GB2的I2环在GABAB受体激活Galpha蛋白能力中的作用。为此，在GB1和GB2的七螺旋结构域(HDs)之间交换了I2环，要么在野生型亚基中，要么在包含GB1的ECD和GB2的HD的嵌合亚单位GB1/2中。还检测了GB2的i3环中的一个额外突变的影响，该突变阻止了异构体受体的耦合。发现感兴趣的组合在细胞表面被正确寻址并组装成异构体。综上所述，我们的数据揭示了关于异构体GABA(B)受体G蛋白偶联的以下新信息：1)GB2 HD内的GB2的i2环是异构体GABAB受体与G蛋白偶联所必需的，而GB1的i2环则不是；2)GB1 HD内GB2的i2环的存在不足以使GB1偶联；3)GB2 HD激活Gq9蛋白，无论它与GB2还是GB1 ECD有关；4)在与两个GB2 HD结合时，每个GB2 HD都能与G蛋白偶联；最后，5)在GB2HD中使用I2、I3或两者的突变为缺乏结构域交换提供了证据，使包括I2和I3在内的区域在亚基之间能够交换。
The gamma-aminobutyrate B (GABA(B)) receptor is the first discovered G-protein-coupled receptor (GPCR) that needs two subunits, GB1 and GB2, to form a functional receptor. The GB1 extracellular domain (ECD) binds GABA, and GB2 contains enough molecular determinants for G-protein activation. The precise role of the two subunits in G-protein coupling is investigated. GB1 and GB2 are structurally related to the metabotropic glutamate, Ca2+-sensing and other family 3 GPCRs in which the second (i2) as well as the third (i3) intracellular loop play important roles in G-protein coupling. Here, the role of the i2 loops of GB1 and GB2 in the GABAB receptor ability to activate Galpha-proteins is investigated. To that aim, the i2 loops were swapped between GB1 and GB2 heptahelical domains (HDs), either in the wild-type subunits or in the chimeric subunits GB1/2 that contain the ECD of GB1 and the HD of GB2. The effect of an additional mutation within the i3 loop of GB2 that prevents coupling of the heteromeric receptor was also examined. Combinations of interest were found to be correctly addressed at the cell surface and to assemble into heteromers. Taken together our data revealed the following new information on the G-protein coupling of the heteromeric GABA(B) receptor: 1) the i2 loop of GB2 within the GB2 HD is required for the heteromeric GABAB receptor to couple to G-proteins, whereas the i2 loop of GB1 is not; 2) the presence of the i2 loop of GB2 within the GB1 HD is not sufficient to allow coupling of GB1; 3) the GB2 HD activates the Gqi9 protein whether it is associated with the GB2 or GB1 ECD; 4) in the combination with two GB2 HDs, each is able to couple to G-proteins; and finally, 5) the use of mutations in i2, i3, or both within the GB2 HD brings evidence for the absence of domain swapping enabling the exchange of region including i2 and i3 between the subunits.