Locking the dimeric GABAB G-protein-coupled receptor in its active state
Locking the dimeric GABAB G-protein-coupled receptor in its active state
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DOI：
10.1523/jneurosci.3141-03.2004
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发表时间：
2004-01-14
影响因子：
5.3
通讯作者：
Pin, JP
中科院分区：
文献类型：
--
作者：
Kniazeff, J;Saintot, PP;Pin, JP
G蛋白偶联受体(GPCRs)在中枢神经系统的细胞间通讯中发挥着重要作用。这些蛋白质在各种非活性构象和活性构象之间振荡，后者由激动剂稳定。虽然突变可以导致结构性活性，但大多数突变都会破坏非活性构象的稳定，并且没有一个会将受体锁定在活性状态。此外，已知GPCRs可以形成二聚体，但每个原型在激活过程中的作用尚不清楚。在这里，我们证明了主要抑制性神经递质--GABA(B)受体的异二聚体GPCR可以通过引入两个半胱氨酸来锁定其活性状态，这两个半胱氨酸有望形成一个二硫键，以保持GABA(B1)亚单位的结合域处于关闭状态。这种固有的活性受体不能被拮抗剂抑制，但它的正常功能、激动剂的激活和拮抗剂的抑制可以在二硫苏糖醇还原后恢复。这些数据表明，GABA(B1)结合域的关闭状态足以启动这种异二聚体受体，并首次说明GPCR可以被锁定在活性构象中。
G-protein-coupled receptors (GPCRs) play a major role in cell-cell communication in the CNS. These proteins oscillate between various inactive and active conformations, the latter being stabilized by agonists. Although mutations can lead to constitutive activity, most of these destabilize inactive conformations, and none lock the receptor in an active state. Moreover, GPCRs are known to form dimers, but the role of each protomer in the activation process remains unclear. Here, we show that the heterodimeric GPCR for the main inhibitory neurotransmitter, the GABA(B) receptor, can be locked in its active state by introducing two cysteines expected to form a disulphide bridge to maintain the binding domain of the GABA(B1) subunit in a closed form. This constitutively active receptor cannot be inhibited by antagonists, but its normal functioning, activation by agonists, and inhibition by antagonists can be restored after reduction with dithiothreitol. These data show that the closed state of the binding domain of GABA(B1) is sufficient to turn ON this heterodimeric receptor and illustrate for the first time that a GPCR can be locked in an active conformation.