Liu Jianfeng, Maurel Damien, Etzol Sébastien, Brabet Isabelle, Ansanay Hervé, Pin Jean-Philippe, Rondard Philippe
Department of Molecular Pharmacology, Laboratory of Functional Genomics, CNRS UPR2580, 141, rue de la Cardonille, F-34094 Montpellier, Cedex 5, France.
J Biol Chem. 2004 Apr 16;279(16):15824-30. doi: 10.1074/jbc.M313639200. Epub 2004 Jan 21.
The gamma-aminobutyric acid type B (GABAB) receptor is an allosteric complex made of two subunits, GABAB1 (GB1) and GABAB2 (GB2). Both subunits are composed of an extracellular Venus flytrap domain (VFT) and a heptahelical domain (HD). GB1 binds GABA, and GB2 plays a major role in G-protein activation as well as in the high agonist affinity state of GB1. How agonist affinity in GB1 is regulated in the receptor remains unknown. Here, we demonstrate that GB2 VFT is a major molecular determinant involved in this control. We show that isolated versions of GB1 and GB2 VFTs in the absence of the HD and C-terminal tail can form hetero-oligomers as shown by time-resolved fluorescence resonance energy transfer (based on HTRF technology). GB2 VFT and its association with GB1 VFT controlled agonist affinity in GB1 in two ways. First, GB2 VFT exerted a direct action on GB1 VFT, as it slightly increased agonist affinity in isolated GB1 VFT. Second and most importantly, GB2 VFT prevented inhibitory interaction between the two main domains (VFT and HD) of GB1. According to this model, we propose that GB1 HD prevents the possible natural closure of GB1 VFT. In contrast, GB2 VFT facilitates this closure. Finally, such inhibitory contacts between HD and VFT in GB1 could be similar to those important to maintain the inactive state of the receptor.
γ-氨基丁酸B型（GABAB）受体是一种由两个亚基，即GABAB1（GB1）和GABAB2（GB2）组成的变构复合物。两个亚基均由一个细胞外捕蝇草结构域（VFT）和一个七螺旋结构域（HD）组成。GB1结合γ-氨基丁酸（GABA），而GB2在G蛋白激活以及GB1的高激动剂亲和力状态中起主要作用。受体中GB1的激动剂亲和力是如何调节的仍不清楚。在此，我们证明GB2的VFT是参与这种调控的主要分子决定因素。我们表明，在没有HD和C末端尾巴的情况下，分离的GB1和GB2的VFT版本可以形成异源寡聚体，时间分辨荧光共振能量转移（基于HTRF技术）显示了这一点。GB2的VFT及其与GB1的VFT的结合以两种方式控制GB1中的激动剂亲和力。首先，GB2的VFT对GB1的VFT施加直接作用，因为它略微增加了分离的GB1的VFT中的激动剂亲和力。其次，也是最重要的，GB2的VFT阻止了GB1的两个主要结构域（VFT和HD）之间的抑制性相互作用。根据这个模型，我们提出GB1的HD阻止了GB1的VFT可能的自然关闭。相反，GB2的VFT促进了这种关闭。最后，GB1中HD和VFT之间的这种抑制性接触可能类似于维持受体无活性状态所重要的那些接触。
