参与GABAB2亚基对GABAB受体激动剂亲和力变构控制的分子决定簇
Molecular determinants involved in the allosteric control of agonist affinity in the GABAB receptor by the GABAB2 subunit
摘要：
γ-氨基丁酸B型（GABAB）受体是由两个亚基GABAB 1（GB 1）和GABAB 2（GB 2）组成的变构复合物。这两个亚基均由细胞外捕蝇草结构域（VFT）和七螺旋结构域（HD）组成。GB 1结合GABA，GB 2在G蛋白活化以及GB 1的高激动剂亲和力状态中起主要作用。GB 1中的激动剂亲和力如何在受体中调节仍然未知。在这里，我们表明，GB 2 VFT是一个主要的分子决定因素参与这种控制。我们发现，在没有HD和C-末端尾部的情况下，GB 1和GB 2 VFT的分离版本可以形成异源寡聚体，如时间分辨荧光共振能量转移（基于HTRF技术）所示。GB 2 VFT及其与GB 1 VFT的关联以两种方式控制GB 1中的激动剂亲和力。首先，GB 2 VFT对GB 1 VFT产生直接作用，因为它略微增加了分离的GB 1 VFT中的激动剂亲和力。第二，也是最重要的，GB 2 VFT阻止了GB 1的两个主要结构域（VFT和HD）之间的抑制性相互作用。根据这一模型，我们建议，GB 1 HD阻止可能的自然关闭GB 1 VFT。相比之下，GB 2 VFT有助于这种闭合。最后，在GB 1中HD和VFT之间的这种抑制性接触可能与维持受体非活性状态的重要接触相似。
英文摘要：
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.
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