Nie J, Lewis D L
Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912, USA.
J Neurosci. 2001 Nov 15;21(22):8758-64. doi: 10.1523/JNEUROSCI.21-22-08758.2001.
The CB1 cannabinoid receptor is a constitutively active receptor that can sequester G(i/o)-proteins and prevent other G(i/o)-coupled receptors from signaling (Bouaboula et al., 1997; Pan et al., 1998; Vasquez and Lewis, 1999). G-protein sequestration occurs because the population of CB1 cannabinoid receptors exists in both an inactive G-protein-precoupled RG(GDP) state and a constitutively active RG(GTP) state. We tested the hypothesis that the distal C-terminal tail acts to prevent G-protein activation. We found that truncation of the distal C-terminal tail of the CB1 receptor (CB1-417) enhanced both the constitutive activity and the ability of the receptor to sequester G-proteins. In addition, we tested the hypothesis that the conserved aspartate (D2.50) in the second transmembrane domain of the CB1 cannabinoid receptor is crucial for constitutive activity and G-protein sequestration. We found that the mutation of aspartate to asparagine (CB1-D164N) abolished G-protein sequestration and constitutive receptor activity without disrupting agonist-stimulated activity. We conclude that the CB1-D164N mutation and the C-terminal truncation shift the population of receptors in opposite directions. The CB1-D164N mutation shifts the receptor into an inactive R state upcoupled from G-proteins, whereas the C-terminal truncation (CB1-417) shifts the receptor into the active RG(GTP) state. Thus the distal C-terminal tail acts to constrain the receptor from activating G-proteins, whereas the aspartate (D2.50) in the second transmembrane domain stabilizes the receptor in both the inactive RG(GDP) state and the active R*G(GTP) state.
CB1大麻素受体是一种组成型活性受体，它可以隔离G(i/o)蛋白，并阻止其他G(i/o)偶联受体发出信号（布阿布拉等人，1997年；潘等人，1998年；瓦斯克斯和刘易斯，1999年）。G蛋白隔离的发生是因为CB1大麻素受体群体同时存在于无活性的G蛋白预偶联RG(GDP)状态和组成型活性RG(GTP)状态。我们测试了远端C末端尾巴起到阻止G蛋白激活作用的假设。我们发现，CB1受体（CB1-417）远端C末端尾巴的截断增强了组成型活性以及受体隔离G蛋白的能力。此外，我们测试了CB1大麻素受体第二个跨膜结构域中保守的天冬氨酸（D2.50）对组成型活性和G蛋白隔离至关重要的假设。我们发现，将天冬氨酸突变为天冬酰胺（CB1-D164N）消除了G蛋白隔离和组成型受体活性，而不会破坏激动剂刺激的活性。我们得出结论，CB1-D164N突变和C末端截断使受体群体向相反方向转变。CB1-D164N突变使受体转变为与G蛋白解偶联的无活性R状态，而C末端截断（CB1-417）使受体转变为活性RG(GTP)状态。因此，远端C末端尾巴起到限制受体激活G蛋白的作用，而第二个跨膜结构域中的天冬氨酸（D2.50）使受体在无活性的RG(GDP)状态和活性R*G(GTP)状态下都保持稳定。
