Woo Anthony Yiu-Ho, Jozwiak Krzysztof, Toll Lawrence, Tanga Mary J, Kozocas Joseph A, Jimenez Lucita, Huang Ying, Song Ying, Plazinska Anita, Pajak Karolina, Paul Rajib K, Bernier Michel, Wainer Irving W, Xiao Rui-Ping
From the Key Laboratory of Chemical Genomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen 518055, China, the Institute of Molecular Medicine, Centers for Life Sciences, Peking University, Beijing 100871, China, the Laboratory of Cardiovascular Science and
the Department of Chemistry, Medical University of Lublin, Lublin, Poland.
J Biol Chem. 2014 Jul 11;289(28):19351-63. doi: 10.1074/jbc.M114.558882. Epub 2014 May 15.
Interaction of a given G protein-coupled receptor to multiple different G proteins is a widespread phenomenon. For instance, β2-adrenoceptor (β2-AR) couples dually to Gs and Gi proteins. Previous studies have shown that cAMP-dependent protein kinase (PKA)-mediated phosphorylation of β2-AR causes a switch in receptor coupling from Gs to Gi. More recent studies have demonstrated that phosphorylation of β2-AR by G protein-coupled receptor kinases, particularly GRK2, markedly enhances the Gi coupling. We have previously shown that although most β2-AR agonists cause both Gs and Gi activation, (R,R')-fenoterol preferentially activates β2-AR-Gs signaling. However, the structural basis for this functional selectivity remains elusive. Here, using docking simulation and site-directed mutagenesis, we defined Tyr-308 as the key amino acid residue on β2-AR essential for Gs-biased signaling. Following stimulation with a β2-AR-Gs-biased agonist (R,R')-4'-aminofenoterol, the Gi disruptor pertussis toxin produced no effects on the receptor-mediated ERK phosphorylation in HEK293 cells nor on the contractile response in cardiomyocytes expressing the wild-type β2-AR. Interestingly, Y308F substitution on β2-AR enabled (R,R')-4'-aminofenoterol to activate Gi and to produce these responses in a pertussis toxin-sensitive manner without altering β2-AR phosphorylation by PKA or G protein-coupled receptor kinases. These results indicate that, in addition to the phosphorylation status, the intrinsic structural feature of β2-AR plays a crucial role in the receptor coupling selectivity to G proteins. We conclude that specific interactions between the ligand and the Tyr-308 residue of β2-AR stabilize receptor conformations favoring the receptor-Gs protein coupling and subsequently result in Gs-biased agonism.
特定的G蛋白偶联受体与多种不同G蛋白之间的相互作用是一种普遍现象。例如，β2肾上腺素能受体（β2-AR）可双重偶联至Gs和Gi蛋白。先前的研究表明，β2-AR的cAMP依赖性蛋白激酶（PKA）介导的磷酸化会导致受体偶联从Gs转换为Gi。最近的研究表明，G蛋白偶联受体激酶，特别是GRK2对β2-AR的磷酸化显著增强了Gi偶联。我们先前已经表明，尽管大多数β2-AR激动剂都会引起Gs和Gi的激活，但（R,R'）-非诺特罗优先激活β2-AR-Gs信号传导。然而，这种功能选择性的结构基础仍然难以捉摸。在这里，我们使用对接模拟和定点诱变，将Tyr-308定义为β2-AR上对Gs偏向性信号传导至关重要的关键氨基酸残基。在用β2-AR-Gs偏向性激动剂（R,R'）-4'-氨基非诺特罗刺激后，Gi破坏剂百日咳毒素对HEK293细胞中受体介导的ERK磷酸化以及对表达野生型β2-AR的心肌细胞的收缩反应均无影响。有趣的是，β2-AR上的Y308F取代使（R,R'）-4'-氨基非诺特罗能够激活Gi并以百日咳毒素敏感的方式产生这些反应，而不会改变PKA或G蛋白偶联受体激酶对β2-AR的磷酸化。这些结果表明，除了磷酸化状态外，β2-AR的内在结构特征在受体与G蛋白的偶联选择性中起着关键作用。我们得出结论，配体与β2-AR的Tyr-308残基之间的特异性相互作用稳定了有利于受体-Gs蛋白偶联的受体构象，随后导致Gs偏向性激动作用。
