Agonist Stereochemistry Determines Beta2-Adrenergic G Protein Coupling

关于受体- g蛋白相互作用的一个基本问题是不同的激动剂是否可以引导受体进入不同的细胞内信号通路。我们之前的研究表明，虽然大多数β - ar激动剂同时激活Gs和Gi蛋白，但β - ar的完全激动剂非诺特罗选择性地激活Gs蛋白。非诺特罗含有两个手性中心，可以以四个立体异构体的形式存在。我们合成了一系列非诺特罗及其衍生物的立体异构体，并表征了它们的受体结合和药理学性质。我们测试了激动剂的立体化学决定受体偶联到不同G蛋白的选择性的假设。我们发现非诺特罗和甲氧基非诺特罗的R，R-异构体比S，R-异构体更能增强心肌细胞收缩。重要的是，虽然R，R-非诺特罗和R，R-甲氧基非诺特罗优先激活Gs信号，但它们的S，R-异构体能够激活Gs和Gi蛋白，这可以从它们对心肌细胞收缩和细胞外信号调节激酶1/2磷酸化的百日咳毒素敏感性中得到证明。通过对G、Gi2和Gi3蛋白的光亲和标记研究，进一步证实了非诺特罗立体异构体的G蛋白选择性差异。R，R-异构体的Gi信号传导效率低下并非由于R，R-异构体无法触发PKA介导的β 2- ar磷酸化，因为R，R-异构体也显著增加了PKA对受体丝氨酸262的磷酸化。我们得出结论，除了受体亚型和磷酸化状态外，给定激动剂的立体化学在决定受体- g蛋白选择性和下游信号事件中起着重要作用。

A fundamental question regarding receptor-G protein interaction is whether different agonists can lead a receptor to different intracellular signaling pathways. Our previous studies have demonstrated that while most beta2-AR agonists activate both Gs and Gi proteins, fenoterol, a full agonist of beta2-AR, selectively activates Gs protein. Fenoterol contains two chiral centers and may exist as four stereoisomers. We have synthesized a series of stereoisomers of fenoterol and its derivatives and characterized their receptor binding and pharmacological properties. We tested the hypothesis that the stereochemistry of an agonist determines selectivity of receptor coupling to different G protein(s). We found that the R,R-isomers of fenoterol and methoxyfenoterol exhibited more potent effects to increase cardiomyocyte contraction than their S,R-isomers. Importantly, while R,R-fenoterol and R,R-methoxyfenoterol preferentially activate Gs signaling, their S,R-isomers were able to activate both Gs and Gi proteins as evidenced by the robust pertussis toxin-sensitivities of their effects on cardiomyocyte contraction and on phosphorylation of extracellular signal-regulated kinase 1/2. The differential G protein selectivities of the fenoterol stereoisomers were further confirmed by photoaffinity labeling studies on Gs, Gi2 and Gi3 proteins. The inefficient Gi signaling with the R,R-isomers is not caused by the inability of the R,R-isomers to trigger the PKA-mediated phosphorylation of the beta2-AR, since the R,R-isomers also markedly increased phosphorylation of the receptor at serine262 by PKA. We conclude that in addition to receptor subtype and phosphorylation status, the stereochemistry of a given agonist plays an important role in determining receptor-G protein selectivity and downstream signaling events.