Agonist Stereochemistry Determines b2-Adrenergic G Protein Coupling Preference

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将受体引导到不同的细胞内信号通路。我们以前的研究表明，虽然大多数b2受体激动剂同时激活Gs和Gi蛋白，但b2受体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，可能以四种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性质。我们检验了一个假设，即激动剂的立体化学决定了受体对不同G蛋白偶联的选择性(S)。我们发现非诺特罗和甲氧基非诺特罗的R，R-异构体比它们的S，R-异构体有更强的促进心肌细胞收缩的作用。重要的是，当R，R-非诺特罗和R，R-甲氧基非诺特罗优先激活Gs信号时，它们的S，R-异构体能够激活Gs和Gi蛋白，这从它们对心肌细胞收缩和细胞外信号调节激酶1/2的磷酸化的强烈的百日咳毒素敏感性得到证明。非诺特罗立体异构体对Gs，GI2和Gi3蛋白的光亲和标记进一步证实了G蛋白的选择性。R，R-异构体的低效GI信号不是由于R，R-异构体不能触发PKA介导的b2-肾上腺素受体的磷酸化引起的，因为R，R-异构体还显著增加了PKA对丝氨酸262受体的磷酸化。我们的结论是，除了受体亚型和磷酸化状态，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方面起着重要作用。

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 b2-adrenoceptor agonists activate both Gs and Gi proteins, fenoterol, a full agonist of b2-adrenoceptor, 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 b2-adrenoceptor, 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.