RGS2 Constitutes A Negative Regulator of Beta2-Adrenergic Gi Signaling

肾上腺素能受体（bAR）刺激是调节能量代谢和心脏功能的最有效手段。密切相关的bAR亚型b1AR和b2AR均在哺乳动物心脏中表达，但它们表现出亚型特异性G蛋白偶联和功能。b1AR与Gs偶联，b2AR与Gs和百日咳毒素（PTX）敏感的Gi蛋白偶联，Gi偶联可抑制Gs介导的收缩反应，促进心脏细胞存活。研究表明，PKA对b2AR的磷酸化促进了受体- gi偶联。然而，抑制b2AR-Gi偶联的机制尚不清楚。在这里，我们将G蛋白信号传导2的调节因子（RGS2）定义为b2AR-Gi偶联的一种新的负调节因子。首先，虽然b1ar诱导的收缩反应保持不变，但在培养24小时的成年小鼠心肌细胞中，在G蛋白表达和两种bAR的密度均未发生改变的情况下，b2AR的收缩反应对PTX产生了深刻的致敏和抗性，这表明b2AR- gi偶联在培养细胞中严重受损。其次，RGS2蛋白在培养细胞中选择性升高，其他主要心脏RGS蛋白包括RGS3、RGS4、RGS5、RGS10和GAIP均未发生变化。第三，在细胞培养过程中，异丙肾上腺素（1.0 nM） bAR刺激完全阻止了RGS2的上调和b2AR-Gi的解偶联，表明b2AR-Gi的偶联是由受体激活诱导的RGS2的下调介导的。这一结论得到了以下事实的证实：即使在没有bAR激动剂刺激的情况下，基因靶向敲除RGS2也能在培养的心肌细胞中维持b2AR-Gi偶联。与此形成鲜明对比的是，RGS2腺病毒过表达在培养的心肌细胞中消除了激动剂诱导的b2AR-Gi偶联。因此，RGS2作为b2ar偶联Gi信号的强大负调节因子，在哺乳动物心脏中敏感b2ar介导的收缩反应。

Beta-adrenergic receptor (bAR) stimulation serves as the most powerful means to regulate energy metabolism and cardiac performance. Both b1AR and b2AR, the closely related bAR subtypes, are expressed in mammalian hearts, but they display subtype-specific G protein coupling and functions. While b1AR couples to Gs, b2AR couples dually to the Gs and pertussis toxin (PTX)-sensitive Gi proteins with the Gi coupling negating the Gs-mediated contractile response and promoting cell survival in the heart. It has been shown that phosphorylation of b2AR by PKA promotes the receptor-Gi coupling. However, the mechanism inhibiting the b2AR-Gi coupling is poorly understood. Here, we define regulator of G protein signaling 2 (RGS2) as a novel negative regulator of the b2AR-Gi coupling. First, while b1AR-induced contractile response remained intact, b2AR contractile response was profoundly sensitized and resistant to PTX in adult mouse cardiomyocytes cultured for 24 h, in the absence of alterations in the expression of G proteins or the density of either bAR subtype, indicating the b2AR-Gi coupling is severely impaired in cultured cells. Second, RGS2 protein was selectively elevated in cultured cells without changes in other major cardiac RGS proteins, including RGS3, RGS4, RGS5, RGS10 and GAIP. Third, both upregulation of RGS2 and b2AR-Gi uncoupling were fully prevented by bAR stimulation with isoproterenol (1.0 nM) during cell culture, suggesting that the b2AR-Gi coupling is mediated by the receptor activation-induced downregulation of RGS2. This conclusion is corroborated by the fact that gene-targeted knockout of RGS2 sustained the b2AR-Gi coupling in cultured cardiomyocytes even in the absence of bAR agonist stimulation. In sharp contrast, RGS2 adenoviral overexpression abrogated the agonist-induced b2AR-Gi coupling in cultured cardiomyocytes. Thus, RGS2 functions as a powerful negative regulator of the b2AR-coupled Gi signaling, sensitizing b2AR-mediated contractile response in mammalian hearts.