ß2-adrenergic receptor transcriptional response in HEK293 wild type, ß-arrestin1/2 KO and Gas KO cells upon isoproterenol stimulation.

The role of nuclear signaling by ß2-adrenergic receptor (ß2AR) through ß-arrestins in the absence of a functional Gas protein has not been evaluated. This has prevented a comprehensive understanding of the relative contribution of Gas and ß-arrestins to the overall ß2AR signaling, thereby limiting our appreciation of how ß-arrestin- or Gas-biased ligands may affect their therapeutic outcomes. To explore the role of Gas and ß-arrestin in nuclear transcriptional programs in a global unbiased approach, we performed RNA sequencing studies in cells lacking either of these two signaling arms downstream from ß2AR, followed by detailed bioinformatics analysis of gene expression signatures. We noticed multiple genes whose individual expression levels were distinct in ß-arrestin1/2 and Gas KO cells. Rather than focusing on the individual gene level, we performed a detailed analysis of gene signatures taking advantage of large datasets that were recently compiled as part of the Molecular Signatures Database (MSigDB). This approach supported that ß-arrestins are not required for the activation of PKA gene signatures, including prior datasets of forskolin and CREB1 targets, while Gas is essential. ß2AR activation also led to significant changes in multiple MAPK signatures, which clearly support the activation of MAPK nuclear transcriptional programs by these receptors. Although individual variations do exist reflecting the complexities of ß-arrestin- and Gas-mediated signaling events, stimulation of these MAPK regulated gene signatures was not significantly reduced in ß-arrestin1/2 KO cells but abolished in Gas KO cells. Overall design: HEK293 wild type, ß-arrestin1/2 KO and Gas KO cells transiently overexpressing wild type ß2-adrenergic receptor were stimulated with isoproterenol or vehicle for 1 h in triplicates. We performed differential gene expression analysis using data obtained from RNAseq. We also obtained enrichment profiles for gene sets related to PKA and MAPK pathways.

在缺乏功能性Gas蛋白的前提下，β2-肾上腺素能受体（β2-adrenergic receptor, β2AR）通过β-阻遏蛋白（β-arrestins）介导核信号转导的作用尚未得到评估。这一研究空白阻碍了学界对Gas蛋白与β-阻遏蛋白在β2AR整体信号转导中相对贡献的全面理解，进而限制了我们对β-阻遏蛋白偏向性配体或Gas偏向性配体如何影响其治疗效果的认知。为以全局无偏的方式探究Gas蛋白与β-阻遏蛋白在核转录程序中的作用，我们分别在缺失β2AR下游两条信号通路之一的细胞中开展了RNA测序（RNA sequencing, RNA-seq）实验，并随后对基因表达特征进行了细致的生物信息学分析。我们观察到，在β-阻遏蛋白1/2敲除（KO）细胞与Gas蛋白敲除细胞中，多个基因的表达水平存在显著差异。相较于聚焦单基因层面，我们借助近期整合入分子特征数据库（Molecular Signatures Database, MSigDB）的大型数据集，对基因特征展开了深入分析。该分析结果表明，尽管Gas蛋白是蛋白激酶A（Protein Kinase A, PKA）基因特征激活所必需的，但β-阻遏蛋白并非激活PKA基因特征所必需，这一结论与福司柯林（forskolin）及CREB1靶基因的既往研究数据集相符。β2AR激活还会引发多个丝裂原活化蛋白激酶（Mitogen-Activated Protein Kinase, MAPK）相关特征的显著变化，这直接证实了该受体可激活MAPK核转录程序。尽管存在反映β-阻遏蛋白与Gas蛋白介导信号转导过程复杂性的个体差异，但这些MAPK调控的基因特征的激活水平在β-阻遏蛋白1/2敲除细胞中并未显著降低，却在Gas蛋白敲除细胞中被完全阻断。实验整体设计：将瞬时过表达野生型β2-肾上腺素能受体的HEK293野生型细胞、β-阻遏蛋白1/2敲除细胞与Gas蛋白敲除细胞，以异丙肾上腺素（isoproterenol）或溶媒对照处理1小时，设置三次生物学重复。我们利用RNA测序（RNA-seq）获得的数据开展了差异基因表达分析，并获取了与PKA及MAPK通路相关的基因集富集分析结果。