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.