Abstract 274: Carvedilol Stimulated Gαi-β-Arrestin Biased β1 Adrenergic Receptor Signaling

Abstract

The β1 and β2 adrenergic receptors (βARs) are the predominant G-protein-coupled receptors (GPCRs) subtypes expressed in the heart. It is now appreciated that ligands can induce multiple distinct “active” receptor conformations with unique downstream functional signaling profiles or efficacies. Our current understanding of GPCR signaling is that ligands can be biased toward activating either a G protein or a β-arrestin-signaling pathway, a concept known as biased ligand signaling. To identify novel biased signaling pathways mediated by β1- and β2ARs, we performed a proteomic interactome study of β1AR and β2AR using stable isotope labeling by amino acids in cell culture (SILAC). We identified several hundred proteins that distinctly bind to the β1AR or β2AR following stimulation with the unbiased full agonist, isoproterenol, or the β-arrestin-biased ligand carvedilol. We found that stimulation by the β-arrestin-biased agonist carvedilol of only β1ARs resulted in the recruitment of Gαi. No of the other ligand tested promoted Gαi recruitment, suggesting that carvedilol may be unique in its ability to activate Gαi-biased signaling. The Gαi inhibitor pertussis toxin blocked β-arrestin-dependent extracellular signal-regulated kinase (ERK) activation and epidermal growth factor receptor (EGFR) transactivation stimulated by carvedilol, suggesting the involvement of Gαi in carvedilol-induced β-arrestin biased signaling of β1ARs. Using β1AR/β2AR chimeric receptors we show that the C-terminal tail of the β1AR is required for carvedilol stimulated Gαi recruitment, but is unable to rescue the lack of Gαi recruitment by the β2AR. Since our current conceptual framework for biased signaling is based on the “bar code hypothesis”, ongoing phosphoproteomic experiments are testing whether recruitment of Gαi by carvedilol induces a distinct phosphorylation pattern on the c-tail of the β1AR. Our study shows that activation of β-arrestin-biased signaling requires G proteins for signaling and provides a new mechanistic understanding of how biased β-blockers can activate signaling.