Loss of Biased Signaling Specificity of the Angiotensin II Type 1 Receptor in Overexpressed Systems.

G protein-coupled receptors (GPCRs) regulate cellular signaling pathways by coupling to two classes of transducers: heterotrimeric G proteins and β-arrestins. [Sarcosine1 Ile4 Ile8 ]-angiotensin II (SII), an analog of the endogenous ligand angiotensin II (AngII) for the angiotensin II type 1 receptor (AT1 R), fails to activate G protein in multiple primary cell lines. Despite this, SII induces phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) through β-arrestin-2-dependent mechanisms. However, studies reliant on exogenous overexpression of the AT1 R indicate that SII is a partial agonist for G protein signaling and lacks β-arrestin-exclusive signaling specificity. We investigated this apparent discrepancy by profiling changes in signaling specificity at the AT--1 R with increasing levels of receptor expression. We hypothesized that overexpression of the AT1 R causes loss of signaling pathway specificity, such that ligands capable of inducing only β-arrestin-mediated responses at lower physiologic receptor expression levels gain the ability to activate G proteins upon receptor overexpression. We established a tetracycline-inducible cellular system for titratable expression of the AT1 R to assess signaling responses at varying levels of receptor expression when stimulated with AngII, SII, and four other AngII analogs with different signaling biases. Unbiased and G protein-biased ligands activated dose-dependent calcium responses at all levels of AT1 R expression. In contrast, β-arrestin-biased ligands induced dose-dependent calcium signaling only at higher levels of receptor overexpression. Using inhibitors of different G proteins, we demonstrated that both Gi and Gq mediated this overexpression-dependent calcium signaling by β-arrestin-biased ligands. All ligands induced ERK1/2 phosphorylation at receptor levels below those required for calcium signaling by β-arrestin-biased ligands. Thus, receptor overexpression causes loss of biased signaling specificity of AT1 R ligands, highlighting the potential risks of using such systems to simulate GPCR ligand behavior in physiologically relevant contexts.

Duke Scholars

Author Robert J. Lefkowitz Medicine, Cardiology

Author Seungkirl Ahn Medicine, Cardiology