(536.10) Loss of Biased Signaling Specificity of the Angiotensin II Type 1 Receptor in Overexpressed Systems

Poster Board Number: B74

Angus Li (Duke University School of Medicine), Samuel Liu (Duke University School of Medicine), Rennica Huang (Duke University School of Medicine), Seungkirl Ahn (Duke University School of Medicine), Robert Lefkowitz (Duke University School of Medicine, Duke University School of Medicine, Duke University School of Medicine)

G protein-coupled receptors (GPCRs) regulate cellular signaling pathways by coupling to two classes of transducers: heterotrimeric G proteins and β-arrestins. [Sarcosine1Ile4Ile8]-angiotensin II (SII), an analog of the endogenous ligand angiotensin II (AngII) for the angiotensin II type 1 receptor (AT1R), 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 AT1R 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 AT­­1R with increasing levels of receptor expression. We hypothesized that overexpression of the AT1R 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 AT1R 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 AT1R 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 AT1R ligands, highlighting the potential risks of using such systems to simulate GPCR ligand behavior in physiologically relevant contexts.

Support or Funding Information

Research support was provided by the Howard Hughes Medical Institute and the National Institutes of Health.