The β-Arrestin Pathway-selective Type 1A Angiotensin Receptor (AT1A) Agonist Sar1Ile4Ile8Angiotensin II Regulates a Robust G Protein-independent Signaling Network

摘要

The angiotensin II peptide analog [Sar¹,Ile⁴,Ile⁸]AngII (SII) is a biased AT1A receptor agonist that stimulates receptor phosphorylation, β-arrestin recruitment, receptor internalization, and β-arrestin-dependent ERK1/2 activation without activating heterotrimeric G-proteins. To determine the scope of G-protein-independent AT1A receptor signaling, we performed a gel-based phosphoproteomic analysis of AngII and SII-induced signaling in HEK cells stably expressing AT1A receptors. A total of 34 differentially phosphorylated proteins were detected, of which 16 were unique to SII and eight to AngII stimulation. MALDI-TOF/TOF mass fingerprinting was employed to identify 24 SII-sensitive phosphoprotein spots, of which three (two peptide inhibitors of protein phosphatase 2A (I1PP2A and I2PP2A) and prostaglandin E synthase 3 (PGES3)) were selected for validation and further study. We found that phosphorylation of I2PP2A was associated with rapid and transient inhibition of a β-arrestin 2-associated pool of protein phosphatase 2A, leading to activation of Akt and increased phosphorylation of glycogen synthase kinase 3β in an arrestin signalsome complex. SII-stimulated PGES3 phosphorylation coincided with an increase in β-arrestin 1-associated PGES3 and an arrestin-dependent increase in cyclooxygenase 1-dependent prostaglandin E2 synthesis. These findings suggest that AT1A receptors regulate a robust G protein-independent signaling network that affects protein phosphorylation and autocrine/paracrine prostaglandin production and that these pathways can be selectively modulated by biased ligands that antagonize G protein activation.