Phosphorylation patterns in the AT1R C-terminal tail specify distinct downstream signaling pathways

Area of Science:

• Pharmacology
• Molecular Biology
• Biochemistry

Background:

• Ligand binding to angiotensin II type 1 receptor (AT1R) induces specific conformations.
• These conformations dictate signaling pathways via G proteins or β-arrestin.
• The "barcode" hypothesis suggests differential C-terminal tail phosphorylation patterns mediate these distinct signaling events.

Purpose of the Study:

• To identify the specific AT1R phosphorylation patterns (barcodes) induced by the endogenous agonist AngII and a β-arrestin-biased agonist.
• To determine the role of these phosphorylation patterns in mediating receptor-transducer interactions and signaling.

Main Methods:

• Mass spectrometry to identify receptor phosphorylation sites.
• Biochemical assays to measure G protein activation and β-arrestin recruitment.
• Molecular dynamics simulations to analyze receptor-β-arrestin interactions.

Main Results:

• AngII and the β-arrestin-biased agonist induced distinct phosphorylation patterns on the AT1R C-terminal tail.
• Specific phosphorylation sites in the proximal and middle tail regions were essential for β-arrestin recruitment and function.
• Distal tail phosphorylation sites had minimal impact on β-arrestin function.
• Molecular dynamics simulations confirmed the importance of proximal and middle phosphorylated residues for stable β-arrestin-receptor binding.

Conclusions:

• Ligand-induced AT1R conformations are encoded by distinct phosphorylation barcodes on the C-terminal tail.
• These phosphorylation barcodes precisely control receptor-transducer engagement, leading to specific signaling outcomes.
• This provides a mechanistic understanding of biased agonism and differential signaling pathway activation.