Distinct Phosphorylation Patterns of AT1R by Biased Ligands and GRK Subtypes

Area of Science:

• Molecular Pharmacology
• G protein-coupled receptor (GPCR) signaling
• Biochemistry

Background:

• G protein-coupled receptors (GPCRs) mediate cellular responses via G proteins and β-arrestins upon agonist binding.
• GPCR phosphorylation by GPCR kinases (GRKs) is crucial for initiating β-arrestin signaling, but mechanisms remain unclear.
• The angiotensin II type 1 receptor (AT1R) is a key model for studying biased ligand and signaling pathway regulation.

Purpose of the Study:

• To investigate how distinct agonists and GRK subtypes differentially phosphorylate the AT1R C-terminus.
• To determine the impact of AT1R phosphorylation patterns on β-arrestin recruitment.
• To elucidate the molecular basis of biased signaling through AT1R phosphorylation.

Main Methods:

• Utilized wild-type and mutant AT1R constructs targeting three C-terminal phosphorylation motifs.
• Employed unbiased (AngII), β-arrestin-biased (TRV026), and G protein-biased (TRV056) agonists with GRK2/3/5/6 subtypes.
• Integrated phosphorylation assays, β-arrestin pull-down, molecular dynamics simulations, and AlphaFold3 predictions.

Main Results:

• GRK2-mediated AT1R phosphorylation depended on Motifs I and II, with Motif II being critical; Gβγ subunits enhanced this.
• GRK5 and GRK6 showed specific phosphorylation patterns, with GRK5 targeting Motif II and GRK6 targeting Motifs I and II depending on the agonist.
• Mutations in Motif II reduced β-arrestin recruitment by GRK5/6, while Motif II phosphorylation facilitated stable β-arrestin interactions, unlike Motif I.

Conclusions:

• Different agonists and GRK subtypes induce distinct AT1R phosphorylation profiles ('barcodes').
• Specific phosphorylation patterns dictate β-arrestin recruitment efficacy, influenced by agonist bias.
• Findings provide a framework for designing AT1R-targeted therapeutics that leverage biased signaling pathways.