Factors modulating the assembly of human β₂-adrenergic receptor-β-arrestin complexes

Area of Science:

• Molecular and Cellular Biology
• Biochemistry
• Pharmacology

Background:

• β-arrestins (βarrs) are key regulators of G protein-coupled receptor (GPCR) signaling, forming complexes with numerous GPCRs.
• Understanding how βarrs form functionally distinct complexes with GPCR tails, cores, or both is a critical research question.

Purpose of the Study:

• To investigate the assembly and mechanical/kinetic stabilities of β₂-adrenergic receptor (β₂AR)-β-arrestin2 (βarr2) complexes.
• To elucidate the role of phospholipid membranes, particularly PIP₂, in modulating βarr2-GPCR complex formation and stability.

Main Methods:

• Single-molecule force spectroscopy
• Molecular dynamics simulations
• Monitoring βarr2 assembly with β₂AR tail, core, and tail-core complexes within phospholipid membranes.

Main Results:

• βarr2 rapidly engages the phosphorylated β₂AR carboxy-terminus (C-tail) within milliseconds, preceding binding to the receptor core.
• Phospholipid membranes significantly contribute to complex stability, with PIP₂ modulating both stability and conformation.
• PIP₂ stabilizes the β₂AR-βarr2 core complex but inhibits simultaneous binding to the phosphorylated β₂AR C-tail.
• βarr2 activation and PIP₂ enhance βarr2-membrane association via insertion of the C-edge and finger loop.

Conclusions:

• PIP₂ is a critical determinant of β₂AR-βarr2 complex assembly, alongside ligand binding and receptor phosphorylation.
• These findings provide mechanistic insights into the regulation of GPCR signaling by β-arrestins and membrane lipids.