Phospho-selective mechanisms of arrestin conformations and functions revealed by unnatural amino acid incorporation and (19)F-NMR

Nat Commun. 2015 Sep 8;6:8202. doi: 10.1038/ncomms9202.

Abstract

Specific arrestin conformations are coupled to distinct downstream effectors, which underlie the functions of many G-protein-coupled receptors (GPCRs). Here, using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance ((19)F-NMR) spectroscopy, we demonstrate that distinct receptor phospho-barcodes are translated to specific β-arrestin-1 conformations and direct selective signalling. With its phosphate-binding concave surface, β-arrestin-1 'reads' the message in the receptor phospho-C-tails and distinct phospho-interaction patterns are revealed by (19)F-NMR. Whereas all functional phosphopeptides interact with a common phosphate binding site and induce the movements of finger and middle loops, different phospho-interaction patterns induce distinct structural states of β-arrestin-1 that are coupled to distinct arrestin functions. Only clathrin recognizes and stabilizes GRK2-specific β-arrestin-1 conformations. The identified receptor-phospho-selective mechanism for arrestin conformation and the spacing of the multiple phosphate-binding sites in the arrestin enable arrestin to recognize plethora phosphorylation states of numerous GPCRs, contributing to the functional diversity of receptors.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Arrestins / genetics
  • Arrestins / metabolism*
  • Binding Sites
  • Blotting, Western
  • Cattle
  • Clathrin / metabolism
  • Escherichia coli
  • Fluorine
  • Fluorine-19 Magnetic Resonance Imaging
  • G-Protein-Coupled Receptor Kinase 2 / metabolism
  • G-Protein-Coupled Receptor Kinases / metabolism
  • HEK293 Cells
  • Humans
  • Microscopy, Confocal
  • Mutation
  • Nuclear Magnetic Resonance, Biomolecular
  • Phosphate-Binding Proteins / metabolism
  • Phosphoproteins / metabolism*
  • Protein Conformation
  • Receptors, G-Protein-Coupled / metabolism*
  • Signal Transduction
  • Tandem Mass Spectrometry
  • Tyrosine / analogs & derivatives
  • Tyrosine / metabolism
  • beta-Arrestin 1
  • beta-Arrestins

Substances

  • ARRB1 protein, human
  • Arrestins
  • Clathrin
  • Phosphate-Binding Proteins
  • Phosphoproteins
  • Receptors, G-Protein-Coupled
  • beta-Arrestin 1
  • beta-Arrestins
  • Fluorine
  • 3,5-difluorotyrosine
  • Tyrosine
  • GRK2 protein, human
  • G-Protein-Coupled Receptor Kinase 2
  • G-Protein-Coupled Receptor Kinases
  • G-protein-coupled receptor kinase 6