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Updated: Jul 16, 2025

Parallel Interrogation of β-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay
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Characterization of the real-time internalization of nine GPCRs reveals distinct dependence on arrestins and G

Thor C Møller1, Ee Von Moo1, Asuka Inoue2

  • 1Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark.

Biochimica Et Biophysica Acta. Molecular Cell Research
|September 15, 2023
PubMed

Insights

This study developed a new assay to track G protein-coupled receptor (GPCR) internalization, revealing diverse pathways independent of receptor class or G protein coupling. The findings offer a versatile platform for studying membrane protein trafficking.

Area of Science:

  • Cellular Biology
  • Molecular Pharmacology
  • Membrane Protein Dynamics

Background:

  • G protein-coupled receptors (GPCRs) mediate cellular responses but require internalization for regulation.
  • Canonical GPCR internalization involves arrestin-dependent, clathrin-mediated endocytosis.
  • Diverse GPCR internalization pathways exist, but inconsistent methodologies hinder clear characterization.

Purpose of the Study:

  • To establish a consistent and efficient method for profiling GPCR internalization pathways.
  • To investigate the internalization mechanisms of nine distinct GPCRs across different classes.
  • To determine the dependence of GPCR internalization on heterotrimeric G proteins and non-visual arrestins.

Main Methods:

  • Utilized a time-resolved Förster resonance energy transfer (TR-FRET) assay for high-throughput internalization studies.
  • Employed clustered regularly interspaced palindromic repeats (CRISPR) engineered knockout cells to assess protein involvement.
  • Examined nine GPCRs from classes A, B, and C with varying G protein coupling profiles.

Main Results:

  • All nine studied GPCRs demonstrated agonist-induced internalization in a concentration-dependent manner.
  • Six of the nine receptors exhibited basal internalization.
  • No correlation was found between GPCR class, primary G protein coupling, and arrestin/G protein dependence for internalization.

Conclusions:

  • The developed TR-FRET assay provides a versatile platform for studying GPCR internalization.
  • GPCR internalization pathways are diverse and not strictly dictated by receptor class or G protein coupling.
  • This methodology can be extended to other membrane proteins and endocytic machinery for comprehensive profiling.
Keywords:
ArrestinsCRISPR/Cas9G protein-coupled receptorInternalizationKnockoutSignaling

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