A luminescent assay for real-time measurements of receptor endocytosis in living cells

Anal Biochem. 2015 Nov 15:489:1-8. doi: 10.1016/j.ab.2015.08.005. Epub 2015 Aug 13.

Abstract

Ligand-mediated endocytosis is a key autoregulatory mechanism governing the duration and intensity of signals emanating from cell surface receptors. Due to the mechanistic complexity of endocytosis and its emerging relevance in disease, simple methods capable of tracking this dynamic process in cells have become increasingly desirable. We have developed a bioluminescent reporter technology for real-time analysis of ligand-mediated receptor endocytosis using genetic fusions of NanoLuc luciferase with various G-protein-coupled receptors (GPCRs). This method is compatible with standard microplate formats, which should decrease work flows for high-throughput screens. This article also describes the application of this technology to endocytosis of epidermal growth factor receptor (EGFR), demonstrating potential applicability of the method beyond GPCRs.

Keywords: Endocytosis; G-protein-coupled receptor; Internalization; NanoLuc; Receptor tyrosine kinase.

Publication types

  • Validation Study

MeSH terms

  • Animals
  • Arthropod Proteins / chemistry
  • Arthropod Proteins / genetics
  • Arthropod Proteins / metabolism*
  • Cell Membrane / drug effects
  • Cell Membrane / enzymology
  • Cell Membrane / metabolism
  • Drug Discovery / methods
  • Endocytosis* / drug effects
  • Fluorescent Dyes / chemistry
  • Genes, Reporter / drug effects
  • HEK293 Cells
  • High-Throughput Screening Assays / methods*
  • Humans
  • Interleukin-6 / chemistry
  • Interleukin-6 / genetics
  • Interleukin-6 / metabolism
  • Kinetics
  • Ligands
  • Luciferases / chemistry
  • Luciferases / genetics
  • Luciferases / metabolism*
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein Sorting Signals / drug effects
  • Receptors, G-Protein-Coupled / agonists
  • Receptors, G-Protein-Coupled / chemistry
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, G-Protein-Coupled / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism

Substances

  • Arthropod Proteins
  • Fluorescent Dyes
  • Interleukin-6
  • Ligands
  • Peptide Fragments
  • Protein Sorting Signals
  • Receptors, G-Protein-Coupled
  • Recombinant Fusion Proteins
  • Luciferases