Cell-free assay of G-protein-coupled receptors using fluorescence polarization

J Biomol Screen. 2008 Jun;13(5):424-9. doi: 10.1177/1087057108318332.

Abstract

A recently developed nanotechnology, the Integral Molecular lipoparticle, provides an essentially soluble cell-free system in which G-protein-coupled receptors (GPCRs) in their native conformations are concentrated within virus-like particles. As a result, the lipoparticle provides a means to overcome 2 common obstacles to the development of homogeneous, nonradioactive GPCR ligand-binding assays: membrane protein solubilization and low receptor density. The work reported here describes the first application of this nanotechnology to a fluorescence polarization (FP) molecular binding assay format. The GPCR chosen for these studies was the well-studied chemokine receptor CXCR4 for which a peptide ligand (T-22) has been previously characterized. The EC50 determined for the CXCR4-T-22 peptide interaction via FP with CXCR4 lipoparticles (15 nM) is consistent with the IC50 determined for the unlabeled T-22 peptide via competitive binding (59 nM).

MeSH terms

  • Cell-Free System
  • Fluorescence Polarization / methods*
  • Nanotechnology / methods*
  • Receptors, G-Protein-Coupled / chemistry*

Substances

  • Receptors, G-Protein-Coupled