Cell-free assay of G-protein-coupled receptors using fluorescence polarization.
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).
Jones
Jessi Wildeson
JW
Biologics and Biomolecular Sciences, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06488, USA.
Greene
Tiffani A
TA
Grygon
Christine A
CA
Doranz
Benjamin J
BJ
Brown
Martha P
MP
D002474:Cell-Free System; D005454:Fluorescence Polarization / Q000379:methods*; D036103:Nanotechnology / Q000379:methods*; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*
D043562:Receptors, G-Protein-Coupled