Cell-Free Assay of G-Protein-Coupled Receptors Using Fluorescence Polarization

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 EC 50 determined for the CXCR4-T-22 peptide interaction via FP with CXCR4 lipoparticles (15 nM) is consistent with the IC 50 determined for the unlabeled T-22 peptide via competitive binding (59 nM). (Journal of Biomolecular Screening 2008:424-429)

A Fluorescence Anisotropy Assay for the Muscarinic M1 G-protein-Coupled Receptor

“…FA assays have been developed for several GPCRs using fluorescently labeled ligands and cellular membranes containing the GPCR of interest. [5][6][7][8][9] In order to measure binding affinity, an FA assay does not require the separation of the receptor-bound fluorescent ligand from the unbound fluorescent ligand. The signal measured in the FA assay derives from the fact that a small, unbound fluorescently labeled ligand ("tracer") can rotate more quickly in solution relative to when bound to a larger receptor.…”

Dynamics of ligand binding to GPCR: Residence time of melanocortins and its modulation

“…This change, detected as fluorescence anisotropy, can be used as a measure of ligand binding and be monitored in real time. The FA method has been successfully 6 used for characterization of ligand binding to peptide receptors, such as chemokine CXC4 [23] and melanocortin MC4 [24], receptors of small molecules, such as muscarinic M1 [25] and serotoninergic 5-HT1A [26], and binding of nucleotides to G proteins [27]. It is important to mention here that the FA assay by nature is ratiometric, and signal can be detected only if the ratio of bound to free fluorescent ligand has significantly changed [28].…”

“…This happens if the concentrations of receptor and ligand used are in a comparable range, and on the level of the dissociation constant of their interaction. Such a high level of receptor binding sites is usually difficult to achieve with natural tissues and therefore first attempts to implement this method have been performed with reconstituted systems [23]. The membranes of baculovirusinfected Sf9 cells demonstrated very reasonable anisotropy signals connected with ligand binding [24], while budded baculoviruses from these cells displaying GPCR on their surfaces are even more suitable targets for this type of assay [29].…”

Recombinant G Protein-Coupled Receptor Expression in Saccharomyces cerevisiae for Protein Characterization

“…Due to the low expression in native systems, the poor quality of heterologous expression, difficult purification, and instability, studying these receptors in vitro is challenging (Allen, Ribeiro, Horuk, & Handel, 2009;Doré et al, 2011;Jones, Greene, Grygon, Doranz, & Brown, 2008;Langelaan, Ngweniform, & Rainey, 2011;McNeely, Naranjo, & Robinson, 2012;Wisedchaisri, Reichow, & Gonen, 2011). The previous purification of functional A 2 aR has shown that in order to retain its α-helical content and ligand-binding activity, the presence of cholesteryl hemisuccinate (CHS) is required when purified in dodecylmaltoside (DDM) (O'Malley et al, 2007).…”