Abstract
Quantitative study of membrane proteins presents considerable technical challenges. Here, we report a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to quantify functional Chemokine (C-C motif) Receptor 5 (CCR5). CCR5 is a seven-transmembrane helical G protein-coupled receptor (GPCR) expressed primarily on immune cells. It is of significant interest due to its role as the co-receptor of R5-tropic HIV-1. The TR-FRET assay exploits energy transfer between a long-lived europium cryptate donor fluorophore and an appropriate acceptor. We have developed a homogeneous sandwich-type immunoassay with labeled antibodies against a conformationally-sensitive epitope on the extracellular domain of CCR5 and an engineered C-terminal 1D4-mAb epitope. The assay yields a quantitative FRET signal corresponding to the total amount receptor. To quantitate “functional” receptor, a labeled anti-HA (hemagglutinin) antibody against an engineered N-terminal HA epitope is used in conjunction with labeled MIP-1a, a chemokine ligand for CCR5. We are also interested in quantifying the degree of chemical labeling of unnatural amino acids incorporated into expressed GPCRs by amber codon suppression technology. The Staudinger-Bertozzi ligation links a commercially-available FLAG-phosphine reagent to p-L-azidophenylalanine. A europium-labeled anti-FLAG antibody can then be used in another version of the sandwich assay. The resulting FRET signals are directly proportional to amount of total, functional, and labeled receptor, but must be calibrated precisely to extract an absolute concentration. Calibration is accomplished by measuring binding of a fluorescent derivative of a small-molecule CCR5 antagonist. The assay is highly specific due to the long lifetime of the europium donor, and nanomolar concentrations of receptor are detectable. This GPCR assay technology can be used to optimize CCR5 reconstitution conditions and can be readily extended to other members of the chemokine receptor family.
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