A Novel Approach to Quantify G-Protein-Coupled Receptor Dimerization Equilibrium Using Bioluminescence Resonance Energy Transfer
Along with other resonance energy transfer techniques, bioluminescence resonance energy transfer (BRET) has emerged as an important method for demonstrating protein–protein interactions in cells. In the field of G-protein-coupled receptors, including chemokine receptors, BRET has been widely used to investigate homo- and heterodimerization, a feature of their interactions that is emerging as integral to function and regulation. While demonstrating the existence of dimers for a given receptor proved to be fairly straightforward, quantitative comparisons of different receptors or mutants are nontrivial because of inevitable variations in the expression of receptor constructs. The uncontrollable parameters of the cellular expression machinery make amounts of transfected DNA extremely poor predictors for the expression levels of BRET donor and acceptor receptor constructs, even in relative terms. In this chapter, we show that properly accounting for receptor expression levels is critical for quantitative interpretation of BRET data. We also provide a comprehensive account of expected responses in all types of BRET experiments and propose a framework for uniform and accurate quantitative treatment of these responses. The framework allows analysis of both homodimer and heterodimer BRET data. The important caveats and obstacles for quantitative treatment are outlined, and the utility of the approach is illustrated by its application to the homodimerization of wild-type (WT) and mutant forms of the chemokine receptor CXCR4.
Key wordsG-protein-coupled receptor Chemokine receptor CXCR4 Dimerization Monomer–dimer equilibrium Bioluminescence resonance energy transfer BRET titration BRET saturation
This work was supported by the National Institute of Health PSI:Biology grants U01 GM094612 (T.M.H. and R.A.) and U54 GM094618 (R.C.S.) and by NIH grants R01 GM081763 (T.M.H.) and R01 GM071872 (R.A.). C.T.G and B.S. were supported by the Cellular and Molecular Pharmacology Training Grant T32 GM007752. D.H. was supported by NIH NRSA grant F32 GM083463. B.W. was supported by NIH grant R01 Al100604 and the grant 11JC1414800 awarded by Science and Technology Commission of Shanghai Municipality. The authors would like to thank Jeffrey Velasquez (TSRI) for help with molecular biology, Tam Trinh and Kirk Allin (TSRI) for help with the baculovirus expression, and M. Bouvier (University of Montreal) for the Rluc and YFP coding sequence containing vectors used to produce all of our BRET constructs. We also thank Pascale Charest (University of Arizona) for valuable discussions regarding the BRET assays and Goran Pljevaljcic (TSRI) for constructive comments on the manuscript.
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