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Along with other resonance energy transfer techniques, bioluminescence resonance energy transfer (BRET) has emerged as animportant method for demonstrating protein–protein interactions in cells. In the field of G-protein-coupled receptors, includingchemokine receptors, BRET has been widely used to investigate homo- and heterodimerization, a feature of their interactionsthat is emerging as integral to function and regulation. While demonstrating the existence of dimers for a given receptorproved to be fairly straightforward, quantitative comparisons of different receptors or mutants are nontrivial because ofinevitable variations in the expression of receptor constructs. The uncontrollable parameters of the cellular expression machinerymake 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 quantitativeinterpretation of BRET data. We also provide a comprehensive account of expected responses in all types of BRET experimentsand propose a framework for uniform and accurate quantitative treatment of these responses. The framework allows analysisof 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 formsof the chemokine receptor CXCR4.