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Methods Mol Biol. 2013;1013:93-127. doi: 10.1007/978-1-62703-426-5_7.

A novel approach to quantify G-protein-coupled receptor dimerization equilibrium using bioluminescence resonance energy transfer.

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1
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA.

Abstract

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.

PMID:
23625495
PMCID:
PMC4091634
DOI:
10.1007/978-1-62703-426-5_7
[Indexed for MEDLINE]
Free PMC Article
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