Highly Accurate Quantification Of The Oligomerization Of The Beta 2 Adrenergic Receptor Using Fret
Biophysical Journal(2012)
Abstract
The oligomerization of G-protein coupled receptors, GPCRs, has in many cases shown to be important for transmembrane protein structure and function [1,2], however achieving quantitative parameters describing the process that governs oligomerization is difficult. Traditionally oligomerization is quantified by preparing distinct samples that carry different concentrations and by monitoring bulk spectroscopic signals e.g FRET efficiencies [3], still the limited amount of samples does not allow very accurate determination of the experimental parameters.Here we developed a nanoscale assay [4,5] that allows a highly accurate quantification of the interaction forces between transmembrane molecules. As a proof of concept we focused on the prototypical human β2-adrenergic receptor, β2AR, reconstituted in lipid vesicles. Our approach allowed us to extract the dimerization equilibrium constant of the β2AR to be −4 ± 0.2 KbT.[1] Bouvier, M. Oligomerization of G-protein-coupled transmitter receptors. Nat Rev Neurosci 2, 274-286 (2001).[2] Smith, N.J. & Milligan, G. Allostery at G protein-coupled receptor homo- and heteromers: uncharted pharmacological landscapes. Pharmacol Rev 62, 701-725 (2010).[3] Fung, J.J. et al. Ligand-regulated oligomerization of beta(2)-adrenoceptors in a model lipid bilayer. EMBO J 28, 3315-3328 (2009).[4] Stamou, D. et al. Amphipathic motifs in BAR domains are essential for membrane curvature sensing. Embo Journal 28, 3303-3314 (2009).[5] Stamou, D. et al. How curved membranes recruit amphipathic helices and protein anchoring motifs. Nature Chemical Biology 5, 835-841 (2009).
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Key words
β2 adrenergic receptor,adrenergic receptor,oligomerization
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