A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer

James JR, Oliveira MI, Carmo AM, Iaboni A, Davis SJ. (2006), Nat Methods. 3, 1001-6

Abstract

Bioluminescence resonance energy transfer (BRET), which relies on nonradiative energy transfer between luciferase-coupled donors and GFP-coupled acceptors, is emerging as a useful tool for analyzing the quaternary structures of cell-surface molecules. Conventional BRET analyses are generally done at maximal expression levels and single acceptor/donor ratios. We show that under these conditions substantial energy transfer arises from random interactions within the membrane. The dependence of BRET efficiency on acceptor/donor ratio at fixed surface density, or expression level at a defined acceptor/donor ratio, can nevertheless be used to correctly distinguish between well-characterized monomeric and oligomeric proteins, including a very weak dimer. The pitfalls associated with the nonrigorous treatment of BRET data are illustrated for the case of G protein-coupled receptors (GPCRs) proposed to form homophilic and/or mixed oligomers on the basis of previous, conventional BRET experiments.

Key figure: Two native class-A GPCRs are monomeric at the cell surface

(a) Both β₂AR and mCannR2, expressed separately as BRET pairs or together as a BRET pair, and β₂AR and CD2 expressed as a BRET pair, give similar low BRET_eff values exhibiting [GFP]/[Luc] ratio independence beyond a [GFP]/[Luc] ratio of 2, with lines representing the best fit to the data, as described in the Figure 3 legend. (b) Variation of the acceptor/donor ratio for β₂AR shows no evidence for oligomerization at three different levels of surface expression, with the highest level being ~2 × 10⁶ molecules/cell. (c) BRET_eff values for a heterodimerizing class-C GPCR, GABA_βR, consisting of GABA_βR1 and GABA_βR2 subunits, exhibit the hyperbolic relationship with acceptor/donor ratio expected for a dimer, as do BRET_eff values obtained for a fusion protein consisting of β₂AR and the heterodimerizing coiled-coil domains of the GABA_βR complex (β₂ARcoil1 and β₂ARcoil2). β₂ARcoil2Δ is a truncated version of β₂ARcoil2 with only half the GABA_βR2 coiled-coil. (d) The decrease in interaction when β₂AR is expressed as a BRET pair with GABA_βR2 is due to the homodimerization of GABA_βR2 at the cell surface, which decreases by half the effective density of molecules present.