BRET Evidence That Β2 Adrenergic Receptors Do Not Oligomerize in Cells

Bioluminescence resonance energy transfer (BRET) is often used to study association of membrane proteins and in particular oligomerization of G protein-coupled receptors (GPCRs). Oligomerization of class A GPCRs is controversial, in part because the methods used to study this question are not completely understood. Here we reconsider oligomerization of the class A β 2 adrenergic receptor (β 2 AR) and reevaluate BRET titration as a method to study membrane protein association. Using inducible expression of the energy acceptor at multiple levels of donor expression we find that BRET between β 2 AR protomers is directly proportional to the density of the acceptor up to ~3,000 acceptors μm −2 and does not depend on the density of the donor or on the acceptor:donor (A:D) stoichiometry. In contrast, BRET between tightly-associating control proteins does not depend on the density of the acceptor, but does depend on the density of the donor and on the A:D ratio. We also find that the standard frameworks used to interpret BRET titration experiments rely on simplifying assumptions that are frequently invalid. These results suggest that β 2 ARs do not oligomerize in cells and demonstrate a reliable method of assessing membrane protein association with BRET.