Förster resonance energy transfer
Förster resonance energy transfer (FRET) is a specific and sensitive technique for detecting the oligomerisation of cytosolic proteins, but using FRET to measure the oligomerisation of membrane proteins is more challenging. Membrane proteins are confined to two dimensions. Many of them cluster in small microdomains, which can lead to spurious FRET at high levels of expression. Those challenges have become apparent in the study of G protein-coupled receptors (GPCRs), where different groups have used FRET efficiencies to conclude variously that class A GPCRs exist as monomers, dimers or tetramers. To examine what led to such markedly different conclusions, we measured FRET efficiencies for membrane proteins that are known to exist as monomers, transient oligomers or stable dimers; we then compared the patterns of efficiencies observed for those controls to the patterns obtained for the M2 muscarinic receptor, a prototypical class A GPCR. In each case, proteins were tagged with GFP2 and eYFP and were expressed transiently in CHO or HEK cells. Stable and constitutive oligomers produced efficiencies that remained high even at low levels of expression and could be described well by a model for a stable dimer. In contrast, efficiencies measured between monomeric CD86 molecules, between M2 receptors, between M2 receptors and Gαi1 proteins, and between M2 receptors and M1 receptors, β2 receptors, CD28 or CD86, all followed a similar pattern that approached zero at low levels of expression and could not be described by a model for a stable dimer. That pattern could be described, however, by a model for bystander FRET or a transient dimer. The view of class A GPCRs as transient oligomers appears to be the most consistent with other biochemical and biophysical data that have been used to quantify GPCR oligomerisation and hitherto have led to a variety of contrasting interpretations.