Utility of FRET in studies of membrane protein oligomerization: the concept of the effective dissociation constant

The activity of many membrane receptors is controlled through their lateral association into dimers or higher order oligomers. While Forster resonance energy transfer (FRET) measurements have been used extensively to characterize the stability of receptor dimers, the utility of FRET in studies of larger oligomers is unclear. Here we show that we can extract an effective equilibrium dissociation constant from FRET measurements for EphA2, a receptor tyrosine kinase (RTK) known to form active oligomers of heterogeneous distributions in response to its ligand ephrinA1-Fc. The newly introduced effective equilibrium dissociation constant has a well-defined physical meaning and biological significance. It denotes the receptor concentration for which half of the receptors are monomeric and inactive, and the other half are associated into oligomers and are active, irrespective of the exact oligomer size. This work illustrates how FRET, along with fluorescence fluctuation techniques which directly measure the oligomer size, can be a very powerful tool in studies of membrane receptor association and signaling in the plasma membrane.