Oligomeric Size of the M2 Muscarinic Receptor in Live Cells as Determined from FRET Efficiencies at the Level of Single Pixels

Abstract

G protein-coupled receptors are known to form oligomers, but their size (n) is unclear. We therefore have developed an approach whereby an explicit value of n can be obtained from the efficiency of fluorescence resonance energy transfer (FRET) measured between fluorophore-tagged receptors at the level of single pixels. That approach has been applied to M2 receptors fused at the N-terminus to enhanced green or yellow fluorescent protein (EGFP2-M2 or EYFP-M2) and coexpressed in Chinese hamster ovary cells. Pixel-level emission spectra were recorded from images captured in a single plane, and the pair-wise FRET efficiency (E) was calculated from the relative contributions of EGFP2-M2 and EYFP-M2 as determined by spectral deconvolution. The number of efficiencies obtained for an oligomer of given size is expected to depend upon the number of combinatorial arrangements of FRET-productive pairs as predicted by the binomial theorem. A dimer will reveal a single efficiency, for example, and a square tetramer will reveal at least three. A sum of three Gaussians was required to describe the distribution of efficiencies obtained from cells coexpressing EGFP2-M2 and EYFP-M2. Also, differences among values of E in an oligomer of specified size can be predicted on the basis of a model in which the apparent FRET efficiency is determined by the pair-wise FRET efficiencies for all combinations of donors and acceptors. With the present data, the observed differences in the mean values of E agree with the predicted differences when n is 4. The M2 receptor therefore can be identified as a tetramer, in agreement with the results of a parallel study in which the oligomeric size has been estimated from intensity-based FRET and fluorescence lifetimes.