Oligomeric Size and Configuration of the M2 Muscarinic and β2 Adrenergic Receptors in Live Cells as Determined by Pixel-Level FRET

Abstract

G protein-coupled receptors are known to form oligomers, but estimates of their size range from dimers to large arrays. We therefore have determined the oligomeric size of fluorophore-tagged M2 and β2 receptors in the plasma membrane of Chinese hamster ovary cells by examining the distribution of FRET efficiencies measured at the level of single pixels. Each receptor was fused at its N-terminus to enhanced green or yellow fluorescent protein and co-expressed as the complementary pair (EGFP2-M2 and EYFP-M2, or EGFP2-β2 and EYFP-β2). Pixel-level emission spectra were recorded from images captured in a single plane; the relative contribution of each fluorophore then was determined by spectral deconvolution and used to calculate the corresponding FRET efficiency. The distribution of efficiencies from each cell was analyzed as a sum of Gaussians. The number of Gaussians and the numeric relationship among the corresponding means (E) is determined by the number of combinatorial arrangements of FRET-productive pairs within a two-dimensional oligomer as predicted by the binomial theorem. A dimer will reveal a single efficiency or Gaussian, and a triangular trimer will reveal two; a square tetramer will reveal three efficiencies, and a rhombus will reveal five. Both the M2 receptor and the β2 receptor required five Gaussians to describe the distributions of efficiencies from several cells taken together. In each case, an efficiency (E) identified as the pair-wise efficiency (Ep) was related to the other four efficiencies in the manner predicted for a rhombus.