Oligomeric Structure of Muscarinic and Adrenergic Receptors in Live Cells

Abstract

We have determined the oligomeric size and configuration of fluorophore-tagged M1, M2, β1, and β2 receptors in the plasma membrane of Chinese hamster ovary (CHO) cells by examining the distribution of FRET efficiencies measured at the level of single pixels. The receptors were fused at the N-terminus to enhanced green or yellow fluorescent protein, and complementary pairs were co-expressed at different ratios of donor to acceptor (i.e., eGFP2-M1 and eYFP-M1, eGFP2-M2 and eYFP-M2, eGFP2-β1 and eYFP-β1, 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 apparent FRET efficiency. The distributions of efficiencies from 20 cells were analyzed in concert as a sum of Gaussians, with the number of components (n) and the relationships among the means taken as predicted for a dimer (n = 1), a triangular trimer (n = 2), and a tetramer configured as a square (n = 3) and a rhombus (n = 5). Distributions from each of the four receptors required 5 Gaussians, the means of which were related in the manner predicted for a rhombus. The inverse agonists atropine and timolol were without effect on the number of components or the relationship among the means detected for the M2 muscarinic receptor and the β2 adrenergic receptor, respectively. Homo-oligomers of the M1, M2, β1, and β2 receptors therefore appear to be rhombic tetramers in CHO cells. The configuration is unaffected by inverse agonists, at least in the case of the M2 and β2 receptors.