Oligomeric Size of the M2 Muscarinic Receptor in Live Cells as Determined by Quantitative Fluorescence Resonance Energy Transfer

Abstract

Fluorescence resonance energy transfer (FRET), measured by fluorescence intensity-based microscopy and fluorescence lifetime imaging, has been used to estimate the size of oligomers formed by the M 2 muscarinic cholinergic receptor. The approach is based on the relationship between the apparent FRET efficiency within an oligomer of specified size (n) and the pairwise FRET efficiency between a single donor and a single acceptor (E). The M 2 receptor was fused at the N terminus to enhanced green or yellow fluorescent protein and expressed in Chinese hamster ovary cells. Emission spectra were analyzed by spectral deconvolution, and apparent efficiencies were estimated by donor-dequenching and acceptor-sensitized emission at different ratios of enhanced yellow fluorescent protein-M 2 receptor to enhanced green fluorescent protein-M 2 receptor. The data were interpreted in terms of a model that considers all combinations of donor and acceptor within a specified oligomer to obtain fitted values of E as follows: n ‫؍‬ 2, 0.495 ؎ 0.019; n ‫؍‬ 4, 0.202 ؎ 0.010; n ‫؍‬ 6, 0.128 ؎ 0.006; n ‫؍‬ 8, 0.093 ؎ 0.005. The pairwise FRET efficiency determined independently by fluorescence lifetime imaging was 0.20 -0.24, identifying the M 2 receptor as a tetramer. The strategy described here yields an explicit estimate of oligomeric size on the basis of fluorescence properties alone. Its broader application could resolve the general question of whether G protein-coupled receptors exist as dimers or larger oligomers. The size of an oligomer has functional implications, and such information can be expected to contribute to an understanding of the signaling process.Much evidence now indicates that G protein-coupled receptors can exist as oligomers (1, 2), a development that has implications for all aspects of GPCR 4 -mediated signaling. Among the many questions prompted by the emergence of such structures is that of oligomeric size. Although commonly referred to as dimers, oligomers of GPCRs have been detected most often by means of coimmunoprecipitation or resonance energy transfer (3). As typically applied, neither technique can distinguish dimers from larger oligomers. The latter have been identified on the basis of their electrophoretic mobility (reviewed in Ref. 4), but the composition of the bands may be unclear, and the size under the conditions of electrophoresis may have little in common with that in the membrane. Larger oligomers also have been identified by approaches in which detection requires the colocalization of three or four proteins, each bearing a different tag (5-11), but such procedures place only a lower limit on the possible size of the array.There have been comparatively few attempts to examine the oligomeric status of a GPCR in a more quantitative and explicit manner. Measurements of BRET at different ratios of acceptor to donor have pointed to dimers of the melatonin receptor (12), the ␤ 1 -and ␤ 2 -adrenergic receptors (13), the M 1 , M 2 , and M 3 muscarinic receptors (14), and the neurotensin receptor (15)....

The Prevalence, Maintenance, and Relevance of G Protein–Coupled Receptor Oligomerization

“…Beginning with conventional pharmacological ligand binding (Park et al, 2002) and biochemical approaches , over a number of years, Wells and colleagues have provided a portfolio of data on this topic for the muscarinic M 2 receptor that are consistent with the potential for tetrameric organization. Most recently, this has derived from application of quantitative FRET (Pisterzi et al, 2010). Once again, these studies provided evidence of tetrameric organization of the receptor, potentially with a rhombic or parallelogram shape, at least in transiently transfected CHO cells.…”

Fluorescence Correlation Spectroscopy Analysis of Serotonin, Adrenergic, Muscarinic, and Dopamine Receptor Dimerization: The Oligomer Number Puzzle

“…For example, RET, FRAP, and TIRF studies of M 1 -, M 2 -, and M 3 -muscarinic receptors have reported monomers (Hern et al, 2010), dimers (Goin and Nathanson, 2006;Hern et al, 2010;Patowary et al, 2013), and tetramers (Pisterzi et al, 2010;McMillin et al, 2011;Patowary et al, 2013). RET and FRAP studies of b 1 -AR and b 2 -AR have reported monomers (James et al, 2006;Dorsch et al, 2009), dimers (Mercier et al, 2002;Dorsch et al, 2009), and higher-order oligomers (Dorsch et al, 2009;Fung et al, 2009).…”

“…Quantitative studies designed to determine the monomeric/oligomeric composition of class A GPCRs have reported the presence of monomers (James et al, 2006;Meyer et al, 2006;Dorsch et al, 2009;Hern et al, 2010;Kasai et al, 2011), dimers (Mercier et al, 2002;Canals et al, 2004;Goin and Nathanson, 2006;Harikumar et al, 2008;Dorsch et al, 2009;HerrickDavis et al, 2012;Knepp et al, 2012;Teitler and Klein, 2012;Patowary et al, 2013), tetramers (Fung et al, 2009;Pisterzi et al, 2010;Patowary et al, 2013), and higher-order oligomers (Guo et al, 2008;Dorsch et al, 2009;Albizu et al, 2010;O'Dowd et al, 2011). Coimmunoprecipitation, resonance energy transfer (RET), fluorescence lifetime imaging (FLIM), and bimolecular fluorescence complementation (BiFC) are commonly used methods to evaluate protein-protein interactions.…”

Coupling of G Proteins to Reconstituted Monomers and Tetramers of the M2 Muscarinic Receptor

“…Although the oligomeric status of muscarinic receptors in heart cells has not been identified directly, a tetramer has been inferred from the binding properties of receptors in native and detergent-solubilized myocardial membranes (1,2,43). Also, a quantitative assessment of FRET efficiencies between fluorophore-tagged protomers has indicated that the M 2 receptor exists wholly or predominantly as a tetramer in CHO cells (13). Clusters of ␤-adrenergic receptors have been identified in cardiac myocytes by means of fluorescent antibodies and near-field scanning optical microscopy (9).…”