Quantitative Analysis of Muscarinic Acetylcholine Receptor Homo- and Heterodimerization in Live Cells

Abstract

Although previous pharmacological and biochemical data support the notion that muscarinic acetylcholine receptors (mAChR) form homo-and heterodimers, the existence of mAChR oligomers in live cells is still a matter of controversy. Here we used bioluminescence resonance energy transfer to demonstrate that M 1 , M 2 , and M 3 mAChR can form constitutive homo-and heterodimers in living HEK 293 cells. Quantitative bioluminescence resonance energy transfer analysis has revealed that the cell receptor population in cells expressing a single subtype of M 1 , M 2 , or M 3 mAChR is predominantly composed of high affinity homodimers. Saturation curve analysis of cells expressing two receptor subtypes demonstrates the existence of high affinity M 1 /M 2 , M 2 /M 3 , and M 1 /M 3 mAChR heterodimers, although the relative affinity values were slightly lower than those for mAChR homodimers. Short term agonist treatment did not modify the oligomeric status of homo-and heterodimers. When expressed in JEG-3 cells, the M 2 receptor exhibits much higher susceptibility than the M 3 receptor to agonistinduced down-regulation. Coexpression of M 3 mAChR with increasing amounts of the M 2 subtype in JEG-3 cells resulted in an increased agonist-induced down-regulation of M 3 , suggesting a novel role of heterodimerization in the mechanism of mAChR long term regulation.Over the last decade, many studies have shown broad evidence that G protein-coupled receptors (GPCR) 3 interact with one another to form constitutive homo-or hetero-oligomers. GPCR dimerization has been implicated in many aspects of receptor pharmacology, such as maturation, ligand binding, activation, signaling, desensitization, endocytosis, and trafficking (1-3). In the early 1990s, several studies showed that muscarinic acetylcholine receptors (mAChR), like other GPCR, might be arranged in dimeric or oligomeric complexes. Complex binding curves for both muscarinic agonists and antagonists were interpreted as evidence for the existence of multiple states of affinity, which is consistent with the notion of binding sites located on dimeric receptor molecules (4, 5). More recent studies on mAChR have shown that such heterogeneity of affinity should rather be attributed to nucleotide-sensitive cooperative effects between interacting sites (6, 7). Because there seems to be one binding site per receptor molecule, cooperativity suggests that mAChR are actually oligomers formed by two or more interacting sites (6).Additional pharmacological evidence that mAChR can form dimers was provided by Maggio et al. (8), who used chimeric ␣ 2 -adrenergic/M 3 muscarinic receptors composed of the first five transmembrane domains (TMD) of one receptor and the last two TMD of the other. No binding or signaling was detected when either fusion protein was expressed alone, but coexpression of both chimeras rescued binding activity and signaling properties of both adrenergic and muscarinic ligands. These results were interpreted as intermolecular interactions between two inactive receptors now eng...

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

“…It follows that eGFP-M 2 and eYFP-trFz1 do not interact at levels of expression that lead to FRET in the case of eGFP-M 2 and eYFP-M 2 , although the possibility that eGFP-M 2 and eYFP-trFz1 form a dark complex in which the dipole of eGFP is perpendicular to that of eYFP cannot be ruled out. The present result is consistent with reports that the M 2 receptor does not coprecipitate with Frizzled-1 (51) or exhibit BRET when coexpressed with the Smoothened receptor, a homologue of Frizzled-1 (14). Heterologous expression was driven by the cytomegalovirus promoter in the latter investigation (14) and in the experiments described here.…”

“…The present result is consistent with reports that the M 2 receptor does not coprecipitate with Frizzled-1 (51) or exhibit BRET when coexpressed with the Smoothened receptor, a homologue of Frizzled-1 (14). Heterologous expression was driven by the cytomegalovirus promoter in the latter investigation (14) and in the experiments described here. These results suggest that the association between eGFP-M 2 and eYFP-M 2 is specific; they argue against the alternative possibility of stochastic interactions and suggest that it is difficult to populate the membrane at a level sufficient for such effects.…”

“…They also may arise from differences in the models themselves. Estimates of n in the earlier studies of GPCRs (12)(13)(14)(15)58) were based on a scheme introduced for gramicidin (16), which incorporates the approximation that the apparent efficiency of energy transfer can be obtained as the product of the pairwise efficiency and the fraction of interacting donors. That assumption is valid only for dimers; in the case of larger oligomers, it disregards the existence of multiple pathways for the de-excitation of donors and for the excitation of acceptors (17).…”

“…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). In each case, however, the data were analyzed in terms of a model developed for gramicidin and based on the notion of a dynamic equilibrium between the monomeric and oligomeric states (16).…”

Serotonin 5-HT2C receptor homodimerization is not regulated by agonist or inverse agonist treatment

“…For example, ligand binding has been reported to increase, decrease or have no effect on receptor dimerization. Somatostatin and gonadotropin-releasing hormone receptors have been reported to be expressed as monomers on the plasma membrane and to undergo ligand-induced dimerization (Rocheville et al, 2000;Cornea et al, 2001), while many other receptors have been reported to be constitutively dimerized on the plasma membrane in the absence of ligand (McVey et al, 2001;Babcock et al, 2003;Dinger et al, 2003;Guo et al, 2003;Terrillon et al, 2003;Berthouze et al, 2005;Goin and Nathanson, 2006). Agonist binding has been reported to have no effect or to promote dissociation of delta opioid receptor homomers (Cvejic et al, 1997;McVey et al, 2001), and thyrotropin homomers (Latif et al, 2002).…”