The muscarinic M3 acetylcholine receptor exists as two differently sized complexes at the plasma membrane

Abstract

The literature on GPCR (G-protein-coupled receptor) homo-oligomerization encompasses conflicting views that range from interpretations that GPCRs must be monomeric, through comparatively newer proposals that they exist as dimers or higher-order oligomers, to suggestions that such quaternary structures are rather ephemeral or merely accidental and may serve no functional purpose. In the present study we use a novel method of FRET (Förster resonance energy transfer) spectrometry and controlled expression of energy donor-tagged species to show that M(3)Rs (muscarinic M(3) acetylcholine receptors) at the plasma membrane exist as stable dimeric complexes, a large fraction of which interact dynamically to form tetramers without the presence of trimers, pentamers, hexamers etc. That M(3)R dimeric units interact dynamically was also supported by co-immunoprecipitation of receptors synthesized at distinct times. On the basis of all these findings, we propose a conceptual framework that may reconcile the conflicting views on the quaternary structure of GPCRs.

Protease-activated Receptor 1 (PAR1) and PAR4 Heterodimers Are Required for PAR1-enhanced Cleavage of PAR4 by α-Thrombin

“…These data will be essential for the development of quantitative studies to examine the stability of the homodimers compared with the heterodimers. An alternative hypothesis is that PAR1 homodimers may interact with PAR4 homodimers to form higher order oligomers as has been described for other GPCRs (36).…”

“…The evidence against this interpretation is mutations that disrupt dimerization also disrupt PAR-1-assisted PAR4 cleavage. This will best be addressed by high resolution time-resolved imaging techniques that are currently being developed (36). A third possibility is that ␣-thrombin mediated the interaction by forming a trimolecular complex with PAR1 and PAR4.…”

The Molecular Basis of Oligomeric Organization of the Human M3Muscarinic Acetylcholine Receptor

“…However, recent studies based on the guanine nucleotide sensitivity of ligand binding to M 2 muscarinic receptors in both reconstituted systems and native sarcolemmal membranes are consistent with the receptor existing and functioning as a tetramer (Redka et al, 2014). Moreover, via application of spectrally resolved, multiphoton fluorescence resonance energy transfer (FRET), Patowary et al (2013) recently identified the predominant oligomeric form of the human M 3 muscarinic receptor (hM 3 R) expressed in a human embryonic kidney (HEK) 293-derived cell line as a tetramer, and that this exists in equilibrium with dimeric species. Atomic level structures of the m-opioid receptor (Manglik et al, 2012) and turkey b 1 -adrenoceptor have identified both a potential protein-protein interface involving residues from each transmembrane domain (TMD) I and helix VIII and an interface involving residues from TMD V with residues from either TMD VI (m-opioid receptor) or TMD IV (b 1 -adrenoceptor).…”

Protease-activated receptors in hemostasis

“…The serotonin 5-HT-2C receptors are primarily dimers that become disrupted when treated with an antagonist and the M3-muscarinic receptors are found as dimers and tetramers. [72][73][74] These studies highlight the potential dynamic nature of GPCR organization within the membrane. PARs are no exception as evidence exists for both constitutive and agonist-induced dimers.…”

“…Future studies that use advanced microscopy such as spatial intensity distribution analysis and spectrally resolved 2-photon microscopy are necessary to shed light on the stoichiometry and dynamics of PAR homo-and hetero-oligomerization as they have for other GPCRs. [72][73][74] The point remains that these highpowered techniques are, for the most part, limited to exogenously expressed receptors and are no substitution for ultimately analyzing receptors in native tissues. Naturally occurring mutations or polymorphisms may provide further evidence of cooperation between PARs.…”