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

Abstract

Background:The allosteric interaction between agonists and guanylyl nucleotides reports on the interaction between G protein-coupled receptors and G proteins. Results: Such allostery differs in kind between reconstituted monomers and tetramers of the M 2 muscarinic receptor. Conclusion: Monomers and tetramers mediate allostery via different mechanisms. Significance: Only tetramers resemble muscarinic receptors in myocardial membranes in the nature of their sensitivity to guanylyl nucleotides.

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

“…Such results have been interpreted as being consistent with the capacity of the receptor to form dimers in a range of ways via a number of distinct interfaces (McMillin et al, 2011;Hu et al, 2012). Recent studies have suggested that rather than being limited to dimers, muscarinic receptors, including both the M 2 (Redka et al, 2014) and M 3 (Patowary et al, 2013) subtypes, may form and potentially function as tetramers. We therefore initiated studies to address such a possibility for structural organization and assess if previous results (McMillin et al, 2011) would be consistent with such a model.…”

“…This may be relevant to the observations both herein and in the studies of McMillin et al (2011) of a clear role for amino acids at the cytoplasmic end of TMD IV, as this is not inherently predicted by the rhombic tetramer models. Organization of other class A GPCRs as tetramers has also been supported by approaches, including reconstitution experiments in artificial lipid bilayers (b 2 -adrenoceptor) (Fung et al, 2009), chemical cross-linking studies (dopamine D 2 ) (Guo et al, 2005), and detailed characterization of ligand binding studies (muscarinic M 2 ) (Park et al, 2002;Redka et al, 2014).…”

“…Many of these studies have assumed that the major organizational structure of such complexes is as a dimer or have been unable to discriminate between the presence of dimers and higher order organization. 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.…”

“…A series of studies has demonstrated the capacity of monomers of muscarinic receptor subtypes to self-associate in both living cells (Goin and Nathanson, 2006;Alvarez-Curto et al, 2010b) and following purification and reconstitution Redka et al, 2014). Many of these studies have assumed that the major organizational structure of such complexes is as a dimer or have been unable to discriminate between the presence of dimers and higher order organization.…”

Graded activation and free energy landscapes of a muscarinic G-protein–coupled receptor

“…Whereas ARC exhibits lower affinity than IXO or QNB as investigated in the present study, certain partial agonists are not necessarily weaker binders compared with full or inverse agonists (16,24). Nevertheless, there appears to be a correlation between efficacy and the breadth of the dispersion of affinities for agonists of the M 2 receptor (16,20,25). The GaMD simulations provided important insights into the binding mechanism of three studied ligands and GPCR graded activation.…”