Cholesterol as a determinant of cooperativity in the M2 muscarinic cholinergic receptor
Introduction
G protein-coupled receptors occur at least partly and perhaps exclusively as dimers or larger aggregates. They appear to be assembled at the time of biosynthesis and transported as such to the plasma membrane, where they may remain intact throughout the signaling process [1], [2], [3]. Although now much in evidence, oligomers of GPCRs went almost unrecognized for many years, and their functional role remains largely unexplored. It has been suggested, however, that they host a complex interplay of cooperative effects whereby signaling is regulated by agonists and guanyl nucleotides [4], [5], [6].
Most GPCRs embody an intriguing heterogeneity that emerges in binding assays as a GTP-sensitive dispersion of affinities for agonists [2], [7]. The effect of guanyl nucleotides is mediated by the G protein and cooperative in nature (e.g., Ref. [8]). It was recognized early on that cooperativity also might account for the heterogeneity detected by agonists (e.g., Refs. [9], [10], [11]), but tests for such effects in the muscarinic receptor proved negative [10]. Furthermore, most biochemical data suggested that GPCRs exist wholly or largely as monomers, at least in solution (e.g., Refs. [12], [13], [14], [15]). There also was widespread acceptance of the view that heterogeneity derives from a transient complex between a monomeric receptor and the G protein [11], [12], although such schemes can be problematic when applied in a quantitative manner [16], [17].
If cooperativity underlies the heterogeneity revealed by agonists, it seems likely to occur in other contexts. It was inferred from the early observation that the net dissociation of [3H]alprenolol from β-adrenergic receptors was faster when initiated by an unlabeled ligand than by dilution [18], [19], although rebinding of the radioligand could not be ruled out as a determinant of the rate following dilution [19]. Negative cooperativity has been invoked more recently to account for kinetic effects in the binding of bradykinin to the B2 bradykinin receptor [20], but it could not be detected in kinetic studies on the D2 dopamine receptor [21].
In studies at equilibrium, Hill coefficients greater than 1 have prompted suggestions of positive cooperativity in the binding of [3H]QNB to muscarinic cholinergic receptors [22], [23] and of some antagonists to receptors labeled by [3H]histamine [24]. Positive cooperativity also is apparent in the observation that one ligand can promote the binding of another in some systems. With M2 muscarinic receptors and G proteins purified as a complex from porcine atria, the binding of [3H]AF-DX 384 was bell-shaped with respect to the concentration of the agonist oxotremorine-M [5]. With heteromers of the δ- and κ-opiate receptors, the apparent affinity of the δ-selective agonist [d-Pen2, d-Pen5]enkephalin was at least 50-fold higher in the presence of the κ-selective agonist U69593 and vice versa [25]. Similarly, β1-selective ligands increased the affinity of β2-selective ligands and vice versa at heteromers of the β1- and β2-adrenergic receptors [26]. Negative cooperativity has been inferred from noncompetitive inhibitory effects at muscarinic [27] and histamine receptors [28], [29]. With heteromers of the CCR5 and CCR2b chemokine receptors, the CCR5-specific ligand MIP-1β was found to inhibit the binding of [125I]MCP-1 to CCR2b and vice versa [30].
A pattern of latent sites and attendant noncompetitive effects has pointed to cooperativity in the binding of antagonists to cardiac muscarinic receptors extracted in cholate–NaCl or Lubrol-PX [5], [31] and to D2 dopamine receptors in membranes from CHO and Sf9 cells [21], [32]. In the case of the muscarinic receptor, those effects can be rationalized quantitatively in terms of cooperativity among at least four interacting sites [31]. That in turn implies a tetramer or larger oligomer, and differently tagged mutants of the M2 muscarinic receptor have been coimmunoprecipitated from extracts of baculoviral-infected Sf9 cells [33], [34]. In preliminary studies, however, the M2 receptor extracted from Sf9 membranes failed to exhibit the cooperative effects found in myocardial extracts under the same conditions.
Manifestations of cooperativity appear to depend upon environmental factors. The cardiac muscarinic receptor exhibits latent sites and noncompetitive binding upon extraction in cholate–NaCl but not in digitonin–cholate [31]. Similar effects at the D2 dopaminergic receptor are lost or much diminished in the presence of sodium ions [32]. It has been suggested that cooperativity in the muscarinic receptor may be regulated by cholesterol [31], which is known to modulate the properties of several GPCRs and other membrane proteins [35]. Levels of cholesterol in the plasma membrane of Sf9 cells are comparatively low [36]. In the present report, M2 muscarinic receptors extracted from cholesterol-supplemented Sf9 membranes are shown to exhibit the cooperative effects found with the receptor from heart.
Section snippets
Ligands, detergents, and other materials
N-[3H]Methylscopolamine was purchased as the chloride salt from PerkinElmer Life Sciences (lots 3406081 and 3474009, 83.5 Ci/mmol; 3436143, 3499213, and 3538031, 81.0 Ci/mmol) and as the bromide salt from Amersham Biosciences (batches B-32, 84.0 Ci/mmol; B-35 and B-36, 81.0 Ci/mmol). Mass spectra provided by the manufacturer indicated that the samples from Amersham Biosciences were devoid of contaminating scopolamine. The purity of material from PerkinElmer Life Sciences has been confirmed
Effect of cholesterol on electrophoretic mobility, apparent capacity and stability
Levels of cholesterol in membranes from Sf9 cells were only about 4% of those in sarcolemmal membranes from porcine atria (Fig. 1). The level in Sf9 membranes increased to 108% of that in sarcolemmal membranes upon treatment of the former with the inclusion complex at a concentration of 0.5 mM with respect to cholesterol. This method was used to prepare the treated Sf9 membranes used in subsequent experiments. Cholesterol-methyl-β-cyclodextrin also was added to growing Sf9 cells, but the complex
Noncompetitive effects induced by cholesterol
Cooperativity in the binding of agonists may play a central mechanistic role in signaling via muscarinic and other GPCRs [4], [5], [6], [22], [29]. Evidence for cooperativity includes a pattern of latent sites and noncompetitive inhibition revealed by antagonists at the muscarinic receptor extracted from porcine atria in cholate–NaCl. The apparent capacity for [3H]QNB is about twice that for [3H]NMS [31], yet comparatively low concentrations of unlabeled NMS inhibit the binding of [3H]QNB at
Acknowledgements
The managers and staff of Quality Meat Packers Limited are gratefully acknowledged for their generous donation of porcine atria. This investigation was supported by the Canadian Institutes of Health Research (MOP43990) and the Heart and Stroke Foundation of Ontario (T4914, T5650).
References (71)
- et al.
Emerging role of homo- and heterodimerization in G protein-coupled receptor biosynthesis and maturation
Trends Pharmacol Sci
(2005) - et al.
Cooperativity manifest in the binding properties of purified cardiac muscarinic receptors
J Biol Chem
(1995) - et al.
Receptor–effector coupling by G proteins
Biochim Biophys Acta
(1990) - et al.
Relationship between the β-adrenergic receptor and adenylate cyclase
J Biol Chem
(1977) - et al.
A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled β-adrenergic receptor
J Biol Chem
(1980) - et al.
Purification of the muscarinic acetylcholine receptor from porcine brain
J Biol Chem
(1985) - et al.
β-Adrenergic receptors: evidence for negative cooperativity
Biochem Biophys Res Commun
(1975) - et al.
Negative cooperativity among β-adrenergic receptors in frog erythrocyte membranes
J Biol Chem
(1976) - et al.
Negative cooperativity in the human bradykinin B2 receptor
J Biol Chem
(1998) - et al.
Guanine nucleotide regulation of a mammalian myocardial muscarinic receptor system. Evidence for homo- and heterotropic cooperativity in ligand binding analyzed by computer-assisted curve fitting
J Biol Chem
(1985)
Guanine nucleotide-induced positive cooperativity in muscarinic–cholinergic antagonist binding
Biochem Biophys Res Commun
Dopamine D2 receptor dimer formation: evidence from ligand binding
J Biol Chem
Nature of the oligomers formed by muscarinic M2 acetylcholine receptors in Sf9 cells
Eur J Pharmacol
Lipid composition of Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn) insect cells used for baculovirus infection
FEBS Lett
Effects of N-ethylmaleimide on conformational equilibria in purified cardiac muscarinic receptors
J Biol Chem
Aggregation of VSV M protein is reversible and mediated by nucleation sites: implications for viral assembly
Virology
C5a receptor oligomerization. I. Disulfide trapping reveals oligomers and potential contact surfaces in a G protein-coupled receptor
J Biol Chem
Organization of the G protein-coupled receptors rhodopsin and opsin in native membranes
J Biol Chem
Transducin activation in electropermeabilized frog rod outer segments is highly amplified, and a portion equivalent to phosphodiesterase remains membrane-bound
J Biol Chem
On the nature of allosteric transitions: a Plausible model
J Mol Biol
Allostery in very large molecular assemblies
Biophys Chem
Cholesterol depletion from the plasma membrane triggers ligand-independent activation of the epidermal growth factor receptor
J Biol Chem
Role of sterols in modulating the human μ-opioid receptor function in Saccharomyces cerevisiae
J Biol Chem
Cholesterol modulates the antagonist-binding function of hippocampal serotonin1A receptors
Biochim Biophys Acta
Cholesterol as stabilizer of the oxytocin receptor
Biochim Biophys Acta
Muscarinic cholinergic signaling in cardiac myocytes: dynamic targeting of M2AChR to sarcolemmal caveolae and eNOS activation
Life Sci
Dynamic targeting of the agonist-stimulated M2 muscarinic acetylcholine receptor to caveolae in cardiac myocytes
J Biol Chem
Rhodopsin-cholesterol interactions in bovine rod outer segment disk membranes
Biochim Biophys Acta-Biomembr
Oligomerization of G protein-coupled receptors
J Cell Sci
Oligomerization of G protein-coupled receptors: past, present, and future
Biochemistry
Effects of adenyl nucleotides and carbachol on cooperative interactions among G proteins
Biochemistry
Cardiac muscarinic receptors. Cooperativity as the basis for multiple states of affinity
Biochemistry
Solubilization and characterization of guanine nucleotide-sensitive muscarinic agonist binding sites from rat myocardium
Br J Pharmacol
The binding of agonists to brain muscarinic receptors
Mol Pharmacol
β-Adrenergic receptors: biochemical mechanisms of physiological regulation
Physiol Rev
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2012, Comprehensive BiophysicsSf9 cells: A versatile model system to investigate the pharmacological properties of G protein-coupled receptors
2010, Pharmacology and TherapeuticsCitation Excerpt :Increasing the cholesterol content of Sf9 cell membranes with a cholesterol-β-cyclodextrin complex enhanced the binding of [125I]-Iodoaminopotentidine ([125I]APT) to the rH2R by up to 169% (Beukers et al., 1997). Another example has been reported for the M2R expressed in Sf9 cells (Colozo et al., 2007). In contrast to the M2R isolated from porcine sarcolemma, M2R extracted from Sf9 cells showed no antagonist binding cooperativity.
Oligomeric size of the M<inf>2</inf> muscarinic receptor in live cells as determined by quantitative fluorescence resonance energy transfer
2010, Journal of Biological ChemistryCitation Excerpt :M2 receptors also can be purified from Sf9 cells as monomers that regroup almost exclusively as tetramers upon reconstitution in phospholipid vesicles (4). Functional studies on the M2 receptor in native preparations and after reconstitution as a tetramer have revealed noncompetitive effects in the binding of muscarinic antagonists, and the pattern can be described quantitatively in terms of cooperative interactions among at least four sites (4, 18–20). Cooperativity indicative of a tetramer also has been reported for the binding of the agonist oxotremorine-M to the muscarinic receptor purified from porcine atria (18).
- 1
Current address: Department of Pharmacology, Case Western Reserve University, 10900 Euclid Avenue, BRB909B, Cleveland, OH 44106-4965, United States.
- 2
Current address: Receptors and Hormone Action Section, Clinical Endocrinology Branch, NIDDK, National Institutes of Health, 50 South Drive, Bethesda, MD 20892-8029, United States.