Elsevier

Biochemical Pharmacology

Volume 74, Issue 2, 15 July 2007, Pages 236-255
Biochemical Pharmacology

Cholesterol as a determinant of cooperativity in the M2 muscarinic cholinergic receptor

https://doi.org/10.1016/j.bcp.2007.04.009Get rights and content

Abstract

M2 muscarinic receptor extracted from Sf9 cells in cholate–NaCl differs from that extracted from porcine sarcolemma. The latter has been shown to exhibit an anomalous pattern in which the capacity for N-[3H]methylscopolamine (NMS) is only 50% of that for [3H]quinuclidinylbenzilate (QNB), yet unlabeled NMS exhibits high affinity for all of the sites labeled by [3H]QNB. The effects can be explained in terms of cooperativity within a receptor that is at least tetravalent [Park PS, Sum CS, Pawagi AB, Wells JW. Cooperativity and oligomeric status of cardiac muscarinic cholinergic receptors. Biochemistry 2002;41:5588–604]. In contrast, M2 receptor extracted from Sf9 membranes exhibited no shortfall in the capacity for [3H]NMS at either 30 or 4 °C, although there was a time-dependent inactivation during incubation with [3H]NMS at 30 °C; also, any discrepancies in the affinity of NMS were comparatively small. The level of cholesterol in Sf9 membranes was only 4% of that in sarcolemmal membranes, and it was increased to about 100% by means of cholesterol-methyl-β-cyclodextrin. M2 receptors extracted from treated Sf9 membranes were stable at 30 and 4 °C and resembled those from heart. Cholesterol induced a marked heterogeneity detected in the binding of both radioligands, including a shortfall in the apparent capacity for [3H]NMS, and there were significant discrepancies in the apparent affinity of NMS as estimated directly and via the inhibition of [3H]QNB. The data can be described quantitatively in terms of cooperative effects among six or more interacting sites. Cholesterol therefore appears to promote cooperativity in the binding of antagonists to the M2 muscarinic 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).

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    Current address: Department of Pharmacology, Case Western Reserve University, 10900 Euclid Avenue, BRB909B, Cleveland, OH 44106-4965, United States.

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    Current address: Receptors and Hormone Action Section, Clinical Endocrinology Branch, NIDDK, National Institutes of Health, 50 South Drive, Bethesda, MD 20892-8029, United States.

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