Evidence for a Tandem Two-site Model of Ligand Binding to Muscarinic Acetylcholine Receptors

Jakubík, Ján; El‐Fakahany, Esam E.; Tuček, Stanislav

doi:10.1074/jbc.m000112200

Cited by 45 publications

(45 citation statements)

References 34 publications

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“…7) showed biphasic association as well as biphasic dissociation with the proportion of slow dissociation increasing with time of association. These results are compatible with at least two receptor-ligand interaction models: 1) isomerization of the receptor-antagonist complex (Järv et al, 1979) and 2) the tandem two-site model (Jakubík et al, 2000). The same pattern of binding kinetics of LMP at the M 3 receptors (Fig.…”

Section: Mechanism Of Methoctramine Binding Discussionsupporting

confidence: 76%

“…Parameters of binding kinetics from FRET measurements were obtained by fitting a tandem two-site model (Jakubík et al, 2000) to the pooled data with subtracted background values using the program COPASI (www.copasi.org) (Hoops et al, 2006). Initial parameter estimates and background values were obtained by fitting two exponential growth and two exponential decay functions, respectively, to the individual data sets using the program Grace.…”

Section: Data Treatmentmentioning

confidence: 99%

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Molecular Mechanisms of Methoctramine Binding and Selectivity at Muscarinic Acetylcholine Receptors

et al. 2014

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Methoctramine (N,-methyl]hexyl]-1,8-octane] diamine) is an M 2 -selective competitive antagonist of muscarinic acetylcholine receptors and exhibits allosteric properties at high concentrations. To reveal the molecular mechanisms of methoctramine binding and selectivity we took advantage of reciprocal mutations of the M 2 and M 3 receptors in the second and third extracellular loops that are involved in the binding of allosteric ligands. To this end we performed measurements of kinetics of the radiolabeled antagonists N-methylscopolamine (NMS) in the presence of methoctramine and its precursors, fluorescence energy transfer between green fluorescent protein-fused receptors and an Alexa-555-conjugated precursor of methoctramine, and simulation of molecular dynamics of methoctramine association with the receptor. We confirm the hypothesis that methoctramine high-affinity binding to the M 2 receptors involves simultaneous interaction with both the orthosteric binding site and the allosteric binding site located between the second and third extracellular loops. Methoctramine can bind solely with low affinity to the allosteric binding site on the extracellular domain of NMSoccupied M 2 receptors by interacting primarily with glutamate 175 in the second extracellular loop. In this mode, methoctramine physically prevents dissociation of NMS from the orthosteric binding site. Our results also demonstrate that lysine 523 in the third extracellular loop of the M 3 receptors forms a hydrogen bond with glutamate 219 of the second extracellular loop that hinders methoctramine binding to the allosteric site at this receptor subtype. Impaired interaction with the allosteric binding site manifests as low-affinity binding of methoctramine at the M 3 receptor.

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Section: Mechanism Of Methoctramine Binding Discussionsupporting

confidence: 76%

Section: Data Treatmentmentioning

confidence: 99%

Molecular Mechanisms of Methoctramine Binding and Selectivity at Muscarinic Acetylcholine Receptors

et al. 2014

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“…A third possibility is that the position of TM 4 in the , located at the N terminus of TM 3. Asp 99 has been proposed to act as a primary contact residue before ligands enter the central binding site (34). This cluster of amino acids may form a peripheral ligand contact site (Fig.…”

Section: Discussionmentioning

confidence: 99%

Transmembrane Domains 4 and 7 of the M1Muscarinic Acetylcholine Receptor Are Critical for Ligand Binding and the Receptor Activation Switch

Lu¹,

Saldanha²,

Hulme³

2001

Journal of Biological Chemistry

106

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Activation of the muscarinic acetylcholine receptors requires agonist binding followed by a conformational change, but the ligand binding and conformationswitching residues have not been completely identified. Systematic alanine-scanning mutagenesis has been used to assess residues 142-164 in transmembrane helix 4 and 402-421 in transmembrane helix 7 of the M 1 muscarinic acetylcholine receptor. Several inward-facing amino acid side chains in the exofacial parts of transmembrane helices 4 and 7 contribute to acetylcholine binding. Alanine substitution of the aromatic residues in this group reduced signaling efficacy, suggesting that they may form part of a charge-stabilized aromatic cage, which triggers rotation and movement of the transmembrane helices. The mutation of adjacent residues modulated receptor activation, either reducing signaling or causing constitutive activation. In the buried endofacial section of transmembrane helix 7, alanine substitution mutants of the conserved NSXXNPXXY motif displayed strongly reduced signaling efficacy, despite having increased or unchanged acetylcholine affinity. These residues may have dual functions, forming intramolecular contacts that stabilize the receptor in the inactive ground state, but that are broken, allowing them to form new intramolecular bonds in the activated state. This conformational rearrangement is critical to produce a G protein binding site and may represent a key mechanism of receptor activation. Muscarinic acetylcholine receptors (mAChRs)1 belong to the rhodopsin-like family of 7-transmembrane (7-TM) receptors. Agonists bind to these receptors at the extracellular end. This leads to the binding and activation of a G protein at the intracellular face. These receptors are characterized by the possession of a particular set of evolutionarily conserved amino acids, mostly located in the 7-TM helices, implying that they may share a common mechanism of activation. However, the topography of the binding pockets and the conformational changes related to agonist-induced receptor activation are incompletely understood. In recent studies on the M 1 mAChR, we have identified ligand contact residues in TM 3 (1), 5 (2), and 6 (3) and inferred a strip of residues in TM 3 contributing to the activated state of the M 1 mAChR by using alanine-scanning mutagenesis, or cysteine-scanning mutagenesis. The three-dimensional crystal structure of the ground state of rhodopsin at 2.8 Å (4) has provided a framework for modeling the mAChRs and allowed us to interpret most of the information obtained by mutagenesis studies on the M 1 mAChR.In the initial projection map of rhodopsin (5), TM 4 appeared as an outlier of the helical bundle, with a large lipid-exposed surface and few polar residues. Consequently, its function has received little attention. However, a recent photo-activated chromophore cross-linking study of rhodopsin has shown a flip-over of the ionone ring from the neighborhood of TM 6 to TM 4 during receptor activation, implying that a substantial movement of TM ...

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“…Bo-PZ association with the fluorescent chimeras is probably also complex as it reveals a biphasic time-course taking place over several hundreds of seconds. Further work will be required to characterize these binding components and to test whether they reflect (i) the existence of distinct conformational states of the M1 receptor (with different affinities for Bo-PZ), (ii) interconversion according to a two-step isomerization process (Järv et al 1979;Luthin and Wolfe 1984) involving, or not, ligand translocation from a peripheral to a central receptor site (Jakubik et al 2000), or (iii) binding of a second ligand molecule according to the two-site tandem model (Jakubik et al 2000). To these ends, the use of a nonseparative Bo-PZ binding assay with real-time monitoring of binding events and, probably, higher sensitivity towards transient binding states certainly represents an advantage over classical filtration radioligand assays.…”

Section: Discussionmentioning

confidence: 99%

“…The availability of new fluorescent muscarinic ligands (agonists and allosteric effectors) should aid characterization of receptor conformational states, and localization and dissection of their modes of interaction with accessory and/ or modulatory sites (Jakubik et al 2000;Birdsall et al 2001) present on the muscarinic M1 receptor protein.…”

Section: Discussionmentioning

confidence: 99%

Fluorescence resonance energy transfer to probe human M1 muscarinic receptor structure and drug binding properties

Ilien

Franchet

Bernard

et al. 2003

Journal of Neurochemistry

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Human M1 muscarinic receptor chimeras were designed (i) to allow detection of their interaction with the fluorescent antagonist pirenzepine labelled with Bodipy [558/568], through fluorescence resonance energy transfer, (ii) to investigate the structure of the N-terminal extracellular moiety of the receptor and (iii) to set up a fluorescence-based assay to identify new muscarinic ligands. Enhanced green (or yellow) fluorescent protein (EGFP or EYFP) was fused, through a linker, to a receptor N-terminus of variable length so that the GFP barrel was separated from the receptor first transmembrane domain by six to 33 amino-acids. Five fluorescent constructs exhibit high expression levels as well as pharmacological and functional properties superimposable on those of the native receptor. Bodipy-pirenzepine binds to the chimeras with similar kinetics and affinities, indicating a similar mode of interaction of the ligand with all of them. From the variation in energy transfer efficiencies determined for four different receptor-ligand complexes, relative donor (EGFP)-acceptor (Bodipy) distances were estimated. They suggest a compact architecture for the muscarinic M1 receptor amino-terminal domain which may fold in a manner similar to that of rhodopsin. Finally, this fluorescence-based assay, prone to miniaturization, allows reliable detection of unlabelled competitors.

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Evidence for a Tandem Two-site Model of Ligand Binding to Muscarinic Acetylcholine Receptors

Cited by 45 publications

References 34 publications

Molecular Mechanisms of Methoctramine Binding and Selectivity at Muscarinic Acetylcholine Receptors

Molecular Mechanisms of Methoctramine Binding and Selectivity at Muscarinic Acetylcholine Receptors

Transmembrane Domains 4 and 7 of the M1Muscarinic Acetylcholine Receptor Are Critical for Ligand Binding and the Receptor Activation Switch

Fluorescence resonance energy transfer to probe human M1 muscarinic receptor structure and drug binding properties

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