Phenotypic classification of mutants: a tool for understanding ligand binding and activation of muscarinic acetylcholine receptors.

GPCRs (G-protein-coupled receptors) such as the M(1) muscarinic receptor have so far proved recalcitrant to direct structure determination. Nevertheless systematic mutagenesis, particularly alanine scanning, has advanced our understanding of their structure-function relationships. GPCRs exhibit multiple conformational states with different affinities for and abilities to activate their cognate G-proteins. Ligand binding alters these conformational equilibria, thus promoting or inhibiting signalling. Alanine-scanning mutagenesis probes the relative contributions of a particular amino acid side chain to the stability of the ground and activated states of the receptor and its complexes. These determine the phenotype of the mutant receptor. Classification of the phenotypes suggests functional roles for particular amino acid side chains, allowing us to group them accordingly. From a rhodopsin-based homology model of the M(1) mAChR, a coherent view emerges of how these clusters of residues function in ligand anchoring, transduction of binding energy, global structural stabilization and selective stabilization of the ground state or the activated state of the receptor. We can identify differences in ligand-binding modes, and suggest inter- and intra-molecular interactions that are weakened or broken, or formed or intensified during acetylcholine-induced activation. In due course, we may be able to extend these insights to activation by unconventional agonists.

The hydration of the neurotransmitter acetylcholine in aqueous solution.

Neutron diffraction augmented with hydrogen isotope substitution has been used to examine the water structure around the acetylcholine molecular ion in aqueous solution. It is shown that the nearest-neighbor water molecules in the region around the trimethylammonium headgroup are located either in a ring around the central nitrogen atom or between the carbon atoms, forming a sheath around the onium group. Moreover the water molecules in this cavity do not bond to the onium group but rather form hydrogen bonds with water molecules in the surrounding aqueous environment. Given that in the bound state the onium headgroup must be completely desolvated, the absence of bonding between the onium headgroup and the surrounding water solvent may be selectively favorable to acetylcholine-binding in the receptor site. Away from the headgroup, pronounced hydrogen-bonding of water to the carbonyl oxygen is observed, but not to the ether oxygen in the acetylcholine chain.

Scanning mutagenesis studies of the M1 muscarinic acetylcholine receptor.

Following the solution of the structure of bovine rhodopsin by X-ray crystallography, it has been possible to build an improved homology model of the M(1) muscarinic acetylcholine receptor. This has been used to interpret the outcome of an extensive series of scanning and point mutagenesis studies on the transmembrane domain of the receptor. Potential intramolecular interactions enhancing the stability of the protein fold have been identified. The residues contributing to the binding site for the antagonist, N-methylscopolamine, and the agonist, acetylcholine have been mapped. The positively charged headgroups of these ligands appear to bind in a charge-stabilized aromatic cage formed by amino acid side chains in transmembrane (TM) helices 3, 6, and 7, while residues in TM 4 may participate in a peripheral docking site. Closure of the cage around the headgroup of acetylcholine may help to transduce binding energy into receptor activation, possibly disrupting a set of Van der Waals interactions between a set of residues underlying the binding site which help to constrain the receptor to the inactive state, in the absence of agonist. This may trigger the reorganization of a hydrogen bonding network between highly conserved residues in the core of the receptor, whose integrity is crucial for activation.

Structure and activation of muscarinic acetylcholine receptors.

A homology model of the M(1) muscarinic acetylcholine receptor, based on the X-ray structure of bovine rhodopsin, has been used to interpret the results of scanning and point mutagenesis studies on the receptor's transmembrane (TM) domain. Potential intramolecular interactions that are important for the stability of the protein fold have been identified. The residues contributing to the binding site for the antagonist, N -methyl scopolamine, and the agonist, acetylcholine, have been mapped. The positively charged headgroups of these ligands probably bind in a charge-stabilized aromatic cage formed by amino acid side chains in TM helices TM3, TM6 and TM7, while residues in TM4 may participate as part of a peripheral docking site. Closure of the cage around the headgroup of acetylcholine may be part of the mechanism for transducing binding energy into receptor activation, probably by disrupting a set of Van der Waals interactions between residues lying beneath the binding site that help to constrain the receptor to the inactive state, in the absence of agonist. This may trigger the reorganization of a hydrogen-bonding network between highly conserved residues in the core of the receptor, whose integrity is crucial for achievement of the activated state.

Transmembrane domains 4 and 7 of the M(1) muscarinic acetylcholine receptor are critical for ligand binding and the receptor activation switch.

Activation of the muscarinic acetylcholine receptors requires agonist binding followed by a conformational change, but the ligand binding and conformation-switching 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.

The conformational switch in muscarinic acetylcholine receptors.

The recently-determined structure of rhodopsin has provided a suitable basis for modeling the three-dimensional structure of the M1 muscarinic acetylcholine receptor. Using this as a framework for interpreting mutagenesis studies, we have been able to suggest most of the contacts which the receptor makes with acetylcholine and many of the intramolecular contacts which are important for the ground-state structure of the receptor. It is possible to outline a mechanism of G-protein interaction.

Scanning mutagenesis identifies amino acid side chains in transmembrane domain 5 of the M(1) muscarinic receptor that participate in binding the acetyl methyl group of acetylcholine.

The exofacial part of transmembrane domain 5 (TMD 5) of the cationic amine-binding subclass of 7-transmembrane receptors is thought to be important in binding the side chain of the agonist. Residues Ile-188 through Ala-196 in TMD 5 of the M(1) muscarinic acetylcholine receptor (mAChR) have been studied by Cys- and Ala-scanning mutagenesis. The results are consistent with a helical conformation for this sequence. The positively charged sulfhydryl reagent N-trimethyl-2-aminoethyl methanethiosulfonate reacted selectively with Phe-190 --> Cys, Thr-192 --> Cys, and Ala-193 --> Cys, indicating that the face of TMD 5 accessible from the binding site crevice is consistent with a recent model by Baldwin and colleagues of the transmembrane domain of the 7-transmembrane receptors. In contrast, the acetylcholine derivative bromoacetylcholine reacted selectively with Thr-192 --> Cys, which forms the focus of a group of amino acids (Ile-188, Thr-189, Thr-192, Ala-196) whose mutation decreased the binding affinity of the transmitter ACh itself. The center of this patch of residues is offset to one side of the binding pocket, suggesting that a rotation of TMD 5, relative to that implied by the Baldwin model, may be necessary to optimize the anchoring of acetylcholine within the binding site of the M(1) mAChR. An induced rotation of TMD 5 could contribute to the formation of the activated state of the receptor.

A network of conserved intramolecular contacts defines the off-state of the transmembrane switch mechanism in a seven-transmembrane receptor.

Activation of the rhodopsin-like 7-transmembrane (7-TM) receptors requires switching interhelical constraints that stabilize the inactive state to a new set of contacts in the activated state, which binds the cognate G-protein. The free energy to drive this is provided by agonist binding, which has higher affinity to the active than to the inactive conformation. We have sought specific interhelical constraint contacts, using the M(1) muscarinic acetylcholine receptor as a model. Histidine substitutions of particular groups of amino acids, in transmembrane domains 3, 6, and 7, created high-affinity Zn(2+) binding sites, demonstrating the close proximity of their side chains in the inactive state. Alanine point substitutions have shown the effect of weakening the individual intramolecular contacts. In each case, the acetylcholine affinity was increased, implying promotion of the activated state. These amino acids are highly conserved throughout the 7-TM receptor superfamily. We propose that they form an important part of a network of conserved interhelical contacts that defines the off-state of a general transmembrane switch mechanism.

ERKI/II regulation by the muscarinic acetylcholine receptors in neurons.

Muscarinic acetylcholine receptors (mAChRs) are known to be involved in learning and memory, but the molecular basis of their involvement is not well understood. The availability of new and specific biochemical tools has revealed a crucial role for the mitogen-activated protein kinase (MAPK) family in learning and memory. Here, we examine the link between mAChRs and MAPK in neurons. Using the MAPK kinase (MEK)-specific inhibitor PD98059, we first demonstrate a necessary role for active ERKI/II in long-term potentiation in vivo. Using phospho-specific antibodies that recognize the activated form of ERKI/II, we find that the level of ERKI/II activation in brain is regulated by mAChRs. Carbachol, a muscarinic agonist, induces prolonged activation of ERKI/II, without effect on the related kinase SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal protein kinase) in primary cortical cultures. ERKI/II activation is Src-dependent and partially phosphoinositide-3 kinase- and Ca(2+)-dependent but is PKC-independent. M1-M4 mAChR subtypes expressed in COS-7 cells can all induce ERKI/II activation using a signal transduction pathway similar to that operating in neurons. The nature of the signal transduction suggests that ERKI/II can serve as a convergence site for mAChR activation and other neurotransmitter receptors.

Alanine-scanning mutagenesis of transmembrane domain 6 of the M(1) muscarinic acetylcholine receptor suggests that Tyr381 plays key roles in receptor function.

Transmembrane domain 6 of the muscarinic acetylcholine (ACh) receptors is important in ligand binding and in the conformational transitions of the receptor but the roles of individual residues are poorly understood. We have carried out a systematic alanine-scanning mutagenesis study on residues Tyr381 to Val387 within the binding domain of the M(1) muscarinic ACh receptor. The seven mutations were then analyzed to define the effects on receptor expression, agonist and antagonist binding, and signaling efficacy. Tyr381Ala produced a 40-fold reduction in ACh affinity and a 50-fold reduction in ACh-signaling efficacy. Leu386Ala had similar but smaller effects. Asn382Ala caused the largest inhibition of antagonist binding. The roles of the hydroxyl group and benzene ring of Tyr381 were probed further by comparative analysis of the Tyr381Phe and Tyr381Ala mutants using three series of ligands: ACh analogs, azanorbornane- and quinuclidine-based ligands, and atropine analogs. These data suggested that the hydroxyl group of Tyr381 is primarily involved in forming hydrogen bond interactions with the oxygen atoms present in the side chain of ACh. We propose that this interaction is established in the ground state and preserved in the activated state of the receptor. In contrast, the Tyr381 benzene ring may form a cation-pi interaction with the positively charged head group of ACh that contributes to the activated state of the receptor but not the ground state. However, the hydroxyl group and benzene ring of Tyr381 both participate in interactions with azanorbornane- and quinuclidine-based ligands and atropine analogs in the ground state as well as the activated state of the receptor.

The conformational switch in 7-transmembrane receptors: the muscarinic receptor paradigm.

The rhodopsin-like superfamily of 7-transmembrane receptors is the largest class of signalling molecules in the mammalian genome. Recently, a combination of mutagenesis, biophysical and modelling studies have suggested a credible model for the alpha-carbon backbone in the transmembrane region of the 7-transmembrane receptors, and have started to reveal the structural basis of the conformational switch from the inactive to the active state. A key feature may be the replacement of a network of radial constraints, centred on transmembrane helix three, which stabilise the inactive ground state of the receptor by a new set of axial interactions which help to stabilise the activated state. Transmembrane helix three may act as a rotary switch in the activation mechanism.

The functional topography of transmembrane domain 3 of the M1 muscarinic acetylcholine receptor, revealed by scanning mutagenesis.

Alanine-scanning mutagenesis has been applied to residues 100-121 in transmembrane domain 3 of the M1 muscarinic acetylcholine receptor. This study complements a previous investigation of the triad Asp122-Arg123-Tyr124 (Lu, Z-L., Curtis, C. A., Jones, P. G., Pavia, J., and Hulme, E. C. (1997) Mol. Pharmacol. 51, 234-241). The results demonstrate the alpha-helical secondary structure of the domain and suggest its orientation with respect to the other transmembrane domains. The C-terminal part of the helix appears to be largely buried within the receptor structure. On its surface, there is a patch of three residues, Val113, Leu116, and Ser120, which may form intramolecular contacts that help to stabilize the inactive ground state of the receptor. Mutagenic disruption of these increased agonist affinity and signaling efficacy. In two cases (L116A and S120A), this led to constitutive activation of the receptor. Parallel to the helix axis and spanning the whole transmembrane region, a distinct strip of residues on one face of transmembrane domain 3 forms intermolecular (acetylcholine-receptor, receptor-G protein) or intrareceptor bonds that contribute to the activated state. The binding of acetylcholine may destabilize the first set of contacts while favoring the formation of the second.

Scanning mutagenesis of transmembrane domain 3 of the M1 muscarinic acetylcholine receptor.

Scanning mutagenesis of transmembrane domain 3 of the M1 muscarinic acetylcholine receptor has revealed a highly-differentiated alpha-helical structure. Lipid-facing residues are distinguished from a patch of residues which selectively stabilise the ground state of the receptor, and from a band of amino acids extending the full length of the helix, which contribute to the active agonist-receptor-G protein complex. The most important residues are strongly conserved in the GPCR superfamily.

The role of the aspartate-arginine-tyrosine triad in the m1 muscarinic receptor: mutations of aspartate 122 and tyrosine 124 decrease receptor expression but do not abolish signaling.

An Asp-Arg-Tyr triad occurs in a majority of rhodopsin-like G protein-coupled receptors. The fully conserved Arg is critical for G protein activation, but the function of the flanking residues is not well understood. We expressed in COS-7 cells m1 muscarinic receptors that were mutated at Asp122 and Tyr124. Most mutations at either position strongly attenuated or prevented the expression of binding sites for the antagonist [3H]N-methylscopolamine. However, sites that were expressed displayed unaltered affinity for the antagonist. Receptor protein, visualized with a carboxyl-terminally directed antibody, was reduced but never completely abolished. The effects of these mutations were partially reversed by the deletion of 129 amino acids from the third intracellular loop of the receptor. In several cases, comparison of immunocytochemistry with binding measurements suggested the presence of substantial amounts of inactive, presumably misfolded, receptor protein. Some of the variants that bound [3H]N-methylscopolamine underwent small changes in their affinities for acetylcholine. All retained nearly normal abilities to mediate an acetylcholine-induced phosphoinositide response. We propose that Asp122 and Tyr124 make intramolecular contacts whose integrity is important for efficient receptor folding but that they do not participate directly in signaling. The role of these residues is completely distinct from that of Arg123, whose mutation abolishes signaling without diminishing receptor expression.

The functional role of the binding site aspartate in muscarinic acetylcholine receptors, probed by site-directed mutagenesis.

Mutation of the Asp in transmembrane domain three of the muscarinic receptors to Asn (M1) or Glu (M1 and M2) strongly reduced the high-affinity component of agonist binding, and the M1 phosphoinositide response. Formation of the acetylcholine-receptor binary complex was also strongly inhibited. In contrast, binary complex formation by other agonists, as well as the antagonist (-)-N-methylscopolamine, was less affected. Ionic bonding between the carboxylate oxyanion and the positively-charged headgroup probably anchors acetylcholine when it is bound in its active conformation, but alternative, non-productive, binding modes, promoted by non-polar forces, may contribute to binary complex formation by other ligands.

The function of a highly-conserved arginine residue in activation of the muscarinic M1 receptor.

Arg123 in the rat muscarinic M1 receptor is part of the highly conserved triad Asp-Arg-Tyr found at the junction of transmembrane helix 3 with the second intracellular loop. Mutation of Arg123 to Lys, Ala, Leu, Glu and Gln had no effect on levels of receptor expression in COS-7 cells, or on affinities for the antagonist N-methylscopolamine. Acetylcholine stimulation of the Lys123 receptor evoked the same maximum phosphoinositide response as the wild type, although the potency was reduced six-fold, but mutation to other residues strongly disrupted receptor function. Mutation of Arg123 always decreased the ratio of the high affinity to the low affinity agonist binding constant, but the absolute effect on the latter varied from a 4-fold increase for the Lys123 to a small decrease for the Leu123 mutation. These results suggest that a positive charge at position 123 is central to the activation of G-proteins by the muscarinic M1 receptor.

Characterization of the rat m3 muscarinic acetylcholine receptor produced in insect cells infected with recombinant baculovirus.

The m3 muscarinic acetylcholine receptor from rat heterologously produced in insect cells after infection with a recombinant baculovirus has an apparent molecular mass of approximately 75 kDa. Polyclonal antibodies raised against a carboxy-terminal nonapeptide that is unique to the m3 subtype can detect the receptors produced in the insect cells by Western blot and can also immunoprecipitate solubilized receptor. Immunofluorescence microscopy as well as electron microscopy revealed that the receptor was located intracellularly, visualized as a ring around the nucleus of the infected insect cells. Solubilization of the receptor was accomplished with digitonin which was added in increments (over 10 min) to a final concentration of 0.8% (mass/vol). The solubilized receptor is unstable when the ligand-binding site is not protected by a ligand. Here the low-affinity ligand propylbenzilylcholine (approximately 10 nM) has demonstrable protective ability during solubilization, but the usefulness of this ligand is limited by a very slow off rate. From the behaviour of the solubilized receptor during DEAE-Sephacel chromatography and lectin-affinity chromatography it can be deduced that the receptor produced in insect cells is heterogeneously glycosylated in the producing insect cells.

The role of charge interactions in muscarinic agonist binding, and receptor-response coupling.

Site-directed mutagenesis has been used to evaluate the roles of the key aspartate and arginine residues in transmembrane domain three of the muscarinic receptors. The results suggest that the formation of an ionic bond between the Asp carboxylate group and the onium headgroup is essential to anchor acetylcholine in its active, bound conformation in both binary agonist-receptor and ternary agonist-receptor-G-protein complexes, but that secondary, non-productive binding modes, promoted by non-polar forces, may contribute to binary complex formation by other ligands. The positive charge of the arginyl side-chain is central to the recognition, and subsequent activation of G-proteins by the agonist-M1 mAChR complex.