Signalling the Future 29 Structure and activation of muscarinic acetylcholine receptors E.C. Hulme* 1 , Z.L. Lu†, J.W. Saldanha‡ and M.S. Bee† *Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London NW7 1AA, U.K., †MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, The University of Edinburgh, Academic Centre, 49 Little France Crescent, Edinburgh EH16 4SB, U.K., and ‡Department of Mathematical Biology, National Institute for Medical Research, Mill Hill, London NW7 1AA, U.K. Abstract 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. Introduction The seven-transmembrane (TM) G-protein-coupled recep- tors are the largest superfamily of TM signalling molecules in the mammalian genome. The muscarinic acetylcholine (ACh) receptors (mAChRs) were among the earliest members of this family to be defined pharmacologically [1]. They share essential sequence motifs with rhodopsin, which is the only seven-TM receptor for which direct three-dimensional structural information has been obtained [2–5]. Mutagenesis, protein labelling and spectroscopic studies suggest that similar mechanisms of activation operate in rhodopsin, several of the cationic amine receptors, including mAChRs, and neuropeptide receptors [6]. The five genetically distinct mAChR subtypes fall into two main groups. The M 1 ,M 3 and M 5 mAChRs couple preferentially to G-proteins of the G q /G 11 class, which leads to phosphoinositide breakdown. In contrast, M 2 and M 4 mAChRs couple primarily to G-proteins of the G i and G o classes, typically leading to adenylate cyclase inhibition and the activation of inward-rectifier potassium conductances. Among a plethora of other possible responses, in a suitable cellular context, all of the mAChR subtypes can activate non-receptor tyrosine kinases, transactivate the epidermal growth factor receptor and activate extracellular signal- related protein kinase cascades [7]. Key words: G-protein, G-protein-coupled receptor, rhodopsin. Abbreviations used: ACh, acetylcholine; mAChR, muscarinic acetylcholine receptor; NMS, N-methyl scopolamine; TM, transmembrane. 1 To whom correspondence should be addressed (ehulme@nimr.mrc.ac.uk). Use of scanning mutagenesis to probe the functions of amino acids in receptor sequences Scanning mutagenesis techniques allow the function of each amino acid side chain within a particular protein sequence to be assessed, relative to its neighbours, and provide information that can be used to interrogate or refine a homology model of the protein, in the absence of direct structural information. Alanine scanning mutagenesis (ala- nine itself is replaced by glycine, although this is not an ideal substitution) deletes the side chain of each residue beyond the β -carbon atom. In principle, this leaves a small hole in the three-dimensional structure of the receptor. In the M 1 mAChR, when expressed in COS-7 cells, this perturbation is surprisingly well tolerated. This technique pinpoints important residues, whose function can be examined by introducing a series of point mutations, or by techniques such as combinatorial histidine- or cysteine-substitution mutagenesis [8–10]. The simplest mechanistic model that is adequate to interpret receptor mutagenesis studies is the extended ternary complex model of agonist–receptor–G-protein interaction, which was first proposed to account for the phenomenon of agonist-independent signalling induced by particular mutations [11]. The primary assumption is that the receptor exists in one of two states – active or inactive – that are in an equilibrium that is governed by an equilibrium constant K (assumed to be ≪1 for the wild-type receptor). Ligands that bind to the receptor may favour either the activated state or the inactive state, or have neutral properties. The activated state of the receptor is postulated to bind the G-protein, and C  2003 Biochemical Society