The X-ray crystal structure of the M3 muscarinic acetylcholine receptor bound to the bronchodilator drug tiotropium is reported; comparison of this structure with that of the M2 muscarinic acetylcholine receptor reveals key differences that could potentially be exploited to develop subtype-selective drugs. The muscarinic acetylcholine receptors (mAChRs) constitute a family of G-protein-coupled receptors. These membrane proteins are targets for treatment of a broad range of conditions, including Alzheimer's disease, schizophrenia and chronic obstructive pulmonary disease. The five mAChR subtypes (M1–M5) share a high degree of sequence homology, but show marked differences in G-protein-coupling preference and physiological function. This pair of papers from Brian Kobilka's group presents the structures of two of the five subtypes. Haga et al. report the X-ray crystal structure of the M2 receptor, which is essential for the physiological control of cardiovascular function; Kruse et al. determine the structure of the M3 receptor, active in the bronchial airways and elsewhere. Comparison of the two structures reveals key differences that could potentially be exploited to develop subtype-selective drugs. Acetylcholine, the first neurotransmitter to be identified1, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1–M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate2,3,4. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences5,6. We describe here the structure of the Gq/11-coupled M3 mAChR (‘M3 receptor’, from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the Gi/o-coupled M2 receptor7, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.

Muscarinic acetylcholine receptors (mAChRs; M₁–M₅) play key roles in regulating the activity of many important functions of the central and peripheral nervous system. Because of the lack of ligands endowed with a high degree of receptor subtype selectivity and the fact that most tissues or cell types express two or more mAChR subtypes, identification of the physiological and pathophysiological roles of the individual mAChR subtypes has proven a difficult task. To circumvent these difficulties, several laboratories recently employed gene-targeting techniques to generate mutant mouse strains deficient in each of the five mAChR subtypes. Phenotyping studies showed that each mutant mouse line displayed characteristic physiological, pharmacological, behavioral, biochemical, or neurochemical deficits. The novel insights gained from these studies should prove instrumental for the development of novel classes of muscarinic drugs.

Impaired functioning of pancreatic β cells is a key hallmark of type 2 diabetes. β cell function is modulated by the actions of different classes of heterotrimeric G proteins. The functional consequences of activating specific β cell G protein signaling pathways in vivo are not well understood at present, primarily due to the fact that β cell G protein-coupled receptors (GPCRs) are also expressed by many other tissues. To circumvent these difficulties, we developed a chemical-genetic approach that allows for the conditional and selective activation of specific β cell G proteins in intact animals. Specifically, we created two lines of transgenic mice each of which expressed a specific designer GPCR in β cells only. Importantly, the two designer receptors differed in their G protein-coupling properties (G _q/11 versus G _s ). They were unable to bind endogenous ligand(s), but could be efficiently activated by an otherwise pharmacologically inert compound (clozapine-N-oxide), leading to the conditional activation of either β cell G _q/11 or G _s G proteins. Here we report the findings that conditional and selective activation of β cell G _q/11 signaling in vivo leads to striking increases in both first- and second-phase insulin release, greatly improved glucose tolerance in obese, insulin-resistant mice, and elevated β cell mass, associated with pathway-specific alterations in islet gene expression levels. Selective stimulation of β cell G _s triggered qualitatively similar in vivo metabolic effects. Thus, this developed chemical-genetic strategy represents a powerful approach to study G protein regulation of β cell function in vivo.

Members of the muscarinic acetylcholine receptor family (M1–M5) are known to be involved in a great number of important central and peripheral physiological and pathophysiological processes. Because of the overlapping expression patterns of the M1–M5 muscarinic receptor subtypes and the lack of ligands endowed with sufficient subtype selectivity, the precise physiological functions of the individual receptor subtypes remain to be elucidated. To explore the physiological roles of the M2 muscarinic receptor, we have generated mice lacking functional M2 receptors by using targeted mutagenesis in mouse embryonic stem cells. The resulting mutant mice were analyzed in several behavioral and pharmacologic tests. These studies showed that the M2 muscarinic receptor subtype, besides its well documented involvement in the regulation of heart rate, plays a key role in mediating muscarinic receptor-dependent movement and temperature control as well as antinociceptive responses, three of the most prominent central muscarinic effects. These results offer a rational basis for the development of novel muscarinic drugs.