Jürgen Wess

G-protein-coupled receptors: molecular mechanisms involved in receptor activation and selectivity of G-protein recognition.

G‐protein‐coupled receptors (GPCRs) play fundamental roles in regulating the activity of virtually every body cell. Upon binding of extracellular ligands, GPCRs interact with a specific subset of heterotrimeric G‐proteins that can then, in their activated forms, inhibit or activate various effector enzymes and/or ion channels. Molecular cloning studies have shown that GPCRs form one of the largest protein families found in nature, and it is estimated that approximately 1000 different such receptors exist in mammals. The molecular mechanisms involved in GPCR function, particularly the molecular modes of receptor activation and G‐pro‐tein recognition and activation, have therefore become the research focus of an ever increasing number of laboratories. This review will summarize and attempt to integrate recent data derived from structural, molecular genetic, biochemical, and biophysical studies that have shed new light on these processes.—Wess, J. G‐protein‐coupled receptors: molecular mechanisms involved in receptor activation and selectivity of G‐protein recognition. FASEB J. 11, 346‐354 (1997)

Structure and dynamics of the M3 muscarinic acetylcholine receptor

Acetylcholine, the first neurotransmitter to be identified, 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 mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. 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 receptor, 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: mutant mice provide new insights for drug development

Muscarinic acetylcholine receptors (mAChRs), M1–M5, regulate the activity of numerous fundamental central and peripheral functions. The lack of small-molecule ligands that can block or activate specific mAChR subtypes with high selectivity has remained a major obstacle in defining the roles of the individual receptor subtypes and in the development of novel muscarinic drugs. Recently, phenotypic analysis of mutant mouse strains deficient in each of the five mAChR subtypes has led to a wealth of new information regarding the physiological roles of the individual receptor subtypes. Importantly, these studies have identified specific mAChR-regulated pathways as potentially novel targets for the treatment of various important disorders including Alzheimers disease, schizophrenia, pain, obesity and diabetes.

Molecular Basis of Receptor/G-Protein-Coupling Selectivity

Molecular cloning studies have shown that G-protein-coupled receptors form one of the largest protein families found in nature, and it is estimated that approximately 1000 different such receptors exist in mammals. Characteristically, when activated by the appropriate ligand, an individual receptor can recognize and activate only a limited set of the many structurally closely related heterotrimeric G-proteins expressed within a cell. To understand how this selectivity is achieved at a molecular level has become the focus of an ever increasing number of laboratories. This review provides an overview of recent structural, molecular genetic, biochemical, and biophysical studies that have led to novel insights into the molecular mechanisms governing receptor-mediated G-protein activation and receptor/G-protein coupling selectivity.

Activation and allosteric modulation of a muscarinic acetylcholine receptor

Despite recent advances in crystallography and the availability of G-protein-coupled receptor (GPCR) structures, little is known about the mechanism of their activation process, as only the β2 adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously bound to the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into the activation mechanism and allosteric modulation of muscarinic receptors.

Identification and Molecular Characterization of m3 Muscarinic Receptor Dimers

Several studies suggest, but do not prove directly, that muscarinic receptors may be able to form dimeric or oligomeric arrays. To address this issue in a more direct fashion, we designed a series of biochemical experiments using a modified version of the rat m3 muscarinic receptor (referred to as m3′) as a model system. When membrane lysates prepared from m3′ receptor-expressing COS-7 cells were subjected to Western blot analysis under non-reducing conditions, several immunoreactive species were observed corresponding in size to putative receptor monomers, dimers, and oligomers. However, under reducing conditions, the monomeric receptor species represented the only detectable immunoreactive protein, consistent with the presence of disulfide-linked m3 receptor complexes. Similar results were obtained when native m3 muscarinic receptors present in rat brain membranes were analyzed. Control experiments carried out in the presence of high concentrations of the SH group alkylating agent,N-ethylmaleimide, suggested that disulfide bond formation did not occur artifactually during the preparation of cell lysates. The formation of m3′ receptor dimers/multimers was confirmed in coimmunoprecipitation studies using differentially epitope-tagged m3′ receptor constructs. In addition, these studies showed that m3′ receptors were also able to form non-covalently associated receptor dimers and that m3′ receptor dimer formation was receptor subtype-specific. Immunological studies also demonstrated that m3′ receptor dimers/multimers were abundantly expressed on the cell surface. Site-directed mutagenesis studies indicated that two conserved extracellular Cys residues (Cys-140 and Cys-220) play key roles in the formation of disulfide-linked m3′ receptor dimers. These results provide the first direct evidence for the existence of muscarinic receptor dimers and highlight the specificity and molecular diversity of G protein-coupled receptor dimerization/oligomerization.

Muscarinic Induction of Hippocampal Gamma Oscillations Requires Coupling of the M1 Receptor to Two Mixed Cation Currents

Oscillatory network activity at gamma frequencies is assumed to be of major importance in cortical information processing. Whereas the synaptic mechanisms of gamma oscillations have been studied in detail, the ionic currents involved at the cellular level remain to be elucidated. Here we show that in vitro gamma oscillations induced by muscarine require activation of M1 receptors on hippocampal CA3 pyramidal neurons and are absent in M1 receptor-deficient mice. M1 receptor activation depolarizes pyramidal neurons by increasing the mixed Na(+)/K(+) current I(h) and the Ca(2+)-dependent nonspecific cation current I(cat), but not by modulation of I(M). Our data provide important insight into the molecular basis of gamma oscillations by unequivocally establishing a novel role for muscarinic modulation of I(h) and I(cat) in rhythmic network activity.

Cholinergic dilation of cerebral blood vessels is abolished in M 5 muscarinic acetylcholine receptor knockout mice

The M5 muscarinic receptor is the most recent member of the muscarinic acetylcholine receptor family (M1-M5) to be cloned. At present, the physiological relevance of this receptor subtype remains unknown, primarily because of its low expression levels and the lack of M5 receptor-selective ligands. To circumvent these difficulties, we used gene targeting technology to generate M5 receptor-deficient mice (M5R−/− mice). M5R−/− mice did not differ from their wild-type littermates in various behavioral and pharmacologic tests. However, in vitro neurotransmitter release experiments showed that M5 receptors play a role in facilitating muscarinic agonist-induced dopamine release in the striatum. Because M5 receptor mRNA has been detected in several blood vessels, we also investigated whether the lack of M5 receptors led to changes in vascular tone by using several in vivo and in vitro vascular preparations. Strikingly, acetylcholine, a powerful dilator of most vascular beds, virtually lost the ability to dilate cerebral arteries and arterioles in M5R−/− mice. This effect was specific for cerebral blood vessels, because acetylcholine-mediated dilation of extra-cerebral arteries remained fully intact in M5R−/− mice. Our findings provide direct evidence that M5 muscarinic receptors are physiologically relevant. Because it has been suggested that impaired cholinergic dilation of cerebral blood vessels may play a role in the pathophysiology of Alzheimers disease and focal cerebral ischemia, cerebrovascular M5 receptors may represent an attractive therapeutic target.

Use of M1-M5 muscarinic receptor knockout mice as novel tools to delineate the physiological roles of the muscarinic cholinergic system

In this review we report recent findings on the physiological role of the five known muscarinic acetylcholine receptors (mAChRs) as shown by gene targeting technology. Using knockout mice for each mAChRs subtype, the role of mAChRs subtypes in a number of physiological functions was confirmed and new activities were discovered. The M1 mAChRs modulate neurotransmitter signaling in cortex and hippocampus. The M3 mAChRs are involved in exocrine gland secretion, smooth muscle contractility, pupil dilation, food intake, and weight gain. The role of the M5 mAChRs involves modulation of central dopamine function and the tone of cerebral blood vessels. mAChRs of the M2 subtype mediate muscarinic agonist-induced bradycardia, tremor, hypothermia, and autoinhibition of release in several brain regions. M4 mAChRs modulate dopamine activity in motor tracts and act as inhibitory autoreceptors in striatum. Thus, as elucidated by gene targeting technology, mAChRs have widespread and manifold functions in the periphery and brain.

Molecular basis of muscarinic receptor function

Abstract Muscarinic acetylcholine receptors play important roles in numerous physiological functions including higher cogmitive processes such as memory and learning. Consistent with the well-documented pharmacological heterogeneity of muscarinic receptors, molecular cloning studies have revealed the existence of five distinct muscarmic receptor proteins (M 1 -M 5 ). Structure-function relationship studies of the cloned receptors have been greatly aided by the high degree of structural homology that muscarinic receptors share with other G protein-coupled receptors. In this review, Jurgen Wess discusses recent mutagenesis studies that have considerably advanced our knowledge of the molecular details underlying muscarinic receptor function.