Acetylcholine (ACh), 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 ACh 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.2–4 Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences.5–6 We describe here the structure of the Gq/11-coupled M3 mAChR 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 new possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows the first 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 raise additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer new 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.

We have produced yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 and YAC72) huntingtin (htt) in a developmental and tissue-specific manner identical to that observed in Huntington's disease (HD). YAC46 and YAC72 mice show early electrophysiological abnormalities, indicating cytoplasmic dysfunction prior to observed nuclear inclusions or neurodegeneration. By 12 months of age, YAC72 mice have a selective degeneration of medium spiny neurons in the lateral striatum associated with the translocation of N-terminal htt fragments to the nucleus. Neurodegeneration can be present in the absence of macro- or microaggregates, clearly showing that aggregates are not essential to initiation of neuronal death. These mice demonstrate that initial neuronal cytoplasmic toxicity is followed by cleavage of htt, nuclear translocation of htt N-terminal fragments, and selective neurodegeneration.

Impaired functioning of pancreatic ␤ cells is a key hallmark of type 2 diabetes. ␤ cell function is modulated by the actions of different classes of heterotrimeric G proteins. The functional consequences of activating specific ␤ cell G protein signaling pathways in vivo are not well understood at present, primarily due to the fact that ␤ cell G protein-coupled receptors (GPCRs) are also expressed by many other tissues. To circumvent these difficulties, we developed a chemicalgenetic approach that allows for the conditional and selective activation of specific ␤ cell G proteins in intact animals. Specifically, we created two lines of transgenic mice each of which expressed a specific designer GPCR in ␤ cells only. Importantly, the two designer receptors differed in their G protein-coupling properties (Gq/11 versus Gs). 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 ␤ cell Gq/11 or Gs G proteins. Here we report the findings that conditional and selective activation of ␤ cell Gq/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 ␤ cell mass, associated with pathway-specific alterations in islet gene expression levels. Selective stimulation of ␤ cell Gs triggered qualitatively similar in vivo metabolic effects. Thus, this developed chemical-genetic strategy represents a powerful approach to study G protein regulation of ␤ cell function in vivo.beta cells ͉ G protein-coupled receptors ͉ transgenic mice ͉ type 2 diabetes T ype 2 diabetes has emerged as one of the major threats to human health in the 21st century (1). Impaired function of pancreatic ␤ cells is one of the key hallmarks of type 2 diabetes, and therapies targeted at improving ␤ cell function are predicted to offer considerable therapeutic benefit (2).␤ Cell function is modulated by the actions of different classes of heterotrimeric G proteins which are the immediate downstream targets of a multitude of G protein-coupled receptors (GPCRs). Like most other cell types, pancreatic ␤ cells are predicted to express many different GPCRs (3-5). Several lines of evidence suggest that activation of G s -coupled receptors expressed by pancreatic ␤ cells, including the glucagon-like peptide (GLP-1) receptor, improves ␤ cell function and can increase in ␤ cell mass via cAMP-dependent mechanisms (5-7). Pancreatic ␤ cells also express several G q/11 -coupled receptors, including the M 3 muscarinic acetylcholine (ACh) receptor (M3R) and GPR40, which can promote insulin release in an agonist-dependent fashion [for recent reviews, see (5,8)].Studies with GLP-1 receptor agonists have yielded detailed information about the beneficial effects of G s signaling on ␤ cell function and whole body glucose homeostasis (note that the GLP-1 receptor is enriched in pancreatic ␤ cells) (5-7). In contrast, much...

Therapeutic strategies that augment insulin release from pancreatic β-cells are considered beneficial in the treatment of type 2 diabetes. We previously demonstrated that activation of β-cell M 3 muscarinic receptors (M3Rs) greatly promotes glucose-stimulated insulin secretion (GSIS), suggesting that strategies aimed at enhancing signaling through β-cell M3Rs may become therapeutically useful. M3R activation leads to the stimulation of G proteins of the G q family, which are under the inhibitory control of proteins known as regulators of G protein signaling (RGS proteins). At present, it remains unknown whether RGS proteins play a role in regulating insulin release. To address this issue, we initially demonstrated that MIN6 insulinoma cells express functional M3Rs and that RGS4 was by far the most abundant RGS protein expressed by these cells. Strikingly, siRNA-mediated knockdown of RGS4 expression in MIN6 cells greatly enhanced M3R-mediated augmentation of GSIS and calcium release. We obtained similar findings using pancreatic islets prepared from RGS4-deficient mice. Interestingly, RGS4 deficiency had little effect on insulin release caused by activation of other β-cell GPCRs. Finally, treatment of mutant mice selectively lacking RGS4 in pancreatic β-cells with a muscarinic agonist (bethanechol) led to significantly increased plasma insulin and reduced blood glucose levels, as compared to control littermates. Studies with β-cell-specific M3R knockout mice showed that these responses were mediated by β-cell M3Rs. These findings indicate that RGS4 is a potent negative regulator of M3R function in pancreatic β-cells, suggesting that RGS4 may represent a potential target to promote insulin release for therapeutic purposes. knockout mice | muscarinic receptor | RGS proteins | G protein-coupled receptor T ype 2 diabetes (T2D) has emerged as a major threat to human health worldwide. Besides peripheral insulin resistance, T2D is usually associated with β-cell dysfunction (1). Thus, the development of new drugs aimed at improving β-cell function, including stimulation of insulin release, is the focus of many laboratories (2).β-cell function is regulated by many hormones and neurotransmitters most of which act on specific G protein-coupled receptors (GPCRs) that are expressed on the surface of pancreatic β-cells (3, 4). Following ligand-induced activation, a specific GPCR interacts with and activates one or more classes of heterotrimeric G proteins (consisting of α, β, and γ subunits), which in turn modulate various intracellular signal transduction pathways (5).Stimulation of the muscarinic cholinergic (parasympathetic) nerves innervating the endocrine pancreas leads to a pronounced increase in glucose-stimulated insulin secretion (GSIS) (4, 6). Studies with pancreatic islets prepared from M 3 muscarinic acetylcholine receptor (M3R) KO mice demonstrated that muscarinic enhancement of GSIS is mediated by the M3R subtype (7,8). The M3R is a member of the muscarinic receptor family (M 1 -M 5 ) that is selectively coupled ...

The amino-terminal domain containing the ligand binding site of the G protein-coupled metabotropic glutamate receptors (mGluRs) consists of two lobes that close upon agonist binding. In this study, we explored the ligand binding pocket of the Group III mGluR4 receptor subtype using site-directed mutagenesis and radioligand binding. The selection of 16 mutations was guided by a molecular model of mGluR4, which was based on the crystal structure of the mGluR1 receptor.