Sung-Jun Han

Conformational changes involved in G-protein-coupled-receptor activation

Little is known about the nature of the conformational changes that convert G-protein-coupled receptors (GPCRs), which bind diffusible ligands, from their resting into their active states. To gain structural insight into this process, various laboratories have used disulfide cross-linking strategies involving cysteine-substituted mutant GPCRs. Several recent disulfide cross-linking studies using the M(3) muscarinic acetylcholine receptor as a model system have led to novel insights into the conformational changes associated with the activation of this prototypical class I GPCR. These structural changes are predicted to involve multiple receptor regions, primarily distinct segments of transmembrane helices III, VI and VII and helix 8. Given the high degree of structural homology found among most GPCRs, it is likely that these findings will be of considerable general relevance. A better understanding of the molecular mechanisms underlying GPCR activation might lead to novel strategies aimed at modulating GPCR function for therapeutic purposes.

Role of the M3 muscarinic acetylcholine receptor in β‐cell function and glucose homeostasis

The release of insufficient amounts of insulin in the presence of elevated blood glucose levels is one of the key features of type 2 diabetes. Various lines of evidence indicate that acetylcholine (ACh), the major neurotransmitter of the parasympathetic nervous system, can enhance glucose‐stimulated insulin secretion from pancreatic β‐cells. Studies with isolated islets prepared from whole body M3 muscarinic ACh receptor knockout mice showed that cholinergic amplification of glucose‐dependent insulin secretion is exclusively mediated by the M3 muscarinic receptor subtype. To investigate the physiological relevance of this muscarinic pathway, we used Cre/loxP technology to generate mutant mice that lack M3 receptors only in pancreatic β‐cells. These mutant mice displayed impaired glucose tolerance and significantly reduced insulin secretion. In contrast, transgenic mice overexpressing M3 receptors in pancreatic β‐cells showed a pronounced increase in glucose tolerance and insulin secretion and were resistant to diet‐induced glucose intolerance and hyperglycaemia. These findings indicate that β‐cell M3 muscarinic receptors are essential for maintaining proper insulin secretion and glucose homeostasis. Moreover, our data suggest that enhancing signalling through β‐cell M3 muscarinic receptors may represent a new avenue in the treatment of glucose intolerance and type 2 diabetes.

Distinct structural changes in a G protein-coupled receptor caused by different classes of agonist ligands.

The activity of G protein-coupled receptors can be modulated by different classes of ligands, including agonists that promote receptor signaling and inverse agonists that reduce basal receptor activity. The conformational changes in receptor structure induced by different agonist ligands are not well understood at present. In this study, we employed an in situ disulfide cross-linking strategy to monitor ligand-induced conformational changes in a series of cysteine-substituted mutant M3 muscarinic acetylcholine receptors. The observed disulfide cross-linking patterns indicated that muscarinic agonists trigger a separation of the N-terminal segment of the cytoplasmic tail (helix 8) from the cytoplasmic end of transmembrane domain I. In contrast, inverse muscarinic agonists were found to increase the proximity between these two receptor regions. These findings provide a structural basis for the opposing biological effects of muscarinic agonists and inverse agonists. This study also provides the first piece of direct structural information as to how the conformations induced by these two functionally different classes of ligands differ at the molecular level. Given the high degree of structural homology found among most G protein-coupled receptors, our findings should be of broad general relevance.

Metabolic Roles of the M3 Muscarinic Acetylcholine Receptor Studied with M3 Receptor Mutant Mice: A Review

The M3 muscarinic acetylcholine (ACh) receptor (M3 mAChR) is expressed in many central and peripheral tissues. It is a prototypic member of the superfamily of G protein-coupled receptors and preferentially activates G proteins of the Gq family. Recent studies involving the use of newly generated mAChR mutant mice have revealed that the M3 mAChR plays a key role in regulating many important metabolic functions. Phenotypic analyses of mutant mice that either selectively lacked or overexpressed M3 receptors in pancreatic β -cells indicated that β -cell M3 mAChRs are essential for maintaining proper insulin release and glucose homeostasis. The experimental data also suggested that strategies aimed at enhancing signaling through β -cell M3 mAChRs might be beneficial for the treatment of type 2 diabetes. Recent studies with whole body M3 mAChR knockout mice showed that the absence of M3 receptors protected mice against various forms of experimentally or genetically induced obesity and obesity-associated metabolic deficits. Under all experimental conditions tested, M3 receptor-deficient mice showed greatly ameliorated impairments in glucose homeostasis and insulin sensitivity, reduced food intake, and a significant elevation in basal and total energy expenditure, most likely due to increased central sympathetic outflow and increased rate of fatty acid oxidation. These findings are of potential interest for the development of novel therapeutic approaches for the treatment of obesity and associated metabolic disorders.

Hepatic Muscarinic Acetylcholine Receptors Are Not Critically Involved in Maintaining Glucose Homeostasis in Mice

OBJECTIVE An increase in the rate of hepatic glucose production is the major determinant of fasting hyperglycemia in type 2 diabetes. A better understanding of the signaling pathways and molecules that regulate hepatic glucose metabolism is therefore of great clinical importance. Recent studies suggest that an increase in vagal outflow to the liver leads to decreased hepatic glucose production and reduced blood glucose levels. Since acetylcholine (ACh) is the major neurotransmitter of the vagus nerve and exerts its parasympathetic actions via activation of muscarinic ACh receptors (mAChRs), we examined the potential metabolic relevance of hepatocyte mAChRs. RESEARCH DESIGN AND METHODS We initially demonstrated that the M3 mAChR is the only mAChR subtype expressed by mouse liver/hepatocytes. To assess the physiological role of this receptor subtype in regulating hepatic glucose fluxes and glucose homeostasis in vivo, we used gene targeting and transgenic techniques to generate mutant mice lacking or overexpressing M3 receptors in hepatocytes only. RESULTS Strikingly, detailed in vivo phenotyping studies failed to reveal any significant metabolic differences between the M3 receptor mutant mice and their control littermates, independent of whether the mice were fed regular or a high-fat diet. Moreover, the expression levels of genes for various key transcription factors, signaling molecules, and enzymes regulating hepatic glucose fluxes were not significantly altered in the M3 receptor mutant mice. CONCLUSIONS This rather surprising finding suggests that the pronounced metabolic effects mediated by activation of hepatic vagal nerves are mediated by noncholinergic signaling pathways.

Neuronal M3 muscarinic acetylcholine receptors are essential for somatotroph proliferation and normal somatic growth

The molecular pathways that promote the proliferation and maintenance of pituitary somatotrophs and other cell types of the anterior pituitary gland are not well understood at present. However, such knowledge is likely to lead to the development of novel drugs useful for the treatment of various human growth disorders. Although muscarinic cholinergic pathways have been implicated in regulating somatotroph function, the physiological relevance of this effect and the localization and nature of the receptor subtypes involved in this activity remain unclear. We report the surprising observation that mutant mice that selectively lack the M3 muscarinic acetylcholine receptor subtype in the brain (neurons and glial cells; Br-M3-KO mice) showed a dwarf phenotype associated with a pronounced hypoplasia of the anterior pituitary gland and a marked decrease in pituitary and serum growth hormone (GH) and prolactin. Remarkably, treatment of Br-M3-KO mice with CJC-1295, a synthetic GH-releasing hormone (GHRH) analog, rescued the growth deficit displayed by Br-M3-KO mice by restoring normal pituitary size and normal serum GH and IGF-1 levels. These findings, together with results from M3 receptor/GHRH colocalization studies and hypothalamic hormone measurements, support a model in which central (hypothalamic) M3 receptors are required for the proper function of hypothalamic GHRH neurons. Our data reveal an unexpected and critical role for central M3 receptors in regulating longitudinal growth by promoting the proliferation of pituitary somatotroph cells.

M1-M3 muscarinic acetylcholine receptor-deficient mice: novel phenotypes.

The five muscarinic acetylcholine receptors (M1-M5 mAChRs) mediate a very large number of important physiological functions (Caulfield, 1993; Caulfield and Birdsall, 1998; Wess, 2004). Because of the lack of small molecule 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 proved to be a challenging task. To overcome these difficulties, we recently generated mutant mouse lines deficient in each of the five mAChR genes (M1R-/- mice, M2R-/- mice, M3R-/- mice, etc. [Wess, 2004]). Phenotyping studies showed that each of the five mutant mouse lines displayed characteristic physiological, pharmacological, behavioral, biochemical, or neurochemical deficits (Wess, 2004). This chapter summarizes recent findings dealing with the importance of the M2mAChR for cognitive processes and the roles of the M1 and M3 mAChRs in mediating stimulation of glandular secretion.

Ligand-specific changes in M3 muscarinic acetylcholine receptor structure detected by a disulfide scanning strategy.

G protein-coupled receptor (GPCR) function can be modulated by different classes of ligands including full and inverse agonists. At present, little is known about the conformational changes that agonist ligands induce in their target GPCRs. In this study, we employed an in situ disulfide cross-linking strategy to monitor ligand-induced structural changes in a series of cysteine (Cys)-substituted mutant M 3 muscarinic acetylcholine receptors. One of our goals was to study whether the cytoplasmic end of transmembrane domain V (TM V), a region known to be critically involved in receptor/G protein coupling, undergoes a major conformational change, similar to the adjacent region of TM VI. Another goal was to determine and compare the disulfide cross-linking patterns observed after treatment of the different mutant receptors with full versus inverse muscarinic agonists. Specifically, we generated 20 double Cys mutant M 3 receptors harboring one Cys substitution within the cytoplasmic end of TM V (L249-I253) and a second one within the cytoplasmic end of TM VI (A489-L492). These receptors were transiently expressed in COS-7 cells and subsequently characterized in pharmacological and disulfide cross-linking studies. Our cross-linking data, in conjunction with a three-dimensional model of the M 3 muscarinic receptor, indicate that M 3 receptor activation does not trigger major structural disturbances within the cytoplasmic segment of TM V, in contrast to the pronounced structural changes predicted to occur at the cytoplasmic end of TM VI. We also demonstrated that full and inverse muscarinic agonists had distinct effects on the efficiency of disulfide bond formation in specific double Cys mutant M 3 receptors. The present study provides novel information about the dynamic changes that accompany M 3 receptor activation and how the receptor conformations induced (or stabilized) by full versus inverse muscarinic agonists differ from each other at the molecular level. Because all class I GPCRs are predicted to share a similar transmembrane topology, the conclusions drawn from the present study should be of broad general relevance.