My section is pursuing two major lines of work both of which deal with a class of cell surface receptors referred to as muscarinic acetylcholine receptors (mAChRs). These receptors are prototypical members of the superfamily of class I (rhodopsin-like) G protein-coupled receptors (GPCRs).? (I) One major focus of my section is to understand how mAChRs function at the molecular level. ? (II) We are also employing gene targeting technology in mice to study the physiological and pathophysiological roles of the individual mAChR subtypes (M1-M5 mAChRs). ? ? (I) RECEPTOR STRUCTURE-FUNCTION STUDIES? GPCRs form one of the largest protein families found in nature, and estimates are that about 50% of drugs in current clinical use act on specific GPCRs or on GPCR-dependent downstream signaling pathways. To understand how these receptors function at a molecular level, we have used the M3 mAChR as a model system. This receptor subtype is selectively coupled to G proteins of the Gq family.? To elucidate the structural changes involved in ligand-dependent GPCR activation, we developed a disulfide cross-linking strategy that allows the formation of intramolecular disulfide cross-links between adjacent Cys residues, with the M3 mAChR present in its native membrane environment (in situ!). Using this strategy, we demonstrated that agonist binding induces a conformational change immediately adjacent to the agonist-binding pocket (Han et al., JBC 280, 34849, 2005). This structural change is predicted to represent one of the early conformational events triggering the more pronounced structural reorganization of the intracellular receptor surface (Ward et al., Biochemistry 45, 676, 2006).? ? ? (II) GENERATION AND ANALYSIS OF MUSCARINIC ACETYLCHOLINE RECEPTOR KNOCKOUT/MUTANT MICE? The precise physiological and pathophysiological roles of the individual mAChRs (M1-M5) are not well understood, primarily due to the lack of receptor subtype-selective ligands. To address this issue, we, in collaboration with Chuxia Deng's lab at NIDDK, used gene targeting technology to generate M1-M5 receptor-deficient mice (KO mice). The M1-M5 mAChR KO mice were then subjected to a series of physiological, pharmacological, behavioral, biochemical, and neurochemical tests. Many of these studies were carried out in collaboration with other laboratories inside and outside of the NIH. This analysis showed that each of the analyzed mAChR KO lines displayed specific functional deficits, indicating that each mAChR subtype mediates distinct physiological functions (for a recent review, see: Wess, Annu. Rev. Pharmacol. Toxicol. 44, 423, 2004).? ? The following key findings were obtained:? ? Central role of beta cell M3 mAChRs in regulating insulin release and blood glucose homeostasis in vivo? By using Cre/loxP technology, we generated mutant mice that selectively lack the M3 mAChR in pancreatic beta cells. We demonstrated that these mutant mice display impaired glucose tolerance and greatly reduced insulin release (Gautam et al., Cell Metabolism 3, 449, 2006). In contrast, transgenic mice selectively overexpressing M3 receptors in pancreatic beta cells show a profound increase in glucose tolerance and insulin release (Gautam et al., Cell Metabolism 3, 449, 2006). Moreover, these mutant mice are resistant to diet-induced glucose intolerance and hyperglycemia. These findings indicate that beta cell M3 mAChRs play a key role in maintaining proper insulin release and glucose homeostasis. Moreover, our data suggest that enhancing signaling through beta cell M3 mAChRs may represent a new avenue in the treatment of obesity and type 2 diabetes.? ? Miscellaneous functions of distinct mAChR subtypes identified by the use of mAChR KO mice ? ? The following results were obtained in collaborative studies:? ? The M3 mAChR mediates increases in synaptic GABA release in the spinal dorsal horn of mice. In contrast, stimulation of presynaptic M2 and M4 mAChRs predominantly attenuates GABAergic inputs to dorsal horn neurons. These findings contribute to a better understanding of the spinal analgesic actions of muscarinic agonists and acetylcholinesterase inhibitors.? (Zhang et al., Mol Pharmacol 69, 1048-55, 2006)? ? The M2 mAChR mediates thromboxane prostanoid receptor-induced changes in lung resistance and airway smooth muscle tension. These findings are of relevance for the treatment of asthma and chronic obstructive pulmonary disease.? (Allen et al., Am J Physiol 290, L526-33, 2006)? ? The M2 mAChR, through PTX-sensitive mechanisms, induces ileal contractions that depend on voltage-dependent calcium entry, especially associated with action potential discharge. The M3 mAChR, through PTX-insensitive mechanisms, induces ileal contractions that depend on voltage-dependent and -independent calcium entry and intracellular calcium release. Given the important roles that mAChRs play in regulating the function of all smooth muscle tissues, these findings are of great general relevance. ? (Unno et al., Br J Pharmacol 146, 98-108, 2005)? ? Vascular M3 mAChRs can be activated by circulating bile acids resulting in peripheral arterial vasodilation. These findings are of considerable clinical interest since serum bile acids are significantly elevated in advanced liver disease.? (Khurana et al., Eur J Pharmacol 517, 103-10, 2005)
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