Studies On Neurotransmitter Receptor Genes

Diabetes, Digestive, Kidney Diseases

Investigators

Linked publications & trials

Abstract

My laboratory pursues two different lines of work which deal with the molecular, biochemical, and physiological analysis of muscarinic acetylcholine and vasopressin receptors. These receptors are prototypical members of the superfamily of G protein-coupled receptors (GPCRs). (I) STRUCTURE-FUNCTION ANALYSIS OF GPCRs 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 different muscarinic acetylcholine and vasopressin receptors as model systems. To elucidate the structural changes involved in ligand-dependent GPCR activation, we developed a disulfide cross-linking strategy that allows the formation of intramolecular Cys-Cys cross-links with the receptor present in its native membrane environment (in situ!). This approach is based on the ability of two cysteine residues that are adjacent to each other in the three-dimensional structure of a protein to form a disulfide bond, either spontaneously or under oxidizing conditions. Disulfide cross-linking experiments revealed that agonist activation of the M3 muscarinic receptor is associated with striking structural changes on the intracellular surface of the receptor protein. Using an analogous approach, we provided evidence that the relative orientations of the transmembrane helices of the M3 receptor differs from that of rhodopsin, the prototypical class I GPCR. To facility structure-function studies, we recently expressed several muscarinic and vasopressin receptor subtypes as well as different G protein alpha subunits in yeast (S. cerevisiae). A great advantage of the yeast expression system is that powerful genetic approaches can be applied to study GPCR structure-function relationships, allowing the screening of large numbers of mutant GPCRs or G proteins in a very efficient manner. To study the structural mechanisms governing M3 receptor activation, we employed an M3 receptor-expressing yeast strain that requires agonist-dependent M3 receptor activation for cell growth. By using receptor random mutagenesis followed by a yeast genetic screen, we identified a point mutation (Q490L) that leads to robust agonist-independent M3 receptor signaling in both yeast and mammalian cells. We then applied a secondary yeast genetic screen to isolate second-site mutations able to suppress the activating effects of the Q490L mutation. This screen led to the identification of 12 amino acids predicted to play key roles in M3 receptor activation and/or receptor/G protein coupling. This strategy should be generally applicable to identify sites in GPCRs that are critically involved in receptor function. (II) GENERATION AND ANALYSIS OF MUSCARINIC ACETYLCHOLINE RECEPTOR KNOCKOUT MICE Most of the important physiological actions of acetylcholine (ACh) are mediated by the binding of ACh to a group of cell surface receptors referred to as muscarinic ACh receptors (mAChRs). The mAChR family consists of five molecularly distinct receptor subtypes (M1-M5) which are abundantly expressed in most tissues or organs. However, primarily due to the lack of receptor subtype-selective ligands, the precise physiological and pathophysiological roles of the individual mAChRs have remained obscure. 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 battery of physiological, pharmacological, behavioral, biochemical, and neurochemical tests. This analysis showed that each of the analyzed mAChR KO lines displayed specific functional deficits, indicating that each mAChR subtype mediates distinct physiological functions. M1 receptor KO mice showed behavioral abnormalities that were reminiscent of human attention deficit-hyperactivity disorder. In addition, ACh-mediated hippocampal gamma oscillations were disrupted in M1 receptor KO mice. M1 receptor KO mice also displayed various biochemical signaling deficits in cerebral cortex and hippocampus. Studies with M2 receptor KO mice revealed that the M2 muscarinic receptor mediates pronounced analgesic effects at both spinal and supraspinal sites and at peripheral nociceptive nerve endings. M2 receptor KO mice also showed cognitive deficits in a passive avoidance test, associated with significant changes in pharmacologically and physiologically evoked ACh release in the hippocampus. In vitro neurotransmitter release studies showed that the M2 receptor subtype is the predominant inhibitory muscarinic autoreceptor in cerebral cortex and hippocampus. The M2 receptor also appears to represent the major muscarinic heteroreceptor mediating inhibition of neurotransmitter release from peripheral sympathetic nerve endings . We previously reported that M3 receptor KO mice displayed a significant decrease in food intake, which was associated with reduced body weight, low serum insulin and leptin levels, and decreased total body fat mass. Recent studies indicated that pancreatic M3 receptors play a key role in ACh-mediated insulin and glucagon release from pancreatic islets. Studies with M2/M4 receptor double KO mice revealed that activation of the M4 receptor subtype (besides the predominant M2 receptor subtype) contributes to the profound analgesia observed after administration of centrally active muscarinic agonists. In vitro neurotransmitter release studies revealed that M4 receptors play a key role in facilitating dopamine release in the striatum. Studies with M4 receptor KO mice also indicated that the M4 receptor subtype is the predominant inhibitory muscarinic autoreceptor in the striatum and several peripheral organs. Initial studies with M5 receptor KO mice indicated that M5 receptors mediate the relaxing effects of ACh on cerebral arteries and arterioles . We subsequently showed that the rewarding effects of morphine and the severity of morphine withdrawal symptoms were substantially reduced in M5 receptor KO mice. Recent studies suggested that M5 receptor activity also modulates cocaine-associated reinforcement and withdrawal. These findings raise the possibility that centrally active M5 receptor antagonists may become therapeutically useful for the treatment of drug addiction. These new findings should be highly relevant for the development of novel muscarinic drugs useful for the treatment of several major pathophysiological conditions including Alzheimer's and Parkinson's disease, drug abuse, and obesity.