Studies On Neurotransmitter Receptor Genes

Diabetes, Digestive, Kidney Diseases

Investigators

Linked publications, trials & patents

Abstract

My laboratory pursues two different lines of work which deal with the molecular, biochemical, and physiological analysis of muscarinic acetylcholine and vasopressin receptors, which 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 signalling 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 recently developed a novel disulfide cross-linking strategy which offers the great advantage that intramolecular Cys-Cys cross-links are generated with the receptor present in its native membrane environment (in situ!). Successful disulfide cross-linking is monitored by cleavage of cross-linked receptors with factor Xa, followed by Western blot analysis carried out under non-reducing conditions (a factor Xa cleavage site was introduced, via site-directed mutagenesis, into one of the intracellular loops of the receptor). Specifically, disulfide cross-linking experiments were carried out with Cys-substituted mutant M3 muscarinic receptors in the absence and in the presence of agonist ligands. This analysis revealed that agonist activation of the M3 receptor is associated with striking structural changes on the intracellular surface of the receptor protein. We recently initiated a novel project that involves the expression of various muscarinic and vasopressin receptor subtypes as well as different G protein a 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. Yeast strains were genetically modified in a fashion such that yeast growth was strictly dependent on productive GPCR/G protein coupling. We first demonstrated that M3 muscarinic and V2 vasopressin receptors retain proper G protein coupling selectivity when expressed in yeast. The two receptors were then subjected to random mutagenesis to generate large libraries of mutant receptor clones. These mutant receptor libraries are currently being screened in yeast in order to isolate mutant receptors with novel/altered functional properties. Using this strategy, we recently isolated a series of mutant V2 vasopressin receptors which displayed novel G protein coupling profiles. Sequence analysis of the recovered mutant receptors shed new light on the structural elements that constrain the G protein coupling selectivity of the V2 receptor. These initial results indicate that yeast expression technology provides a powerful novel tool to study the structural elements that govern GPCR function. (II) GENERATION AND ANALYSIS OF MUSCARINIC ACETYLCHOLINE RECEPTOR KNOCKOUT MICE Another major focus of my laboratory is the use of gene targeting technology to elucidate the physiological roles of the individual muscarinic receptor subtypes (M1-M5). Muscarinic receptors are critically involved in many fundamental physiological processes. However, since most tissues express multiple muscarinic receptors and the individual receptor subtypes are very difficult to distinguish by classical pharmacological tools, it remains unclear in most cases which specific muscarinic receptors are involved in mediating the diverse muscarinic actions of acetylcholine. To overcome these difficulties, we, in collaboration with Chuxia Deng's lab at NIDDK, employed gene targeting approaches to generate mutant mouse lines deficient in M1, M2, M3, M4, or M5 muscarinic receptors. Recently, we also obtained M2/M4 and M1/M3 receptor double knockout (KO) mice. These mutant animals are currently being analyzed by using a multidisciplinary approach, involving physiological, behavioral, pharmacological, biochemical, electrophysiological, and neurochemical studies and several collaborators inside and outside of the NIH. Pharmacological analysis of M2 receptor KO mice indicated that the M2 subtype plays a key role in muscarinic receptor-mediated tremor, hypothermia, and analgesia. Moreover, muscarinic receptor-dependent reduction in heart rate was absent in M2 receptor KO mice. The M4 receptor KO mice showed a significant increase in basal locomotor activity. Moreover, the stimulatory locomotor effects observed after administration of selective D1 dopamine receptor agonists were greatly enhanced in M4 receptor KO mice. This latter observation suggests that M4 receptors may function physiologically to mediate inhibition of D1 receptor-mediated locomotor stimulation, probably at the level of striatal projection neurons where both receptors are co-expressed at high levels. The release of acetylcholine, like that of many other neurotransmitters, is regulated by release-inhibiting autoreceptors located on cholinergic nerve terminals. Neurochemical studies with M2 and M4 receptor KO mice showed that autoinhibition of ACh release is mediated primarily by M2 receptors in hippocampus and cerebral cortex, but predominantly by M4 receptors in the striatum . These findings demonstrate the new concept that different muscarinic receptor subtypes can act as inhibitory autoreceptors in different regions of the brain. Interestingly, M3 receptor KO mice displayed a significant decrease in food intake, reduced body weight and peripheral fat deposits, and very low serum leptin and insulin levels. More detailed pharmacological and molecular studies suggested that hypothalamic M3 receptors play a role in stimulating food intake by regulating the activity of a subpopulation of neurons (which express the appetite-stimulating peptide, melanin-concentrating hormone) located in the lateral hypothalamus. These findings suggest the existence of a novel cholinergic pathway that participates in the regulation of appetite and food intake. M1 receptor KO mice showed a pronounced increase in locomotor activity in a variety of behavioral tests. Additional behavioral studies suggested that M1 receptors appear to be less critical for cognitive processes than previously assumed. (III) Generation of a mouse model of X-linked nephrogenic diabetes insipidus Inactivating V2 vasopressin receptor mutations are known to be the cause of X-linked nephrogenic diabetes insipidus (XNDI), a disease characterized by massive polyuria and excessive thirst. To generate an animal model of XNDI, we used gene targeting technology to create a mouse line lacking functional V2 receptors. Whereas V2 receptor?deficient hemizygous male pups died within the first week after birth due to hypernatremic dehydration, female mice heterozygous for the V2 receptor mutation were viable and displayed an XNDI-like phenotype, characterized by reduced urine concentrating ability of the kidney, polyuria, and polydipsia. The V2 receptor mutant mice should serve as useful tools for the development of novel therapeutic strategies for the treatment of XNDI and the dissection of the molecular pathways involved in renal urine concentration.