G protein-coupled receptors (GPCRs) form one of the largest protein families found in nature. To understand how these receptors function at a molecular level, we have used a combined molecular genetic/biochemical approach. For these studies, different muscarinic acetylcholine (M1-M5) and vasopressin receptor subtypes (V1, V2) served as model systems. GPCR assembly: We recently reported that coexpression of muscarinic or vasopressin receptor fragments -obtained by splitting these receptors (via site-directed mutagenesis) in various intracellular and extracellular loops- results in the reconstitution of functional receptor complexes. Based on these findings, we have developed a sandwich ELISA that provides a sensitive system for monitoring fragment association. This system is currently being used to identify residues in different muscarinic and vasopressin recepors that are essential for proper receptor assembly. These experiments should lead to novel information about the molecular mechanisms involved in GPCR assembly and folding. Receptor/G protein coupling selectivity: We recently succeeded in expressing various muscarinic and vasopressin receptor subtypes as well as different G protein alpha-subunits in yeast. A great advantage of this heterologous expression system is that powerful genetic approaches can be applied to analyze structure- function relationships. Genetically engineered yeast strains will be employed that require agonist-dependent GPCR/G protein activation for cell growth. We could show that the various receptors maintain their G protein coupling preferences (as determined in mammalian expression systems) in yeast. We are currently in the process of transforming yeast with mutant receptor and G protein libraries in order to isolate mutant receptors or G proteins with specific coupling properties. This approach should greatly enhance our knowledge about the structural determinants regulating receptor/G protein coupling selectivity. Structural basis of GPCR activation: We have prepared mutant muscarinic and vasopressin receptors that lack most native cysteine (Cys) residues. Cys residues will be introduced into defined positions of these mutant receptor proteins, followed by their modification with Cys-specific modifying agents carrying environment-sensitive reporter groups such as fluorescence markers or spin labels. In addition, the Cys-free mutant receptors are also being used as background for disulfide-cross-linking studies, following introduction of an internal protease cleavage site and systematic reintroduction of pairs of Cys residues. These approaches should lead to novel insights into GPCR structure and the dynamic processes that accompany GPCR activation. Generation and analysis of muscarinic receptor receptor and V2 vasopressin receptor knock-out mice: We have applied gene targeting technology to create mutant mouse lines lacking individual muscarinic receptor subtypes (M1-M5). Pharmacological, physiological, and behavioral analysis of these animals will reveal the roles that the M1- M5 receptor subtypes play in vivo. Recently, we have completed an initial analysis of mouse lines lacking functional M2 or M4 receptors. We found that the M2 receptor subtype, besides mediating the well- documented bradycardic effect of acetylcholine, plays a key role in mediating muscarinic agonist-dependent tremor, temperature control, and analgesia, three of the most prominent central muscarinic effects. Behavioral analysis of M4 receptor knock-out mice indicated that M4 receptors exert an inhibitory effect on the increase in locomotor activity following D1 receptor activation. These results provide a rational basis for the development of novel muscarinic drugs. In addition, we have used gene targeting technology to generate a mouse line lacking functional V2 vasopressin receptors (V2 receptors play a key role in the maintenance of a normal body water balance). Preliminary studies suggest that the V2 receptor mutant mice will serve as an excellent model system for human X-linked nephrogenic diabetes insipidus. - G protein-coupled receptors muscarinic receptors vasopressin receptors structure-function analysis split receptors knock-out mice
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