The five muscarinic acetylcholine (ACh) receptors(ml-m5) are members of the superfamily of G protein-coupled receptors. The primary focus of this research project is to elucidate the molecular mechanisms underlying muscarinic receptor function (ligand binding, G-protein coupling, etc.) and to define the regulatory genetic elements controlling muscarinic receptor expression. Based on the high degree of structural homology found among all G protein-coupled receptors, these studies should be of general importance for the entire receptor family. Mutational analysis of the m3 muscarinic receptor has led to the identification of a series of threonine and tyrosine residues (which are conserved among all muscarinic receptors) which are critically involved in ACh binding. These residues are predicted to define a plane in the plasma membrane in which ACh binding to the muscarinic receptors occurs. Binding studies with a series of ACh derivatives suggest that some of the conserved threonine and tyrosine residues are primarily involved in the recognition of the ACh ester bond (e.g., by hydrogen bonding). In addition, two of these amino acids were found to be critically involved in agonist-induced receptor activation. Another mutagenesis study focussed on amino acid residues which are highly conserved among all G protein-coupled receptors. A series of four conserved proline residues were identified that play key roles in receptor expression, ligand binding and receptor function. Coexpression in COS-7 cells of N- and C- terminal domains of the m2 and m3 muscarinic receptors resulted in """"""""reconstituted"""""""" receptors that displayed ligand binding and functional properties similar to those of the wild type receptors, indicating that muscarinic receptors may behave in a fashion analogous to multiple-subunit receptors. The human m4 receptor gene was expressed in transgenic mice, however, the expression pattern of the transgene in heart and skeletal muscle was not fully consistent with the distribution of the native mouse m4 receptor. Additional lines of transgenic mice are needed to elucidate the molecular mechanisms underlying this phenomenon. Muscarinic drugs have considerable therapeutic potential in a variety of pathological conditions including Alzheimer's and Parkinson's diseases. The delineation of a detailed molecular model of the ligand-receptor-G protein interaction should allow a more rational approach toward the development of novel therapeutic agents, as well as contributing to basic understanding of cellular signal transduction processes.
