Muscarinic cholinergic receptors have important physiological functions throughout the body and are potential sites for therapeutic drug intervention in pathological states such as chronic obstructive pulmonary disease, spastic colon disease, and Alzheimer's disease. Recently, molecular cloning techniques have demonstrated the existence of at least five distinct genes encoding for the muscarinic receptors. Individual subtypes of these receptors display different affinities for muscarinic cholinergic drugs and are differentially expressed in various organs of the body. These receptors couple with varying selectivity, via several G regulatory proteins, to three major second messenger systems: adenylyl cyclase-cAMP, phosphatidyl inositol turnover, and Ca++ and K+ channels. Insight into the different functions of the individual muscarinic receptors at the molecular level would prove valuable for the design of specific cholinergic drugs. The primary goals of the this project are, first, to define the amino acid residues involved in ligand binding and , second, to analyze those sequence domains involved in second messenger coupling and those governing receptor desensitization and internalization. The framework of these goals will allow us to test the hypothesis that dynamic rearrangements of the seven membrane spanning regions found in these receptor molecules determine whether the receptors are active or inactive. Our experimental approach will be to introduce a series of mutations into the muscarinic receptor genes and test the effects of each mutation on the functional state of the receptor after transfection and expression of the mutated genes in suitable mammalian target cells. To facilitate interpretation of the results, a combination of site specific mutagenesis, deletion mutation, and chimera formation will be employed in an iterative fashion. Our results will be compared to data previously obtained from other members of the some superfamily of receptors capable of coupling to second messengers via G proteins, particularly the adrenergic receptors. The project will provide a better understanding of the domains of the muscarinic cholinergic receptors involved in ligand binding, coupling, and regulation.
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