The long-term goal of this research is to elucidate the mechanisms involved in the expression and function of muscarinic acetylcholine receptors (mAChR) in the nervous system. Muscarinic receptors play a key role in the central nervous system, where they are involved in such functions as memory, learning, and control of movement; This proposal will combine biochemical and molecular biological approaches to study the molecular mechanisms regulating the function and localization of the mAChRs. Newly synthesized mAChR initially exhibit a diminished ability to mediate functional responses. This proposal will test the hypothesis that the mAChR itself is synthesized in an immature form which is slowly modified to a form with increased functional activity. Different subtypes of mAChR may be differentially localized in a variety of cell types, and preliminary evidence suggests that mAChR are differentially localized in polarized epithelial cells. This proposal will determine the functional consequences of this differential localization, identify the regions of the receptors responsible for this differential localization, and determine the mechanisms responsible for generation of mAChR asymmetry. Recent evidence with other G-protein linked receptors suggest that their native conformation constrains the receptors from being in an active confornation, and that this constraint can be removed either by the binding of agonist or by certain mutations in key regions of the receptors. This proposal will use saturation mutagenesis combined with several different selection and screening procedures to isolate constitutive mutations which result in activation of the m1 and m2 mAChRs in the absence of agonist. Different mAChR subtypes have been suggested to have different functions in the central nervous system, but one can not unequivocally implicate a mAChR subtype with a given function. This proposal will continue recently begun studies to use gene targeting by homologous recombination to determine the role of specific receptor subtypes in signal transduction pathways in the nervous system. The experiments described here should provide new and exciting information on the regulation and function of muscarinic acetylcholine receptors. The identification of the factors responsible for the regulation localization, and function of the macromolecules involved in synaptic transmission is a problem of fundamental interest in cell and neurobiology. The identification of constitutive mutations will provide important information on the structural constraints for mAChR function, and may be of clinical importance in the identification of potential oncogenic mutations in the mAChR. In addition, this research is of clinical interest because of the role of mAChR in learning, memory, the cognitive defects in Alzheimer's disease, and the movement disorders in Parkinson's disease. The elucidation of the factors involved in the regulation and function of mAChR receptors may aid in understanding the etiology and possible treatment of a variety of mental abnormalities.
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