Studies of the cellular and molecular aspects of Alzheimer's disease have focused upon cytoskeletal proteins, particularly the microtubule associated protein, Tan, which makes up neurofibrillary tangles. Neurotransmitter dysfunction has also been implicated in the Alzheimer's disease brain and such dysfunction is likely to result in or originate from some aspect of G protein-mediated signal transduction. This project represents an attempt to correlate the changes in G-protein mediated signaling with polymerization of Tau in the hope that clear linkage might be made between the cytoskeletal changes and the neurochemical changes of Alzheimer's disease, especially in relation to neurotransmitter responsiveness. This laboratory has been engaged, for the past several years, in efforts to demonstrate that cytoskeletal proteins, especially tubulin, interact directly with specific G proteins to regulate neurotransmitter- responsive enzymes such as adenylyl cyclase and phospholipase C. Tubulin regulation of phospholipase is biphasic. At low concentrations, tubulin activates Gq (the G protein which activates phospholipase Cbeta1) by forming a complex and transferring GTP directly. At high concentrations, tubulin inhibits the enzymes activation both by disrupting the coupling between muscarinic acetylcholine receptors and Gq as well as by binding the substrate, PIP2, directly. Thus, increased tubulin dimer concentration, resulting from increased calcium, could """"""""feedback inhibit"""""""" phospholipase C. In the Alzheimer's brain, this regulatory process may be disrupted by Tau and Tau polymers, since it appears that PIP2 may be crucial for the assembly of Tau as well. This project will attempt to test the interaction between Gq and tubulin and how Tau or PIP2 might modify that process. This will be done by studying the binding of the species (fluorescence and microscopy) and the activation by GTP transfer (fluorescence, photoaffinity and binding studies). Experiments will be done with purified protein components as well as with SF9 cells infected with baculovirus containing cDNA for (in various combinations) ml receptors, Gq and phospholipase Cbeta1. Effects of Tau and tubulin on the enzyme activity will be determined in the hope that a logical pathway for this richly articulated regulation can be described.
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