Alterations in muscarinic receptor neurotransmission in the central nervous system have been observed in Alzheimer's disease, narcolepsy, affective disorders, Parkinsonism and as a consequence of aging. As a result, significant effort has been expended in the design and development of muscarinic ligands that might be of potential therapeutic value. To date, five subtypes of muscarinic cholinergic receptors (mAChRs) have been described, three of which (m1, m3, m5) couple to a phosphoinositide-specific phospholipase C, with the attendant formation of inositol trisphosphate and diacylglycerol. Knowledge of the mechanisms underlying the regulation of mAChRs is important for evaluating the role played by mAChRs in signal transduction and in the rational design of therapeutic agents that could target this signaling pathway. In this proposal, we plan to extend our ongoing studies of the regulation of mAChR signaling to phosphoinositide turnover by examining the molecular mechanism underlying agonist-induced internalization of the receptor in human SH-SY5Y neuroblastoma cells. Specifically, the hypothesis to be tested is that the activity of phosphatidylinositol 4- kinase (PI4K), is an absolute requirement for the occurrence of receptor endocytosis. We will evaluate this hypothesis by the following approaches. By pharmacological means, we will determine the ability of known inhibitors of PI4K to inhibit mAChR endocytosis. By means of biochemical, immunological and cell biological approaches, we will determine the specific isoform(s) of PI4K that are involved in receptor endocytosis and their site of action within the endocytic cycle. To evaluate cause and effect, molecular genetic approaches will be employed to examine the effects of depletion and over-expression of PI4K on the extent of receptor internalization. Although phosphoinositides have been implicated in membrane trafficking events, most emphasis has been placed on the 3'-phosphoinositides. The present proposal focuses on the potential role played the quantitatively major 4'-phosphoinositides in this process, evidence for which is beginning to accumulate in both normal and pathological states. This proposal presents strong preliminary evidence linking PI4P synthesis to the internalization of mAChRs. It is likely that the knowledge gained from these studies will be of fundamental importance to understanding the regulation of cell signaling events in neural cells initiated not only by mAChRs, but also by other G-protein coupled receptors.
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