Protein phosphorylation is widely accepted as one of the principle mechanisms in the control of almost all cellular processes. Studies over the last two decades have provided evidence that protein phosphorylation plays a major role in the regulation of neuronal function. A newly discovered unique class of protein kinases which exclusively phosphorylates tyrosine residues of their substrate proteins has recently been shown to be abundant in neurons. However, the role of protein tyrosine kinases in the regulation of neuronal function is not known. The nicotinic acetylcholine receptor (AChR) is a neurotransmitter gated ion channel which is the most well-characterized neurotransmitter receptor and ion channel in biology. Recent studies have demonstrated that the AChR is phosphorylated on tyrosine residues. To study the role of tyrosine phosphorylation in the regulation of synaptic transmission, the tyrosine phosphorylation of the AChR will be studied and used as a model system for the regulation of neurotransmitter receptors and ion channels by protein tyrosine kinases.
The specific aims of this research proposal are to identify the protein tyrosine kinase that phosporylates the AChR and the phosphotyrosine phosphatase that dephosphorylates the receptor and to characterize the functional consequences of tyrosine phosphorylation of the AChR. To accomplish these goals the protein tyrosine kinases and the phosphotyrosine phosphatases from postsynaptic membranes enriched in the AChR will be purified and biochemically characterized. The purified AChR will be phosphorylated on tyrosine residues and reconstituted into phospholipid vesicles and the single channel properties of the reconstituted phosphorylated AChR will be analyzed in detail. In addition the tyrosine phosphorylation of the AChR in muscle cell cultures as well as in intact muscle will be studied and the regulation of this phosphorylation by innervation and denervation will be investigated. The functional effects of tyrosine phosphorylation in muscle will also be analyzed. The proposed research will provide a better understanding of the role of a basic regulatory mechanism in the modulation of synaptic function. Such knowledge is essential for the understanding of both normal and abnormal neuronal function.
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