The principal aim of this proposal is the exploration of the role of protein tyrosine phosphorylation in the regulation of secretory processing of the amyloid precursor protein (APP) of Alzheimer's disease (AD). Previous results indicated that cleavage and release of APP is stimulated by neurotransmitter receptors. In human embryonic kidney (HEK) 293 cell lines transfected with the genes for different muscarinic receptor subtypes, carbachol stimulated APP release in cells expressing m1 and m3 receptors, but not in cells expressing m2 and m4 receptors. The former are coupled to phosphatidylinositol turnover, and the latter are not. These results, and those from other laboratories, are consistent with a role for protein kinase C (PKC) in the regulation of APP processing. This application is based on preliminary evidence demonstrating that protein tyrosine phosphorylation may also be involved in the regulation of APP release. Three agonists have been identified that stimulate both APP release and tyrosine phosphorylation in HEK cells expressing m3 receptors: carbachol, the PKC activating phorbol esters, and the protein tyrosine phosphatase inhibitor pervanadate (see """"""""Preliminary Results""""""""). Their effects on APP release are blocked by inhibitors of tyrosine kinase. The experiments proposed in this grant are designed to establish a causal relationship between tyrosine phosphorylation and APP release. Time- courses, dose-response characteristics, and susceptibility to PKC and tyrosine kinase antagonists, of the responses to agonists will be examined. The proteins phosphorylated in response to these agonists will be identified by micro-sequencing, or by immunoprecipitation with specific antibodies to candidate proteins. The mechanism by which receptors with intrinsic tyrosine kinase activity stimulate APP release will be studied in wild-type Swiss 3T3 fibroblasts. The involvement of PKC in the regulatory pathways in both HEK cells and fibroblasts will be tested. Signal transduction pathways regulating APP release in fibroblasts from human control and AD donors will be compared. Abnormalities in tyrosine phosphorylation and PKC activity in AD have been described. The proposed experiments may enumerate mechanisms by which these abnormalities are related to the alterations in APP processing and release implicated in the pathology of AD.
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