Alzheimer's disease (AD) is characterized by abnormal protein phosphorylation and aberrant protein catabolism. A major goal of Project 2 is to define and characterize the biological consequences which may be attributed to changes in the state of phosphorylation of several AD-related phosphoproteins. Specifically, these proteins are: (i) the Alzheimer amyloid precursor protein (APP); (ii) APPL, the Drosophila homologue of APP; and (iii) the APP promoter-binding homeoproteins. Studies of other integral membrane phosphoproteins have yielded information which may be applicable to APP and APPL. Many cell-surface receptors are phosphorylated in their cytodomains, either in response to the binding of a ligand or as a mechanism of integrating several signals from multiple cytoplasmic pathways (e.g., cAMP-stimulated phosphorylation of the acetylcholine receptor modifies its sensitivity to ligand-stimulated ion flux). The intracellular trafficking of certain cell surface molecules is another function modulated by changes in the phosphorylation state of integral membrane proteins. The epidermal growth factor receptor (EGFR) and interleukin-2 receptor (IL-2R) are internalized in response to phosphorylation in their respective cytodomains. The phosphorylation site of APP which we are studying is located in a position highly homologous to those of EGFR and IL-2R. Further, the protein phosphorylating enzyme implicated in regulating the phosphorylation state of APP, EGFR and IL-2R is identical. Thus, testing the possibility that APP phosphorylation regulates its internalization in one goal of this Project. Studies of APP trafficking in general are also likely to provide important information for defining how an amyloidogenic portion of APP can be released from its usual transmembrane location. On a molecular level, changes in protein phosphorylation state typically exert effects by inducing alterations in the conformation of the substrate protein. Biologically, this may translate into a modification of the protein-protein interactions involving that phosphoprotein. For APP and APPL, this may mean an altered interaction with a proteolytic enzyme, with a cytoskeletal protein or with some other component of the cell. We propose to test these possibilities directly, using in vitro approaches as well as studies in intact cells. Our strategy includes experiments designed to identify protein-protein interactions involving APP either on the cell surface or within the cytoplasm. At the transcriptional level, signal transduction has been reported to regulate APP mRNA expression. One possible mechanism for this phenomenon is that the activity of APP promoter-binding transcription factors are regulated by their state of phosphorylation. Several members of the homeoprotein class of transcription factors are known to be phosphoproteins which bind the APP promoter. We propose to characterize the effect of this binding on the regulation of APP transcription. The biological consequences of phosphorylation of AD-related proteins are not known but several hypotheses have been proposed. Experiments in Project 2 are designed to test some of those hypotheses, and the data which result should offer insights into the pathobiology of AD and may identify targets for therapy.
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