General goals of the Program include the characterization of signal transduction pathways which regulate the biology of the Alzheimer amyloid percussor protein (APP), its Drosophila homologue, APPL, and the APP promoter-binding homeoproteins. Other Projects have focussed on the regulation and biological consequences of the phosphorylation of these substrates. Project 4 proposes to study in detail the regulation by protein phosphorylation of the transcription of APP and APPL mRNA, using several approaches. The first approach is to use specific treatments which activate protein phosphorylation and analyze for their effects on APP transcription. This will be performed in intact cells, using biochemical and molecular biological techniques. First messengers (neurotransmitters, neuropeptides, growth factors), and membrane permeable second messengers and direct enzyme activating agents will be applied to cells and RNA extracted from those cells will be assayed for APP expression. This approach, though convenient, is limited by the availability of relatively inaccessible to stimulation by permeable agents. In order to cause stimulated activity of isolated pathways, cDNAs for individual enzymes will be transfected into cells and the effect on APP mRNA expression will be measured. In related experiments, homeoproteins and homeoprotein kinases or phosphatases (identified by Project 1) will be cotransfected and effects on APP expression will be compared with that observed in cells transfected with either alone. In order to study APPL, flies will be transformed with protein kinases, including protein kinase C and calcium/calmodulin-dependent protein kinase II. In addition to morphologic examination for phenotypic abnormalities, these flies will be studied for expression of APPL. Primary cultures, derived from these flies, will also be studied: this Project will study APPL expression while Project 1 will study APPL phosphorylation. Because of its predicted structure as an integral transmembrane protein, the normal function of APP (currently unknown) may include the transduction of signals across the plasma membrane. In order to address this possibility, we propose in Project 4 to prepare, express and analyze chimeric molecules which are composed of a domain of APP joined to a complementary domain of a characterized signal transducing protein. Several classes of chimeras will be prepared, joining APP to the complementary domain of the epidermal growth factor receptor, CD4 receptor or neural cell adhesion molecule (N-CAM). For all three, there exist detailed data concerning the biochemical mechanisms responsible for their ability to transduce. Chimeras will be assayed for the ability of an APP domain to successfully substitute for the corresponding domain of EGFR, CD4 or N-CAM. Successful chimeras bearing the APP ectodomain will form the basis of assays for endogenous """"""""ligands"""""""" for APP. Experiments defining the role of protein phosphorylation in the expression of Alzheimer's disease (AD)-related proteins may yield data relevant to the pathobiology and possible therapy of AD. Chimeric analysis may suggest physiological functions for APP, and these functions may also hold etiologic and clinical implications.
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