Having detected an 80 kDa cellular protein (80 K) which interacts with the cytoplasmic domain of APP (APPc) in vitro; we now propose l) to identify and characterize this protein by isolating the corresponding cDNA, 2) to study its interaction with APPc in vivo and in vitro, and 3) most importantly, to study its function with respect to APP cell biology. APP is a ubiquitously expressed, highly conserved protein whose precise physiologic function is uncertain. Normal cells process APP, alternately, into secreted amyloid beta-protein (Abeta), or into a secreted form cleaved in the middle of the Abeta domain (APPsalpha), or degrade APP completely within lysosomes. The mechanisms and regulation of APP processing at a molecular level are quite unclear. Mutations flanking the Abeta domain of APP co-segregate with familial Alzheimer's disease (AD) suggesting that abnormal APP processing is sufficient to cause AD. Evidence exists in the literature demonstrating that the cytoplasmic domain of APP (APPc) plays a role in regulating the processing of APP and possibly also its cellular localization and function. Specifically, changes in the secretion of Abeta and APPsalpha and the N-terminal sequence of secreted Abeta have been reported with truncations or mutations of APPc. APP was also shown to interact with the cytoskeleton, presumably via its cytoplasmic domain. By analogy, the cytoplasmic domains of many integral membrane proteins are involved in regulating diverse properties of these proteins, including secretory processing, endocytosis, subcellular localization, and regulation of intracellular signalling pathways. Characterizing the interaction of 80 K and APPc will increase our understanding both the normal function and pathologic processes of this complex protein. Specifically, we propose to optimize our affinity purification to obtain quantities of 80 K sufficient for partial amino acid sequence determination to clone the cDNA. In addition to identifying 80 K, obtaining the cDNA will enable us to perform gene transfer studies using vaccinia virus vectors with 80 K (and subfragments) to test the function of 80 K and its in vivo interactions with APPc. In vitro characterization of the interaction of 80 K with APPc will enable us to design mutant APP and 80 K molecules to further probe the in vivo function of this interaction. Specifically, we will investigate the effect of 80 ....APPc complexes on secretion of APP and Abeta, on cell-associated APP derivatives and on the subcellular localization of APP. As APP processing is known to vary between cell types and due to changes in protein phosphorylation, we will evaluate the function of 80 K in a variety of cell types and in relation to protein phosphorylation cascades. These studies will investigate a novel finding and a new aspect of the regulation of APP processing and should have a direct impact on understanding APP processing and possibly AD pathogenesis.
