The transition metal ion copper(II) has a critical role in chronic neurologic diseases. The amyloid precursor protein (APP) of Alzheimer's disease or a synthetic peptide representing its copper-binding site reduced bound copper(II) to copper(I). This copper ion-mediated redox reaction led to disulfide bond formation in APP, which indicated that free sulfhydryl groups of APP were involved. Neither superoxide nor hydrogen peroxide had an effect on the kinetics of copper(II) reduction. The reduction of copper(II) to copper(I) by APP involves an electron-transfer reaction and could enhance the production of hydroxyl radicals, which could then attack nearby sites. Thus, copper-mediated toxicity may contribute to neurodegeneration in Alzheimer's disease.

We reported previously that the carbohydrate domain of the amyloid precursor protein is involved in amyloid precursor protein (APP)-APP interactions. Functional in vitro studies suggested that this interaction occurs through the collagen binding site of APP. The physiological significance remained unknown, because it is not understood whether and how APP dimerization occurs in vivo. Here we report that cellular APP exists as homodimers matching best with a two-site model. Consistent with our published crystallographic data, we show that a deletion of the entire sequence after the kunitz protease inhibitor domain did not abolish APP homodimerization, suggesting that two domains are critically involved but that neither is essential for homodimerization. Finally, we generated stabilized dimers by expressing mutant APP with a single cysteine in the ectodomain juxtamembrane region. Mutation of Lys 624 to cysteine produced ϳ6 -8-fold more A␤ than cells expressing normal APP. Our results suggest that amyloid A␤ production can in principle be positively regulated by dimerization in vivo. We suggest that dimerization could be a physiologically important mechanism for regulating the proposed signal activity of APP.

Previously it has been shown that the extracellular domain of transmembrane BA4 amyloid precursor protein (APP) includes binding sites for zinc(I1) and for molecules of the extracellular matrix such as collagen, laminin and the heparin sulfate chains of proteoglycans (HSPGs). Here we report that APP also binds copper ions. A copper type I1 binding site was located within residues 135-I 55 of the cysteine-rich domain of APP,,, which is present in all eight APP splice isoforms known so far. The two essential histidines in the type II copper binding site of APP are conserved in the related protein APLP2. Copper(I1) binding is shown to inhibit homophilic APP binding. The identification of a copper(I1) binding site in APP suggests that APP and APLP2 may be involved in electron transfer and radical reactions.

The specific binding of the amyloid precursor protein (APP) to extracellular matrix molecules suggests that APP regulates cell interactions and has a function as a cell adhesion molecule and/or substrate adhesion molecule. On the molecular level APP has binding sites for collagen, laminin, and glycosaminoglycans which is a characteristic feature of cell adhesion molecules. We have examined the interactions between the APP and collagen types I and IV and identified the corresponding binding sites on APP and collagen type I.We show that APP bound most efficiently to collagen type I in a concentration-dependent and specific manner in the native and heat-denatured states, suggesting an involvement of a contiguous binding site on collagen. This binding site was identified on the cyanogen bromide fragment ␣1(I)CB6 of collagen type I, which also binds heparin. APP did not bind to collagen type I-heparin complexes, which suggests that there are overlapping binding sites for heparin and APP on collagen. We localized the site of APP that mediates collagen binding within residues 448 -465 of APP 695 , which are encoded by the ubiquitously expressed APP exon 12, whereas the high affinity heparin binding site of APP is located in exon 9. Since a peptide encompassing this region binds to collagen type I and inhibits APP-collagen type I binding in nanomolar concentrations, this region may comprise the major part of the collagen type I binding site of APP. Moreover, our data also indicate that the collagen binding site is involved in APP-APP interaction that can be modulated by Zn(II) and heparin. Taken together, the data suggest that the regulation of APP binding to collagen type I by heparin occurs through the competitive binding of heparin and APP to collagen.

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