A better understanding of precisely how AβPP is processed to generate the Aβ peptide and other, poorly understood proteolytic products has become a priority in Alzheimer's research. In the November 1 Journal of Neuroscience, Ayae Kinoshita, working with Brad Hyman at Massachusetts General Hospital and colleagues, reports new insight on this question gained by using biophysics technology that has been adapted for the study of cell biological problems.
The method is Fluorescence Resonance Energy Transfer (FRET) coupled with confocal microscopy. FRET involves tagging two proteins with different fluorophors and measuring how the color or intensity of the emitted fluorescence changes when the two proteins come to within 10 nm of each other. With this method, scientists can detect whether two proteins interact in a living cell and where in the cell those contacts occur. By adding molecular biology, they can also probe exactly which domains of the proteins make contact.
In this study, Kinoshita et al. found interactions between AβPP and low-density lipoprotein receptor-related protein (LRP) in cultured human glioma cells. Soluble and membrane-bound versions of APP interacted in different ways but generally speaking, AβPP and LRP-both membrane-spanning proteins-directly interacted with their ligand-binding domains outside of the membrane, as well as indirectly with their intracellular tails. This two-pronged interaction occurred both on the cell membrane and, unexpectedly, in intracellular organelles that comprise the cell's secretory pathway, including the Golgi apparatus.
This could be important because previous work has shown LRP to be responsible for endocytosis of soluble AβPP (Kounnas et al. 1995). Moreover, inhibiting LRP-AβPP interactions prevents AβPP's delivery to intracellular compartments of the endocytic pathway, where most Aβ is made. Co-author Dudley Strickland of the Holland Laboratory at the American Red Cross, Rockville, Maryland, showed last year, also with Hyman and colleagues, that disturbing the AβPP-LRP interaction with receptor-associated protein (RAP) reduces Aβ synthesis by up to 80% in the same cell type (Ulery PG et al. 2000).
The intracellular interaction between AβPP and LRP is probably indirect, Kinoshita et al. report. Previous work had suggested that the cyotosolic adapter protein Fe65 might form a trimeric complex with the cytoplasmic tails of AβPP and LRP (Trommsdorff et al., 1998). Indeed, FRET supported this model by showing that the N-terminus of Fe65 interacts with LRP and the C-terminus of Fe65 interacts with APP. Last July, Fe65 attracted attention for being involved in gene expression mediated by the cytoplasmic APP tail that is released after γ-secretase cleavage and then proceeds to the nucleus (see related news item).—Gabrielle Strobel
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