Increasing evidence has implicated the low density lipoprotein receptor-related protein (LRP) and the adaptor protein FE65 in Alzheimer's disease pathogenesis. We have shown previously that LRP mediates ␤-amyloid precursor protein (APP) processing and affects amyloid ␤-protein and APP secretion and APP-c-terminal fragment generation. Furthermore, LRP mediates APP processing through its intracellular domain. Here, we set out to examine whether this interaction is of direct or indirect nature. Specifically, we asked whether adaptor proteins such as FE65 influence the LRP-mediated effect on APP processing by forming a protein complex. In coimmunoprecipitation experiments, we confirmed the postulated APP-FE65 and the LRP-FE65 interaction. However, we also showed an LRP-FE65-APP trimeric complex using pull-down techniques. Because FE65 alters APP processing, we investigated whether this effect is LRP dependent. Indeed, FE65 was only able to increase APP secretion in the presence of LRP. In the absence of LRP, APP secretion was unchanged compared with the LRP knock-out phenotype. Using RNA short interference techniques against FE65, we demonstrated that a reduction in FE65 protein mimics the LRP knock-out phenotype on APP processing. These results clearly demonstrate that FE65 acts as a functional linker between APP and LRP.

The low density lipoprotein receptor-related protein 1 (LRP1) emerges to play fundamental roles in cellular signaling pathways in the brain. One of its prominent ligands is the serine proteinase tissue-type plasminogen activator (tPA), which has been shown to act as a key activator of neuronal mitogen-activated protein kinase pathways via the N-methyl-D-aspartate (NMDA) receptor. However, here we set out to examine whether LRP1 and the NMDA receptor might eventually act in a combined fashion to mediate tPA downstream signaling. By blocking tPA from binding to LRP1 using the receptor-associated protein, we were able to completely inhibit NMDA receptor activation. Additionally, inhibition of NMDA receptor calcium influx with MK-801 resulted in dramatic reduction of tPA-mediated downstream signaling. This indicates a functional interaction between the two receptors, since both experimental approaches resulted in strongly reduced calcium influx and Erk1/2 phosphorylation. Additionally, we were able to inhibit Erk1/2 activation by competing for the LRP1 C-terminal binding motif with a truncated PSD95 construct resembling its PDZ III domain. Furthermore, we identified the distal NPXY amino acid motif in the C terminus of LRP1 as the crucial element for LRP1-NMDA receptor interaction via the adaptor protein PSD95. These results provide new insights into the mechanism of a tPA-induced, LRP1-mediated gating mechanism for NMDA receptors.LRP1 (low density lipoprotein receptor-related protein) is a member of the low density lipoprotein receptor gene family with its highest expression in liver and brain. Following its synthesis in the endoplasmic reticulum as a 600-kDa type I transmembrane glycoprotein, LRP1 is cleaved in the Golgi compartment by furin, producing two subunits, 515 and 85 kDa in size. These two subunits remain noncovalently associated during their transport to the cell surface (1). Via a receptor-recycling pathway, LRP1 is responsible for the endocytosis of more than 30 different extracellular ligands (2, 3). In neurons, where it is highly expressed and predominantly localized in neuronal cell bodies and dendritic processes (4), LRP1 is a major receptor for apoE/lipoprotein-containing particles and tissue-type plasminogen activator (tPA) 2 (5). In addition to its role in endocytosis of various ligands, LRP1 has been implicated to play a crucial role in cell signaling. The observation that the LRP1 C terminus undergoes rapid tyrosine phosphorylation after tPA binding corroborates its function as a signal transmitter (6). Several adaptor proteins can bind to the LRP1 tail, some of which seem to play a role in neuronal calcium, platelet-derived growth factor, or MAPK signaling (7-9). Beyond its known function of lipid uptake, LRP1 has also been attributed to be the mediator of an LTP-enhancing effect of exogenously added tPA in hippocampal slices from tPA-deficient mice (5). Further evidence supporting the hypothesis of an involvement in synaptic plasticity results from neuron-specific LRP1 knock-out mice, which...

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