Interaction of Cytosolic Adaptor Proteins with Neuronal Apolipoprotein E Receptors and the Amyloid Precursor Protein

Marion Trommsdorff,Jean-Paul Borg,Benjamin Margolis,Joachim Herz

doi:10.1074/jbc.273.50.33556

Abstract

Apolipoprotein E, alpha2-macroglobulin, and amyloid precursor protein (APP) are involved in the development of Alzheimer's disease. All three proteins are ligands for the low density lipoprotein (LDL) receptor-related protein (LRP), an abundant neuronal surface receptor that has also been genetically linked to Alzheimer's disease. The cytoplasmic tails of LRP and other members of the LDL receptor gene family contain NPxY motifs that are required for receptor endocytosis. To investigate whether these receptors may have functions that go beyond ligand internalization, e.g. possible roles in cellular signaling, we searched for proteins that might interact with the cytoplasmic tails of the receptors. A family of adaptor proteins containing protein interaction domains that can interact with NPxY motifs has previously been described. Using yeast 2-hybrid and protein coprecipitation approaches in vitro, we show that the neuronal adaptor proteins FE65 and mammalian Disabled bind to the cytoplasmic tails of LRP, LDL receptor, and APP, where they can potentially serve as molecular scaffolds for the assembly of cytosolic multiprotein complexes. FE65 contains two distinct protein interaction domains that interact with LRP and APP, respectively, raising the possibility that LRP can modulate the intracellular trafficking of APP. Tyrosine-phosphorylated mammalian Disabled can recruit nonreceptor tyrosine kinases, such as src and abl, to the cytoplasmic tails of the receptors to which it binds, suggesting a molecular pathway by which receptor/ligand interaction on the cell surface could generate an intracellular signal.

Highlights

Apolipoprotein E, ␣2-macroglobulin, and amyloid precursor protein (APP) are involved in the development of Alzheimer’s disease
Using yeast 2-hybrid and protein coprecipitation approaches in vitro, we show that the neuronal adaptor proteins FE65 and mammalian Disabled bind to the cytoplasmic tails of LRP, low density lipoprotein (LDL) receptor, and APP, where they can potentially serve as molecular scaffolds for the assembly of cytosolic multiprotein complexes
The present results have shown that the cytosolic adaptor protein mDab binds to the cytoplasmic tails of the multifunctional receptor LRP, the LDL receptor, and APP, presumably by interacting with the NPxY motifs present in the tails

Summary

EXPERIMENTAL PROCEDURES

Bait sequences were amplified by PCR and sequenced after cloning. The Generation of GST-Fusion Proteins and the Construction of mDab Expression Plasmid—GST-fusion plasmids of the PID domains of p96, X11, FE65, and Numb have been described previously [19]. The mDab and FE65 PI domains and the LDL receptor and LRP tail sequences were amplified by PCR, cloned into pGEX-2T vector (Pharmacia), and verified by sequencing. Cells were harvested and lysed in Buffer A (20 mM Tris-HCl, pH 7.5, 2 mM CaCl2, 2 mM MgCl2, 150 mM NaCl, and 1% Triton X-100) in the presence of a protease inhibitor mixture (Boehringer Mannheim) by forcing them through a 22-gauge needle 10 times. Lysates were incubated with 50 ␮l of glutathione-agarose and 10 ␮g of the respective purified GST-fusion protein for 6 h at 4 °C. Proteins were separated by electrophoresis on 4 –15% SDS-polyacrylamide gel electrophoresis under nonreducing conditions and analyzed by immunoblotting using specific antibodies and ECL detection

RESULTS

Interaction of Adaptor Proteins with the LDLR Gene Family

DISCUSSION