Biol. Bull. 207: 167. (October 2004)
© 2004 Marine Biological Laboratory
Use of the Split-Ubiquitin Two-Hybrid System to Identify Proteins Interacting With the Alzheimer Proteins APP and LRP
Rebecca Vitale and
Joseph D. Buxbaum
Mount Sinai School of Medicine, New York, New York
Two proteins that have been strongly implicated in the pathology of Alzheimer’s disease are the Alzheimer amyloid protein precursor (APP) and the LDL receptor-related protein (LRP). Both of these transmembrane proteins undergo cleavage in the intra-membranous domain by a presenilin/
-secretase complex, releasing a cytoplasmic peptide. With APP this cleavage contributes to the release of the Aß peptide, the peptide found in the extracellular plaques characteristic of Alzheimer’s disease. LRP is a large (approximately 600 kDa) endocytic receptor with four clusters of ligand-binding repeats. The function of the cytoplasmic peptides cleaved from APP and LRP is still unclear. Notch is another transmembrane protein that undergoes cleavage in the intra-membranous domain by the presenilin/-secretase complex. The cytoplasmic peptide released from Notch goes to the nucleus where it interacts with an obligate binding partner from the CSL family of DNA-binding proteins to activate transcription. We are using a novel (split-ubiquitin) yeast two-hybrid approach, which allows for the use of transmembrane proteins as baits, to identify proteins interacting with the cytoplasmic domains of APP and LRP. Traditional yeast two-hybrid approaches have identified the FE65 family of proteins as APP-interacting proteins. Using the split-ubiquitin system, we confirmed that these proteins could be isolated from a mouse brain library as APP-interacting proteins. We are now characterizing additional clones from the APP and LRP screens to identify novel interacting proteins that may contribute to the function and/or processing of these proteins.
