| The primary pathogenic cause of Alzheimer's disease (AD) is widely believed to be the amyloid beta peptide (AR), which is proteolytically derived from the amyloid precursor protein (APP). Extensive research has been carried out to explore the roles of this protein in the context of AD; however, little information about its normal physiologic activities has been elucidated. Therefore, a crosslinking technique was developed to identify proteins interacting with APP on the plasma membrane of cells growing in culture. Following crosslinking, four APP-containing complexes were detected by Western blot in cell lysates. Crosslinked complexes containing presenilin 1 (PSI), beta-site APP-cleaving enzyme 1 (BACE1), and possibly β1-integrin were also observed, and the masses of these complexes were consistent with the possibility that any of these APP-interacting proteins might be components of APP-containing crosslinked complexes. Neither the intracellular APP-binding protein Fe65 nor the extracellular Aβ-binding protein α2-macroglobulin was detected in the novel crosslinked complexes. Immunopurification using anti-APP antibodies covalently linked to agarose pulled down a doublet of APP-containing crosslinked complexes, which did not contain immunoreactive PS1 or BACE1. The observation that the quantity of APP-containing crosslinked complexes was inversely correlated to cell density and the suggestion that β1-integrin was present in a high molecular mass complex support the growing evidence that APP participates in cell adhesion processes. Establishing an important technical foundation for future exploration of transmembrane protein-protein interactions, the crosslinking procedure described herein generated four APP-containing protein complexes that can further the investigation of APP function. |
