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Polarized sorting of beta-amyloid precursor protein and its proteolytic products in MDCK cells is regulated by two
1Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115.
Abstract:
Progressive cerebral deposition of the amyloid (A beta) beta-protein is an early and invariant feature of Alzheimer's disease. A beta is derived by proteolysis from the membrane-spanning beta-amyloid precursor protein (beta APP). beta APP is processed into various secreted products, including soluble beta APP (APPs), the 4-kD A beta peptide, and a related 3-kD peptide (p3). We analyzed the mechanisms regulating the polarized basolateral sorting of beta APP and its proteolytic derivatives in MDCK cells. Deletion of the last 32 amino acids (residues 664-695) of the beta APP cytoplasmic tail had no influence on either the constitutive approximately 90% level of basolateral sorting of surface beta APP, or the strong basolateral secretion of APPs, A beta, and p3. However, deleting the last 42 amino acids (residues 654-695) or changing tyrosine 653 to alanine altered the distribution of cell surface beta APP so that approximately 40-50% of the molecules were inserted apically. In parallel, A beta was now secreted from both surfaces. Surprisingly, this change in surface beta APP had no influence on the basolateral secretion of APPs and p3. This result suggests that most beta APP molecules which give rise to APPs in MDCK cells are cleaved intracellularly before reaching the surface. Consistent with this conclusion, we readily detected intracellular APPs in carbonate extracts of isolated membrane vesicles. Moreover, ammonium chloride treatment resulted in the equal secretion of APPs into both compartments, as occurs with other non-membranous, basolaterally secreted proteins, but it did not influence the polarity of cell surface beta APP. These results demonstrate that in epithelial cells two independent mechanisms mediate the polarized trafficking of beta APP holoprotein and its major secreted derivative (APPs) and that A beta peptides are derived in part from beta APP holoprotein targeted to the cell surface by a signal that includes tyrosine 653.
Insights
Alzheimer's disease involves amyloid beta deposition. This study reveals that specific changes in beta-amyloid precursor protein (beta APP) affect its cell surface targeting and amyloid beta secretion in epithelial cells.
Area of Science:
- Neuroscience
- Cell Biology
- Molecular Biology
Background:
- Alzheimer's disease is characterized by progressive cerebral deposition of amyloid beta (A beta) protein.
- A beta is proteolytically derived from the beta-amyloid precursor protein (beta APP).
- beta APP is processed into secreted products like soluble beta APP (APPs), A beta, and p3 peptides.
Purpose of the Study:
- To investigate the mechanisms regulating the polarized sorting of beta APP and its derivatives in epithelial cells (MDCK).
- To understand how alterations in the beta APP cytoplasmic tail affect its cell surface distribution and secretion of proteolytic products.
Main Methods:
- Utilized Madin-Darby Canine Kidney (MDCK) cells as an epithelial model system.
- Performed deletion mutagenesis on the cytoplasmic tail of beta APP to assess its role in protein trafficking.
- Analyzed the localization and secretion of beta APP, APPs, and A beta peptides using biochemical and cell biological techniques.
Main Results:
- Deletion of the C-terminal 32 amino acids of beta APP did not affect its basolateral sorting or secretion of derivatives.
- Deletion of the C-terminal 42 amino acids or mutation of tyrosine 653 resulted in altered apical insertion of beta APP and dual-surface secretion of A beta.
- Secretion of APPs and p3 remained predominantly basolateral, suggesting intracellular cleavage of beta APP for these products.
Conclusions:
- Two independent mechanisms govern the polarized trafficking of beta APP holoprotein and its secreted derivative APPs in epithelial cells.
- Tyrosine 653 in the beta APP cytoplasmic tail is crucial for targeting the holoprotein to the cell surface, influencing A beta peptide generation.
- Most APPs are generated from intracellular cleavage of beta APP, independent of cell surface targeting signals.