Haass C, Koo E H, Capell A, Teplow D B, Selkoe D J
Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115.
J Cell Biol. 1995 Feb;128(4):537-47. doi: 10.1083/jcb.128.4.537.
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.
淀粉样β蛋白（Aβ）在大脑中的进行性沉积是阿尔茨海默病的一个早期且恒定的特征。Aβ是通过跨膜β淀粉样前体蛋白（βAPP）的蛋白水解作用产生的。βAPP被加工成各种分泌产物，包括可溶性βAPP（APPs）、4-kD Aβ肽和一种相关的3-kD肽（p3）。我们分析了在MDCK细胞中调节βAPP及其蛋白水解衍生物极化基底外侧分选的机制。删除βAPP细胞质尾巴的最后32个氨基酸（残基664 - 695）对表面βAPP约90%的组成性基底外侧分选水平，或APPs、Aβ和p3的强烈基底外侧分泌均无影响。然而，删除最后42个氨基酸（残基654 - 695）或将酪氨酸653突变为丙氨酸会改变细胞表面βAPP的分布，使得约40 - 50%的分子插入到顶端。同时，Aβ现在从两个表面分泌。令人惊讶的是，表面βAPP的这种变化对APPs和p3的基底外侧分泌没有影响。这一结果表明，在MDCK细胞中产生APPs的大多数βAPP分子在到达表面之前就在细胞内被切割。与这一结论一致的是，我们在分离的膜泡的碳酸盐提取物中很容易检测到细胞内的APPs。此外，氯化铵处理导致APPs在两个区室中均等分泌，这与其他非膜性的、基底外侧分泌的蛋白质情况相同，但它不影响细胞表面βAPP的极性。这些结果表明，在上皮细胞中，两种独立的机制介导βAPP全蛋白及其主要分泌衍生物（APPs）的极化运输，并且Aβ肽部分来源于通过包含酪氨酸653的信号靶向细胞表面的βAPP全蛋白。
