THE TRIPLET OF LYSINE RESIDUES (LYS724-LYS725-LYS726) OF ALZHEIMERS AMYLOID PRECURSOR PROTEIN PLAYS AN IMPORTANT ROLE IN MEMBRANE ANCHORAGEAND PROCESSING

One of the pathological changes of Alzheimer's disease is the deposit of beta/A4 protein, which is derived f rom Alzheimer amyloid precursor protein (APP). In the secretory pathway, APP is cleaved at an interna l region of beta/A4 protein by a hypothetical enzyme ''secretase.'' Ou r previous study showed that the site of cleavage of APP by secretase is determined by the length from the membrane-spanning region. To inve stigate the role of the transmembrane region in APP secretion, we cons tructed the mutations of triplet lysine residues (Lys724-LyS725-LyS726 ), which are located just in the carboxyl region after the proposed me mbrane domain. The mutations were as follows: VVK, Val724-Val725-LyS72 6; LLI, Leu724-Leu725-Ile726 ; and EEE, Glu724 -Glu725-Glu726. Wild-ty pe APP and mutant APPs were expressed transiently in COS-1 cells by cD NA transfection. The hydrophobic mutant VVK and LLI were processed and secreted in a way similar to that of the wild-type APP, although the rate of secretion was decreased. The acidic mutant EEE was not secrete d into medium. Proteinase K treatment and cell surface biotinylation o f the COS-1 cells expressing APP revealed that APP was located in the plasma membrane with a short intracellular carboxyl region. However, E EE was completely digested by proteinase K treatment, which suggested that the whole residues of this mutant are located at the outer surfac e of the cell, including its proposed membrane domain and carboxyl reg ion. This mutant was not cleaved at all by secretase. These findings s uggested that the triplet lysine residues of APP after the predicted m embrane spanning domain play an important role in the membrane anchora ge. In addition, the membrane anchorage was also important for the nor mal processing by secretase.