The Triplet of Lysine Residues (Lys 724 ‐Lys 725 ‐Lys 726 ) of Alzheimer's Amyloid Precursor Protein Plays an Important Role in Membrane Anchorage and Processing

One of the pathological changes of Alzheimer's disease is the deposit of β/A4 protein, which is derived from Alzheimer amyloid precursor protein (APR). In the secretory pathway, APR is cleaved at an internal region of β/A4 protein by a hypothetical enzyme “secretase.” Our previous study showed that the site of cleavage of APR by secretase is determined by the length from the membrane‐spanning region. To investigate the role of the transmem‐ brane region in APR secretion, we constructed the mutations of triplet lysine residues (Lys 724 ‐Lys 725 ‐Lys 726 ), which are located just in the carboxyl region after the proposed membrane domain. The mutations were as follows: VVK, Val 724 ‐Val 725 ‐Lys 726 ; LLI, Leu 724 ‐Leu 725 ‐lle 726 ; and EEE, Glu 724 ‐Glu 725 ‐Glu 726 . Wild‐type APR and mutant APPs were expressed transiently in COS‐1 cells by cDNA trans‐fection. The hydrophobic mutant VVK and LLI were processed and secreted in a way similar to that of the wild‐ type APR, although the rate of secretion was decreased. The acidic mutant EEE was not secreted into medium. Proteinase K treatment and cell surface biotinylation of the COS‐1 cells expressing APR revealed that APR was located in the plasma membrane with a short intracellular carboxyl region. However, EEE was completely digested by proteinase K treatment, which suggested that the whole residues of this mutant are located at the outer surface of the cell, including its proposed membrane domain and carboxyl region. This mutant was not cleaved at all by secretase. These findings suggested that the triplet lysine residues of APR after the predicted membrane spanning domain play an important role in the membrane anchorage. In addition, the membrane anchorage was also important for the normal processing by secretase.