Mechanism of the cleavage specificity of Alzheimer's disease gamma-secretase identified by phenylalanine-scanning mutagenesis of the transmembrane domain of the amyloid precursor protein

Proteolytic processing of the amyloid precursor protein by beta-secretase y ields A4CT (C99), which is cleaved further by the as yet unknown gamma-secr etase, yielding the beta-amyloid (A beta) peptide with 40 (A beta(40)) or 4 2 residues (A beta(42)). Because the position of gamma-secretase cleavage i s crucial for the pathogenesis of Alzheimer's disease, we individually repl aced all membrane-domain residues of A4CT outside the A beta domain with ph enylalanine, stably transfected the constructs in COS7 cells, and determine d the effect of these mutations on the cleavage specificity of gamma-secret ase (A beta(42)/A beta(40) ratio). Compared with wild-type A4CT, mutations at Val-JJ, Ile-47, and Val-50 led to decreased A beta(42)/A beta(40) ratios , whereas mutations at Thr-43, Ile-45, Val-46, Leu-49, and Met-51 led to in creased A beta(42)/A beta(40) ratios. A massive effect was observed for I45 F (34-fold increase) making this construct important for the generation of animal models for Alzheimer's disease. Unlike the other mutations, A4CT-V44 F was processed mainly to A beta(38), as determined by mass spectrometry, O ur data provide a detailed model for the active site of gamma-secretase: ga mma-secretase interacts with A4CT by binding to one side of the a-helical t ransmembrane domain of A4CT. Mutations in the transmembrane domain of A4CT interfere with the interaction between gamma-secretase and A4CT and, thus, alter the cleavage specificity of gamma-secretase.