Mechanism of the cleavage specificity of Alzheimer's disease γ-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 yields A4CT (C99), which is cleaved further by the as yet unknown gamma-secretase, yielding the beta-amyloid (A beta) peptide with 40 (A beta(40)) or 42 residues (A beta(42)). Because the position of gamma-secretase cleavage is crucial for the pathogenesis of Alzheimer's disease, we individually replaced all membrane-domain residues of A4CT outside the A beta domain with phenylalanine, stably transfected the constructs in COS7 cells, and determined the effect of these mutations on the cleavage specificity of gamma-secretase (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 increased A beta(42)/A beta(40) ratios. A massive effect was observed for I45F (34-fold increase) making this construct important for the generation of animal models for Alzheimer's disease. Unlike the other mutations, A4CT-V44F was processed mainly to A beta(38), as determined by mass spectrometry, Our data provide a detailed model for the active site of gamma-secretase: gamma-secretase interacts with A4CT by binding to one side of the a-helical transmembrane 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.