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 β-secretase yields A4CT (C99), which is cleaved further by the as yet unknown γ-secretase, yielding the β-amyloid (Aβ) peptide with 40 (Aβ₄₀) or 42 residues (Aβ₄₂). Because the position of γ-secretase cleavage is crucial for the pathogenesis of Alzheimer's disease, we individually replaced all membrane-domain residues of A4CT outside the Aβ domain with phenylalanine, stably transfected the constructs in COS7 cells, and determined the effect of these mutations on the cleavage specificity of γ-secretase (Aβ₄₂/Aβ₄₀ ratio). Compared with wild-type A4CT, mutations at Val-44, Ile-47, and Val-50 led to decreased Aβ₄₂/Aβ₄₀ ratios, whereas mutations at Thr-43, Ile-45, Val-46, Leu-49, and Met-51 led to increased Aβ₄₂/Aβ₄₀ 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 Aβ₃₈, as determined by mass spectrometry. Our data provide a detailed model for the active site of γ-secretase: γ-secretase interacts with A4CT by binding to one side of the α-helical transmembrane domain of A4CT. Mutations in the transmembrane domain of A4CT interfere with the interaction between γ-secretase and A4CT and, thus, alter the cleavage specificity of γ-secretase.