Proteolysis of chimeric beta-amyloid precursor proteins containing the Notch transmembrane domain yields amyloid beta-like peptides.

gamma-Secretase is an unusual intramembranous protease that has been reported to cleave the beta-amyloid precursor protein (APP) near the middle of its transmembrane domain (TMD) but cleave Notch near the cytoplasmic end of its TMD. To ascertain whether the TMD sequence of the substrate determines where gamma-secretase cleaves and whether the region just before the TMD participates in recognition by the enzyme, we expressed chimeric human APP molecules containing either the TMD or pre-TMD regions of Notch or other transmembrane proteins. APP chimeras bearing either the Notch or the amyloid precursor-like protein-2 TMD released similar amounts of approximately 4-kDa amyloid beta-peptide (Abeta)-like peptides as did intact APP. Mass spectrometry revealed that the principal Abeta-like peptide ended at residue 40, indicating cleavage at the middle of the Notch TMD in the chimera. Generation of Abeta-like peptides was significantly decreased when the APP TMD was replaced by those of SREBP-1 or human epithelial growth factor receptor 3. Replacement of the APP pre-TMD region (Abeta 10-28) with that of SREBP-1 increased generation of Abeta-like peptides, while those of human epithelial growth factor receptor 3 or amyloid precursor-like protein-2 decreased it. We conclude that gamma-secretase can cleave near the middle of the Notch TMD, that Abeta-like peptides may arise during Notch processing, and that the pre-TMD sequence of the substrate influences recognition or binding by the enzyme.