Generation and characterization of mutant cell lines defective in gamma-secretase processing of Notch and amyloid precursor protein.
Several type I integral membrane proteins, such as the Notch receptor or the amyloid precursor protein, are cleaved in their intramembrane domain by a gamma-secretase enzyme, which is carried within a multiprotein complex. These cleavages generate molecules that are involved in intracellular or extracellular signaling. At least four transmembrane proteins belong to the gamma-secretase complex: presenilin, nicastrin, Aph-1, and Pen-2. It is still unclear whether these proteins are the only components of the complex and whether a unique complex is involved in the different gamma-secretase cleavage events. We have set up a genetic screen based on the permanent acquisition or loss of an antibiotic resistance depending on the presence of an active gamma-secretase able to cleave a Notch-derived substrate. We selected clones deficient in gamma-secretase activity using this screen on mammalian cells after random mutagenesis. We further analyzed two of these clones and identified previously undescribed mutations in the nicastrin gene. The first mutation abolishes nicastrin production, and the second mutation, a point mutation in the ectodomain, abolishes nicastrin maturation. In both cases, gamma-secretase activity on Notch and APP is impaired.
Olry
Annie
A
Unité de Signalisation Moléculaire et Activation Cellulaire, URA 2582, CNRS, Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France.
Chastagner
Patricia
P
Israël
Alain
A
Brou
Christel
C
D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism*; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000172:deficiency / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D010450:Endopeptidases; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000172:deficiency / Q000378:metabolism*; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D051381:Rats; D051880:Receptors, Notch; D011994:Recombinant Proteins / Q000378:metabolism
D016564:Amyloid beta-Protein Precursor; D017931:DNA Primers; D008565:Membrane Proteins; D051880:Receptors, Notch; D011994:Recombinant Proteins; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human