Impaired trafficking and activation of tumor necrosis factor-alpha-converting enzyme in cell mutants defective in protein ectodomain shedding.
Protein ectodomain shedding is a specialized type of regulated proteolysis that releases the extracellular domain of transmembrane proteins. The metalloprotease disintegrin tumor necrosis factor-alpha-converting enzyme (TACE) has been convincingly shown to play a central role in ectodomain shedding, but despite its broad interest, very little is known about the mechanisms that regulate its activity. An analysis of the biosynthesis of TACE in mutant cell lines that have a gross defect in ectodomain shedding (M1 and M2) shows a defective removal of the prodomain that keeps TACE in an inactive form. Using LoVo, a cell line that lacks of active furin, and alpha1-Antitrypsin Portland, a protein inhibitor of proprotein convertases, we show that TACE is normally processed by furin and other proprotein convertases. The defect in M1 and M2 cells is due to a blockade of the exit of TACE from the endoplasmic reticulum. The processing of other zinc-dependent metalloproteases, previously suggested to participate in activated ectodomain shedding is normal in the mutant cells, indicating that the component mutated is highly specific for TACE. In summary, the characterization of shedding-defective somatic cell mutants unveils the existence of a specific mechanism that directs the proteolytic activation of TACE through the control of its exit from the ER.
Borroto
Aldo
A
Laboratori de Recerca Oncològica, Servei d'Oncologia Mèdica, Hospital Universitari Vall d'Hebron, Psg. Vall d'Hebron 119-129, Barcelona 08035, Spain.
Ruiz-Paz
Soraya
S
de la Torre
Teresa Villanueva
TV
Borrell-Pages
Maria
M
Merlos-Suarez
Anna
A
Pandiella
Atanasio
A
Blobel
Carl P
CP
Baselga
Josep
J
Arribas
Joaquin
J
D051722:ADAM Proteins; D000072198:ADAM17 Protein; D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004789:Enzyme Activation; D006023:Glycoproteins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008666:Metalloendopeptidases / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D016211:Transforming Growth Factor alpha / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000378:metabolism
D018076:DNA, Complementary; D006023:Glycoproteins; D016211:Transforming Growth Factor alpha; D014409:Tumor Necrosis Factor-alpha; D000515:alpha 1-Antitrypsin; C113421:alpha 1-antitrypsin Portland; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human; D015032:Zinc