Mutagenesis of a Gly-Ser cleavage site in MUC1 inhibits ectodomain shedding.
MUC1 mucin is a type 1 transmembrane glycoprotein dimer of extracellular and membrane-bound subunits. The two non-covalently associated subunits are produced from a single polypeptide chain by proteolysis at a Gly-Ser peptide bond in the endoplasmic reticulum prior to localization on the cell surface. However, once expressed on the surface, the extracellular subunit is shed from cells in the absence of the membrane-associated subunit. Previous studies implicated a cellular metalloproteinase mediating MUC1 ectodomain shedding, but no reports have delineated the site of metalloproteinase cleavage or directly assessed the role of the Gly-Ser bond in shedding. Therefore, we performed site-directed mutagenesis of the Gly-Ser site and determined the effects on MUC1 proteolysis and shedding. Ser-->Ala substitution blocked MUC1 cleavage and inhibited shedding. Equal amounts of wild type and mutant MUC1 were expressed on the cell surface, indicating that lack of shedding of the mutant molecule was not due to reduced surface localization. We conclude that the Gly-Ser peptide bond is required for MUC1 shedding.
Lillehoj
Erik P
EP
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, 20 N Pine St, Baltimore, MD 21201, USA.
Han
Feng
F
Kim
K Chul
KC
D000595:Amino Acid Sequence; D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism; D019556:COS Cells; D005998:Glycine / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism; D051379:Mice; D018396:Mucin-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
D018396:Mucin-1; D012694:Serine; D005998:Glycine