Structural Requirements for the Ectodomain Cleavage of Human Cell Surface Macrophage Colony-stimulating Factor

One form of human macrophage colony-stimulating factor (CSF-1256, M-CSFα) is a member of a restricted set of cell surface transmembrane proteins, which is selected to undergo proteolytic ectodomain cleavage. To determine the substrate requirements for this cleavage, we have constructed a series of m...

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Bibliographic Details
Published in:The Journal of biological chemistry 1996-07, Vol.271 (27), p.16338-16343
Main Authors: Deng, Ping, Rettenmier, Carl W., Pattengale, Paul K.
Format: Article
Language:English
Subjects:
3T3 Cells
Allosteric Regulation
Amino Acid Sequence
Animals
Brefeldin A
Calcimycin - pharmacology
Cell Membrane - metabolism
Chloroquine - pharmacology
Cyclopentanes - pharmacology
DNA, Complementary
Humans
Macrophage Colony-Stimulating Factor - biosynthesis
Macrophage Colony-Stimulating Factor - chemistry
Macrophage Colony-Stimulating Factor - metabolism
Methionine - metabolism
Mice
Models, Structural
Molecular Sequence Data
Mutagenesis, Site-Directed
Polymerase Chain Reaction
Protein Conformation
Protein Synthesis Inhibitors - pharmacology
Recombinant Proteins - biosynthesis
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Sequence Deletion
Sulfur Radioisotopes
Tetradecanoylphorbol Acetate - pharmacology
Transfection
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Summary:One form of human macrophage colony-stimulating factor (CSF-1256, M-CSFα) is a member of a restricted set of cell surface transmembrane proteins, which is selected to undergo proteolytic ectodomain cleavage. To determine the substrate requirements for this cleavage, we have constructed a series of mutations in the cytoplasmic tail, transmembrane domain, and juxtamembrane region of CSF-1256 and stably expressed the mutated genes in NIH 3T3 cells. Our results demonstrate that membrane association of the CSF-1 precursor is required for cleavage of its growth factor ectodomain and furthermore that the juxtamembrane region Pro161-Gln162-Leu163-Gln164-Glu165 (PQLQE) (residues 161–165 of the ectodomain) is an essential determinant of cell surface CSF-1256 cleavage and that the cleavage site is partially sequence-specific. Furthermore, a mechanism of steric hindrance, which likely involves interference with protease accessibility, is postulated to explain the observed decreases in the cleavage efficiency in certain CSF-1 mutants. Finally, our results strongly suggest that the CSF-1 ectodomain is cleaved at or very near the cell surface by a membrane-associated proteolytic system.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.271.27.16338