Structural requirements for the ectodomain cleavage of human cell surface macrophage colony-stimulating factor.
One form of human macrophage colony-stimulating factor (CSF-1(256), M-CSFalpha) 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-1(256) 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-1(256) 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.
Deng
P
P
Department of Pathology, Children's Hospital of Los Angeles and University of Southern California School of Medicine, Los Angeles, California 90027, USA.
Rettenmier
C W
CW
Pattengale
P K
PK
D016475:3T3 Cells; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D020126:Brefeldin A; D000001:Calcimycin / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002738:Chloroquine / Q000494:pharmacology; D003517:Cyclopentanes / Q000494:pharmacology; D018076:DNA, Complementary; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013462:Sulfur Radioisotopes; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D003517:Cyclopentanes; D018076:DNA, Complementary; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D020126:Brefeldin A; D000001:Calcimycin; D016173:Macrophage Colony-Stimulating Factor; D002738:Chloroquine; D008715:Methionine; D013755:Tetradecanoylphorbol Acetate