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.