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- CSF alpha) is a member of a restricted set of cell surface transmembra ne proteins, which is selected to undergo proteolytic ectodomain cleav age. To determine the substrate requirements for this cleavage, we hav e constructed a series of mutations in the cytoplasmic tail, transmemb rane domain, and juxtamembrane region of CSF-1(256) and stably express ed the mutated genes in NIH 3T3 cells. Our results demonstrate that me mbrane association of the CSF-1 precursor is required for cleavage of its growth factor ectodomain and furthermore that the juxtamembrane re gion Pro(161)-Gln(162)-Leu(163)-Gln(164)-Glu(165) (PQLQE) (residues 16 1-165 of the ectodomain) is an essential determinant of cell surface C SF-1(256) cleavage and that the cleavage site is partially sequence-sp ecific. Furthermore, a mechanism of steric hindrance, which likely inv olves interference with protease accessibility, is postulated to expla in the observed decreases in the cleavage efficiency in certain CSF-1 mutants. Finally, our results strongly suggest that the CSF-1 ectodoma in is cleaved at or very near the cell surface by a membrane-associate d proteolytic system.