Multiple factors determine the selection of the ectodomain cleavage site of human cell surface macrophage colony-stimulating factor

Human cell surface macrophage colony-stimulating factor (CSF-1(256), M-CSF alpha) is converted to a soluble growth factor by a regulated proteolytic c leavage process at amino acid residues 157-159. We have previously shown th at multiple factors specified by the juxtamembrane region determine the cle avage efficiency [Deng, P., Rettenmier, C. W,, and Pattengale, P. K. (1996) J. Biol, Chem. 271, 16338-16343], In the present paper, we studied the eff ect of various deletion, insertion, and substitution mutations at or near t he cleavage site on both the number and size of cleaved CSF-1(256) products to identify the mechanisms by which the cleavage sites are selected. Delet ion of regions 161-162 or 163-165, C-terminal to the cleavage site, as well as deletion of region 150-156, N-terminal to the cleavage site, each yield ed a single cleavage product that was smaller than that derived from the wi ld type (WT). In these experiments cleavage apparently occurred at a specif ic distance from the transmembrane domain, Insertion of three additional re sidues between the normal cleavage site and the transmembrane domain yielde d one major product that was the same size as the processed form of WT CSF- 1(256) In this case the selection of the cleavage site was apparently deter mined by the amino acid sequence of the juxtamembrane region rather than by the distance from the transmembrane domain. Other amino acid substitutions at the cleavage site caused changes in cleavage site selection, providing additional evidence for the role of amino acid sequence in cleavage site se lection. Finally, a comparison of cleavage site selection in the presence a nd absence of tunicamycin treatment showed that N-glycosylation of certain mutant forms of CSF-1(256) sterically interfered with protease accessibilit y, which in turn had an effect on the selection of the site used for cleava ge. Taken together, these results indicate that cleavage site selection is determined by the amino acid sequence of the juxtamembrane region, the dist ance of the site from the transmembrane domain, and steric accessibility of the protease.