IDENTIFICATION AND MUTATION OF PRIMARY AND SECONDARY PROTEOLYTIC CLEAVAGE SITES IN MURINE STEM-CELL FACTOR CDNA YIELDS BIOLOGICALLY-ACTIVE,CELL-ASSOCIATED PROTEIN

Phenotypic abnormalities of melanocytes, germ cells, and hematopoietic cells of Steel mice demonstrate the critical role of stem cell factor (SCF) in development. Production of SCF in the hematopoietic microenv ironment as either a membrane-associated or soluble factor leads to pl eiotropic effects on hematopoietic stem and progenitor cells and signi ficant effects on the production of erythroid cells. Although the prod uction of these two forms of SCF is highly regulated, the physiologic role(s) of membrane-associated and soluble SCF remain unclear. We have demonstrated that the generation of soluble murine SCF by murine stro mal cells derived from the fetal hematopoietic microenvironment is dep endent on two distinct proteolytic cleavage sites. The primary site in exon 6 is preferentially utilized in these cells. The secondary site located in exon 7 is utilized only in the absence of the primary site. Proteolytic processing at this secondary site appears to be species-s pecific, since the human protein sequence diverges at this site, and p rotein expressed from the hilman cDNA encoding this site in murine str omal cells remains largely membrane-associated. Site-directed mutagene sis of the murine SCF cDNA encoding both proteolytic cleavage sites le ads to the generation of membrane-associated and biologically active S CF on murine stromal cells. These results suggest that the regulation of processing of the secondary proteolytic cleavage site could play a critical role in the function of membrane-associated SCF protein.