BIOCHEMICAL AND GENETIC-CHARACTERIZATION OF MULTIPLE SPLICE VARIANTS OF THE FLT3 LIGAND

We have performed a comprehensive analysis of cell lines and tissues t o compare and contrast the expression patterns of Flt3 ligand (FL), c- Kit ligand (KL), and macrophage colony-stimulating factor as well as t heir receptors, Flt3, c-Kit, and c-Fms. The message for FL is unusuall y ubiquitous, whereas that of its receptor is quite restricted, appare ntly limiting the function of the ligand to fetal development and earl y hematopoiesis. We have also sequenced a mouse FL genomic clone, reve aling how the three splice variant FL mRNAs that we have isolated aris e. The chromosomal location of the FL gene has been mapped, by in situ hybridization, to chromosome 7 in mouse and chromosome 19 in human. N atural FL protein has been purified from a stromal cell line and shown to be a 65 kD nondisulfide-linked homodimeric glycoprotein comprised of 30 kD subunits, each containing 12 kD of N- and O-linked sugars. Pu lse-chase experiments show that one of the splice variants (T110) is r esponsible for producing the bulk of soluble FL, but only after it has first been expressed at the cell surface as a membrane-bound form. Th e other splice-variant forms produce molecules that are either obligat orily soluble (T169) or membrane-bound but released only very slowly ( T118). Finally, even though most cell lines express some amount of FL mRNA, we found that very little FL protein is actually made, with T ce lls and stromal cells being the major producers. The data suggests tha t FL plays its roles over Very short distances, perhaps requiring cell -cell contact. (C) 1996 by The American Society of Hematology.