Identification of fetal liver tyrosine kinase 3 (flt3) ligand domain required for receptor binding and function using naturally occurring ligand isoforms.

We used a comparative approach to identify the fetal liver tyrosine kinase 3 (flt3) ligand structure required for binding and function. Two conserved bovine flt3 ligand isoforms, which differ in a defined region within the extracellular domain, were identified and shown to be uniformly transcribed in individuals with diverse MHC haplotypes. Notably, at the amino acid level, the extracellular domain of the bovine flt3 ligand isoform 1 is 81 and 72% identical with the extracellular domains of the human and murine flt3 ligands, respectively, whereas isoform-2 has a deletion within this domain. Bovine flt3 ligand isoform 1, but not 2, bound the human flt3 receptor and stimulated murine pro B cells transfected with the murine flt3 receptor. This retention of binding and function allowed definition of key residues by identifying sequences conserved among species. We have shown that a highly conserved, 18 aa sequence within the flt3 ligand extracellular domain is required for flt3 receptor binding and function. However, a peptide representing this sequence is insufficient for receptor binding as demonstrated by its failure to inhibit the bovine flt3 ligand isoform 1 binding to the human flt3 receptor. The requirement for flanking structure was confirmed by testing bovine flt3 ligand isoform 1 constructs truncated at specific residues outside the 18 aa sequence. Overall, the flt3 ligand structure required for function is markedly similar to that of the related hemopoietic growth factors, CSF-1 and steel factor. This definition of the required flt3 ligand structure will facilitate development of agonists to enhance dendritic cell recruitment for vaccines and immunotherapy.