Purification of B16-F1 melanoma autocrine motility factor and its receptor

Tumor autocrine motility factor (AMF) is a cytokine which stimulates both random and directed cell migration by self-producing cells. AMF has been detected in and purified from serum-free conditioned medium of murine B16-F1 melanoma cells. Under nonreducing conditions AMF migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band of Mr 55,000, whereas under reducing conditions it migrates as a single polypeptide of Mr 64,000. Two-dimensional polyacrylamide gel electrophoresis of the purified AMF resolved two polypeptides with isoelectric points of 6.35 (major) and 6.4 (minor). No carbohydrate side chains were detected in the B16-F1 AMF. Purified AMF stimulated B16-F1 cell migration in a dose-dependent fashion and bound directly in a protein-protein-binding assay to the AMF receptor, a cell surface glycoprotein of Mr 78,000 [glycoprotein (gp) 78]. The involvement of gp78 in AMF-stimulated function was demonstrated by motility assays. These results suggest that AMF is the natural ligand for the gp78-AMF receptor.