| Abstract
| - Midkine (MK), a retinoic acid-inducible heparin-binding protein, is a mitogen which initiatesa cascade of intracellular protein tyrosine phosphorylation mediated by the JAK/STAT pathway afterbinding to its high affinity p200+/MKR cell surface receptor in the G401 cell line [Ratovitski, E. A.(1998) J. Biol. Chem. 273, 3654−3660]. In this study, we determined the biophysical characteristics ofpurified recombinant murine MK and analyzed the requirements for ligand multimerization and cell surfaceproteoglycan binding for the G401 cell mitogenic activity of MK. Our studies indicate that the secretedform of MK (M = 13 kDa) exists in solution as an asymmetric monomer with a frictional coefficient of1.48 and a Stokes radius of 23.7 Å. By constructing bead models of MK using the program AtoB and theprogram HYDRO to predict the hydrodynamic properties of each model, our data suggest that MK hasa dumb-bell shape in solution composed of independent N- and C-terminal domains separated by anextended linker. This asymmetric MK monomer is a biologically active ligand with mitogenic activity onG401 cells in vitro. Neither heparin-induced formation of noncovalent MK multimers nor tissuetransglutaminase II covalent multimerization of MK enhanced MK mitogenic activity in this system.Since neither heparin competition nor cell treatment with chondroitinase ABC or heparinase III abolishedthe mitogenic effects of MK on G401 cells, cell-surface proteoglycan binding by MK does not appear tobe a requirement for its observed mitogenic effects. These results provide strong evidence that the MK-specific p200+/MKR has distinctive biochemical properties which distinguish it from the receptor tyrosinephosphatase cell-surface proteoglycan PTPζ/RPTPβ and support the hypothesis that the diverse biologicaleffects of MK are mediated by multiple cell-specific signal transduction receptors.
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