Glycosaminoglycans differentially bind HARP and modulate its biological activity

Heparin affin regulatory peptide (HARP) is a polypeptide belonging to a fam ily of heparin binding growth/differentiation factors. The high affinity of HARP for heparin suggests that this secreted polypeptide should also bind to heparan sulfate proteoglycans derived from cell surface and extracellula r matrix defined as extracellular compartments. Using Western blot analysis , we detected HARP bound to heparan sulfate proteoglycans in the extracellu lar compartments of MDA-MB 231 and MC 3T3-E1 as well as NIH3T3 cells overex pressing HARP protein. Heparitinase treatment of EEL cells inhibited HARP-i nduced cell proliferation, and the biological activity of HARP in this syst em was restored by the addition of heparin, We report that heparan sulfate, dermatan sulfate, and to a lesser extent, chondroitin sulfate A, displaced HARP bound to the extracellular compartment, Binding analyses with a biose nsor showed that HARP bound heparin with fast association and dissociation kinetics (k(ass) = 1.6 x 10(6) M-1 s(-1); k(diss) = 0.02 s(-1)), yielding a K-d value of 13 nM; the interaction between HARP and dermatan sulfate was characterized by slower association kinetics (k(ass) = 0.68 x 10(6) M-1 s(- 1)) and a lower affinity (K-d = 51 nM). Exogenous heparin, heparan sulfate, and dermatan sulfate potentiated the growth-stimulatory activity of HARP, suggesting that corresponding proteoglycans could be involved in the regula tion of the mitogenic activity of HARP.