ALTERNATIVE SPLICING DETERMINES THE BINDING OF PLATELET-DERIVED GROWTH-FACTOR (PDGF-AA) TO GLYCOSAMINOGLYCANS

We have shown previously that the platelet-derived growth factor (PDGF ) and a synthetic oligopeptide, corresponding to the basic carboxyl-te rminal amino acid extension of the long PDGF-A isoform, bind to hepari n. Here, we have expressed the long (rA(125)) and the short (rA(109)) variants of PDGF A-chains in Escherichia coli and produced the functio nal homodimers. Surface plasmon resonance analyses showed that while t he dimeric rA(125) bound with high affinity to low molecular weight he parin, the rA(109), lacking the basic extension, did not. This strongl y indicated that high affinity binding is due to the carboxyl-terminal extension. Investigations of kinetics and thermodynamics suggested an allosteric binding mechanism. Thus, dimeric rA(125) contains two equi valent binding sites. Following low affinity binding of heparin to one binding site, the dimer undergoes a conformational change, increasing the affinity for heparin about 40 times. This positive cooperativity requires the basic amino acid extension in both monomers of the dimeri c PDGF molecule. Thermodynamics of the reaction, showing an entropy-dr iven endothermic process, suggest the involvement of hydrophobic inter actions in this rearrangement. Three amino acids in the basic carboxyl -terminal extension were essential for the interaction: the basic resi dues Arg(111) and Lys(116), and the polar Thr(125). We also found that other glycosaminoglycan species, corresponding to those produced by h uman arterial smooth muscle cells, bound to dimeric rA(125) and that h eparan sulfate showed the highest affinity.