Alternative Splicing Determines the Binding of Platelet-Derived Growth Factor (PDGF-AA) to Glycosaminoglycans

Abstract

We have shown previously that the platelet-derived growth factor (PDGF) and a synthetic oligopeptide, corresponding to the basic carboxyl-terminal amino acid extension of the long PDGF-A isoform, bind to heparin. Here, we have expressed the long (rA₁₂₅) and the short (rA₁₀₉) variants of PDGF A-chains in Escherichia coli and produced the functional homodimers. Surface plasmon resonance analyses showed that while the dimeric rA₁₂₅ bound with high affinity to low molecular weight heparin, the rA₁₀₉, lacking the basic extension, did not. This strongly indicated that high affinity binding is due to the carboxyl-terminal extension. Investigations of kinetics and thermodynamics suggested an allosteric binding mechanism. Thus, dimeric rA₁₂₅ contains two equivalent 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 dimeric PDGF molecule. Thermodynamics of the reaction, showing an entropy-driven endothermic process, suggest the involvement of hydrophobic interactions in this rearrangement. Three amino acids in the basic carboxyl-terminal extension were essential for the interaction: the basic residues Arg¹¹¹ and Lys¹¹⁶, and the polar Thr¹²⁵. We also found that other glycosaminoglycan species, corresponding to those produced by human arterial smooth muscle cells, bound to dimeric rA₁₂₅ and that heparan sulfate showed the highest affinity.