STRUCTURAL ROLE OF EXTRACELLULAR DOMAIN-1 OF ALPHA-PLATELET-DERIVED GROWTH-FACTOR (PDGF) RECEPTOR FOR PDGF-AA AND PDGF-BB BINDING

The purpose of this study was to bacterially express, purify, and refo ld combinations of the extracellular immunoglobulin (Ig)-like domains (2-3, 1-3, and 1-5) of the human alpha-platelet-derived growth factor receptor (alphaPDGFR) to characterize molecular interactions with its ligand, platelet-derived growth factor (PDGF). The far UV circular dic hromism spectroscopy of the alpha-PDGFR extracellular domains (ECDs) r evealed a predominantly beta-sheet protein, with a structure consisten t with folded Ig-like domains. The addition of PDGF-BB to these ECD ty pes changed the conformation of all three types with a decrease in mea n residue ellipticity in the following rank order: 1-5=1-3 greater tha n 2-3. In striking contrast, addition of PDGF-AA to these ECD types ma rkedly changed the conformation of ECD 2-3, by an increased mean resid ue ellipticity but no changes were observed for ECDs 1-3 and 1-5. PDGF -AA bound to the immobilized ECD types 2-3, 1-3, and 1-5 at concentrat ions of 20, 11, and 7.5nM, respectively. In contrast, PDGF-BB bound th e ECD types 2-3, 1-3, and 1-5 at concentrations of 3,3, and 2.2 nM, re spectively. Scatchard analysis of binding studies using labeled ECDs i ndicated that PDGF-BB bound ECD 1-3 and ECD 2-3 with K-D values of 74 and 72 nM, respectively. While, PDGF-AA bound ECD 1-3 and ECD 2-3 with K-D values of 33 and 87 nM, respectively. Therefore, our results indi cated that the loss of ECD 1 impaired the binding affinity of alphaPDG FR ECD 1-3 toward PDGF-AA without having a similar effect on PDGF-BB b inding. Together all of our data suggest that ECD 1 is differentially required for proper orientation of PDGF-AA but not PDGF-BB binding det erminant within ECDs 2 and 3