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 refold combinations of the extracellular immunoglobulin (Ig)-like domains (2-3, 1-3, and 1-5) of the human alpha-platelet-derived growth factor receptor (alpha PDGFR) to characterize molecular interactions with its ligand, platelet-derived growth factor (PDGF). The far UV circular dichroism spectroscopy of the alpha-PDGFR extracellular domains (ECDs) revealed a predominantly beta-sheet protein, with a structure consistent with folded Ig-like domains. The addition of PDGF-BB to these ECD types changed the conformation of all three types with a decrease in mean residue ellipticity in the following rank order: 1-5 = 1-3 > 2-3. In striking contrast, addition of PDGF-AA to these ECD types markedly changed the conformation of ECD 2-3, by an increased mean residue 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 concentrations of 20, 11, and 7.5 nM, respectively. In contrast, PDGF-BB bound the ECD types 2-3, 1-3, and 1-5 at concentrations of 3, 3, and 2.2 nM, respectively. Scatchard analysis of binding studies using labeled ECDs indicated that PDGF-BB bound ECD 1-3 and ECD 2-3 with KD values of 74 and 72 nM, respectively. While, PDGF-AA bound ECD 1-3 and ECD 2-3 with KD values of 33 and 87 nM, respectively. Therefore, our results indicated that the loss of ECD 1 impaired the binding affinity of alpha PDGFR ECD 1-3 toward PDGF-AA without having a similar effect on PDGF-BB binding. Together all of our data suggest that ECD 1 is differentially required for proper orientation of PDGF-AA but not PDGF-BB binding determinant within ECDs 2 and 3.
D000077214:Becaplermin; D001665:Binding Sites; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D005110:Extracellular Space / Q000502:physiology; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008961:Models, Structural; D010982:Platelet-Derived Growth Factor / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017510:Protein Folding; D020574:Proto-Oncogene Proteins c-sis; D011869:Radioligand Assay; D020796:Receptor, Platelet-Derived Growth Factor alpha; D017479:Receptors, Platelet-Derived Growth Factor / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D007457:Iodine Radioisotopes; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins; C075278:platelet-derived growth factor A; D000077214:Becaplermin; D020796:Receptor, Platelet-Derived Growth Factor alpha; D017479:Receptors, Platelet-Derived Growth Factor