Abstract
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.
Highlights
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 ␣-platelet-derived growth factor receptor (␣PDGFR) to characterize molecular interactions with its ligand, platelet-derived growth factor (PDGF)
These findings indicated that high affinity binding of mAb ␣R1 to ␣PDGFR requires proper folding of determinants within Ig-like domain 2
In this report we describe bacterial expression, purification, and refolding of the ␣-PDGFR extracellular domains (ECDs) 2–3, 1–3, and 1–5 to evaluate structural features that contribute to PDGF binding
Summary
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 ␣-platelet-derived growth factor receptor (␣PDGFR) to characterize molecular interactions with its ligand, platelet-derived growth factor (PDGF). Heterodimer (AB) have been isolated from serum [5] and bind with high affinities to either or both cell surface-glycosylated receptors designated as ␣ [6] and  [7] of 180 and 185 kDa, respectively. Both receptors are highly homologous, the three PDGF isoforms bind the ␣-PDGFR [8] while the -PDGFR primarily binds the PDGF-BB form [4]. The tyrosine-phosphorylated receptor becomes a target for binding Src homology region 2 (SH2) domains of a number of signaling molecules, which include phosphatidylinositol-3 kinase, GTPase-activating protein, phospholipase C␥ (PLC␥), Src, Grb, Nck, and the tyrosine phosphatase Syp (for a review, see Ref. 11), which activate a number of inter-linked downstream signaling pathways
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