Mechanism of ligand : receptor complex assembly of platelet-derived growth factor (PDGF)

血小板衍生生长因子是一种二聚体蛋白，通过酪氨酸激酶α和β受体发挥作用。重组α受体结构域1-4(αRd1-4)、1-3(αRd1-3)和1-2(αRd1-2)用于研究可溶性配体-受体复合体的组装。在微摩尔浓度下，αRd1-3和αRd1-4都形成了摩尔组成为1：2的复合体，即一个二聚体PDGF分子与两个可溶性受体结合。与αRd1-4相比，αRd1-3在配体过剩的条件下形成了可检测到的1：1络合物。在限制配体浓度的条件下，αRD1-4与αRD1-3相比，形成1：2络合物的能力增强。因此，我们得出结论，Ig-4介导的受体-受体相互作用稳定了该复合体。由于αrd1-4和αrd1-3在阻断亚纳摩尔浓度的血小板衍生生长因子与细胞表面受体的结合方面是相同的，我们还得出结论：…更多的这种作用主要是通过形成Ig样结构域4-独立的1：1配体-受体复合体来实现的。最后，由于αRd1-2以高亲和力结合PDGF1-BB，而PDGF1-AA仅以低亲和力结合，我们得出结论：α-受体的Ig-3含有对PDGFAA结合具有特殊重要性的表位，并且大多数与PDGF1-BB结合的表位位于Ig-1和2GST中。我们构建了αRd1-4和βRd1-3(αRd1-4-GST和βRd1-3-Gst)的重组GST融合蛋白，并对其阻断细胞表面受体的能力进行了分析。在α-和β-受体的情况下，与裂解形式相比，GST-融合蛋白的浓度需要低100-1,000倍。αRd1-4-Gst和βRd1-3-Gst与αRd1-4和βRd1-3相反，是以非配体二聚体形式存在的。与αRd1-4相比，共价交联的αRd1-4二聚体的效价提高了50倍。因此，我们得出结论，可溶性受体的二聚性是产生高拮抗效力的原因。较少

Platelet-derived growth factor (PDGF) is a dimeric protein that exerts its effects through tyrosine kinase α-and β-receptors. The extracellular part of each receptor is composed of five Ig-like domains.Recombinant α-receptor domains 1-4 (αRD1-4), 1-3 (αRD1-3), and 1-2 (αRD1-2) were prepared and used to study the assembly of soluble ligand-receptor complexes. With micromolar concentrations of PDGF, both αRD1-3 and αRD1-4 formed complexes of 1: 2 molar composition, i.e. one dimeric PDGF molecule bound two soluble receptors. αRD1-3, in contrast to αRD1-4, formed detectable 1:1 complexes under conditions of ligand excess. αRD1-4 displayed an increased ability to form 1:2 complexes as compared with αRD1-3 under conditions of limiting concentrations of ligand. We thus conclude that Ig-4-mediated receptor-receptor interactions stabilize the complex. Since αRD1-4 and αRD1-3 were equipotent in blocking binding of subnanomolar concentrations of PDGF to cell-surface receptors, we also conclude th … More at this effect is predominantly achieved through formation of Ig-like domain 4-independent 1:1 ligand-receptor complexes. Finally, since αRD1-2 bound PDGF-BB with high affinity, whereas PDGF-AA was bound only with low affinity, we conclude that Ig-3 of the PDGF α-receptor contains epitopes of particular importance for PDGF-AA binding and that most of the PDGF-BB-binding epitopes reside in Ig-1 and 2.Recombinant GST-fusion proteins of αRD1-4 andβRD1-3 (αRD1-4-GST and βRD1-3-GST) were generated and analyzed with regard to their ability to block PDGF binding to cell surface receptors. Both in the case of the α- and β-receptors, 100-1,000 fold lower concentration of the GST-fusion protein were required, as compared to the cleaved forms. αRD1-4-GST and βRD1-3-GST, in contrast to αRD1-4 and βRD1-3, were shown to occur as ligand independent dimers. Covalently cross-linked αRD1-4 dimers displayed a 50-fold increased potency as compared to αRD1-4. We thus conclude that the dimeric nature of the soluble receptors is responsible for the high antagonistic potency. Less