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

血小板源性生长因子（PDGF）是一种二聚体蛋白，通过酪氨酸激酶α-和β-受体发挥其作用。每个受体的胞外部分由5个Ig样结构域组成，制备了重组α受体结构域1-4（α RD 1 -4）、1-3（α RD 1 -3）和1-2（α RD 1 -2），并用于研究可溶性配体-受体复合物的组装。在微摩尔浓度的PDGF下，α RD 1 -3和α RD 1 -4均形成1：2摩尔组成的复合物，即一个二聚体PDGF分子结合两个可溶性受体。与α RD 1 -4相反，α RD 1 -3在配体过量的条件下形成可检测的1：1复合物。α RD 1 -4与α RD 1 -3相比，在限制配体浓度的条件下，形成1：2复合物的能力增强。因此，我们得出结论，IG-4介导的受体-受体相互作用稳定复合物。由于α RD 1 -4和α RD 1 -3在阻断亚纳摩尔浓度的PDGF与细胞表面受体结合方面是等效的，我们也得出结论， ...更多信息 这种作用主要是通过形成Ig样结构域4非依赖性1：1配体-受体复合物来实现的。最后，由于α RD 1 -2以高亲和力结合PDGF-BB，而PDGF-AA仅以低亲和力结合，我们的结论是，PDGF α受体的IG-3含有对PDGF-AA结合特别重要的表位，并且大多数PDGF-BB结合表位位于IG-1和2中。制备了α RD 1 -4-GST和β RD 1 -3-GST，并分析了它们阻断PDGF与细胞表面受体结合的能力。在α-和β-受体的情况下，与切割形式相比，需要100- 1，000倍低浓度的GST融合蛋白。与α RD 1 -4和β RD 1 -3不同，α RD 1 -4-GST和β RD 1 -3-GST以配体非依赖性二聚体形式存在。与α RD 1 -4相比，共价交联的α RD 1 -4二聚体的效价增加了50倍。因此，我们得出结论，可溶性受体的二聚体性质是高拮抗效力的原因。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