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

配体机制：血小板衍生生长因子（PDGF）受体复合物组装

• 批准号: 10680602
• 负责人: MIYAZAWA Keiji
• 金额: $ 1.86万
• 依托单位: Tokyo Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680602/
• 关键词: growth factor; receptor; antagonist; アンタゴニスト; プロテアーゼ

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

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

项目成果

Leppanen O.-P.et al.: "Predimerization of recombinant platelet-derived growth factor extracellular domains increases antagonistic potency"Biochemistry. (印刷中). (2000)

Leppanen O.-P. 等人：“重组血小板衍生生长因子胞外结构域的预二聚化增加了拮抗效力”（出版中）。

Miyazawa, K., Wang, Y., Minoshima, S., Shimizu, N., and Kitamura, N.: "Structural organization and chromosomal localization of the human hepatocyte growth factor activator gene. Phylogenetic and functional relationship with blood coagulation factor XII, u

Miyazawa, K.、Wang, Y.、Minoshima, S.、Shimizu, N. 和 Kitamura, N.：“人肝细胞生长因子激活剂基因的结构组织和染色体定位。与凝血因子 XII 的系统发育和功能关系

Shimomura, T., Denda, K., Kawaguchi, T., T., Matusmoto, K., Miyazawa, K., and Kitamura, N.: "Multiple sites of proteolytic cleavage to release soluble forms of hepatocyte growth factor activator inhibitor type 1 from a transmembrance form"J. Biochem.. 126

Shimomura, T.、Denda, K.、Kawaguchi, T.、T.、Matusmoto, K.、Miyazawa, K. 和 Kitamura, N.：“多个蛋白水解位点释放可溶形式的肝细胞生长因子激活剂抑制剂

Leppanen, O.-P, et al.: "Predimerization of rcombinant platelet-derived growth factor receptor extracellular domains increases antagonistic potency"Biochemistry. (印刷中). (2000)

Leppanen，O.-P 等人：“重组血小板衍生生长因子受体胞外结构域的预二聚化增加了拮抗效力”《生物化学》（出版中）。

Leppanen, O-P., Miyazawa, K., Backstrom, G., Pietras, K., Sjoblom, T., Heldin, C.-H., and Ostman, A.: "Predimerization of recombinant platelet-derived growth factor receptor extracellular domains increases antagonistic potency"Biochemistry. (in press). (2

Leppanen, O-P.、Miyazawa, K.、Backstrom, G.、Pietras, K.、Sjoblom, T.、Heldin, C.-H. 和 Ostman, A.：“重组血小板衍生生长因子受体胞外预二聚化