Leal S M, Huang S S, Huang J S
Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.
J Biol Chem. 1999 Mar 5;274(10):6711-7. doi: 10.1074/jbc.274.10.6711.
High affinity insulin-like growth factor-binding proteins (IGFBP-1 to -6) are a family of structurally homologous proteins that induce cellular responses by insulin-like growth factor (IGF)-dependent and -independent mechanisms. The IGFBP-3 receptor, which mediates the IGF-independent growth inhibitory response, has recently been identified as the type V transforming growth factor-beta receptor (TbetaR-V) (Leal, S. M., Liu, Q. L., Huang, S. S., and Huang, J. S. (1997) J. Biol. Chem. 272, 20572-20576). To characterize the interactions of high affinity IGFBPs with TbetaR-V, mink lung epithelial cells (Mv1Lu cells) were incubated with 125I-labeled recombinant human IGFBPs (125I-IGFBP-1 to -6) in the presence of the cross-linking agent disuccinimidyl suberate and analyzed by 5% SDS-polyacrylamide gel electrophoresis and autoradiography. 125I-IGFBP-3, -4, and -5 but not 125I-IGFBP-1, -2, and -6 bound to TbetaR-V as demonstrated by the detection of the approximately 400-kDa 125I-IGFBP.TbetaR-V cross-linked complex in the cell lysates and immunoprecipitates. The analyses of 125I-labeled ligand binding competition and DNA synthesis inhibition revealed that IGFBP-3 was a more potent ligand for TbetaR-V than IGFBP-4 or -5. Most of the high affinity 125I-IGFBPs formed dimers at the cell surface. The cell-surface dimer of 125I-IGFBP-3 preferentially bound to and was cross-linked to TbetaR-V in the presence of disuccinimidyl suberate. IGFBP-3 did not stimulate the cellular phosphorylation of Smad2 and Smad3, key transducers of the transforming growth factor-beta type I/type II receptor (TbetaR-I.TbetaR-II) heterocomplex-mediated signaling. These results suggest that IGFBP-3, -4, and -5 are specific ligands for TbetaR-V, which mediates the growth inhibitory response through a signaling pathway(s) distinct from that mediated by the TbetaR-I and TbetaR-II heterocomplex.
高亲和力胰岛素样生长因子结合蛋白（IGFBP - 1至 - 6）是一类结构同源的蛋白质家族，它们通过胰岛素样生长因子（IGF）依赖和非依赖机制诱导细胞反应。介导IGF非依赖生长抑制反应的IGFBP - 3受体，最近被鉴定为V型转化生长因子 - β受体（TbetaR - V）（Leal, S. M., Liu, Q. L., Huang, S. S., and Huang, J. S. (1997) J. Biol. Chem. 272, 20572 - 20576）。为了表征高亲和力IGFBPs与TbetaR - V的相互作用，将貂肺上皮细胞（Mv1Lu细胞）与125I标记的重组人IGFBPs（125I - IGFBP - 1至 - 6）在交联剂辛二酸二琥珀酰亚胺酯存在下孵育，并通过5% SDS - 聚丙烯酰胺凝胶电泳和放射自显影进行分析。如在细胞裂解物和免疫沉淀中检测到约400 kDa的125I - IGFBP.TbetaR - V交联复合物所示，125I - IGFBP - 3、 - 4和 - 5与TbetaR - V结合，而125I - IGFBP - 1、 - 2和 - 6不结合。125I标记的配体结合竞争和DNA合成抑制分析表明，IGFBP - 3是比IGFBP - 4或 - 5更强效的TbetaR - V配体。大多数高亲和力的125I - IGFBPs在细胞表面形成二聚体。在辛二酸二琥珀酰亚胺酯存在下，125I - IGFBP - 3的细胞表面二聚体优先结合并与TbetaR - V交联。IGFBP - 3不刺激Smad2和Smad3的细胞磷酸化，Smad2和Smad3是转化生长因子 - β I型/II型受体（TbetaR - I.TbetaR - II）异源复合物介导信号传导的关键转导因子。这些结果表明，IGFBP - 3、 - 4和 - 5是TbetaR - V的特异性配体，TbetaR - V通过与TbetaR - I和TbetaR - II异源复合物介导的信号传导途径不同的信号传导途径介导生长抑制反应。
