Nam T J, Busby W H, Rees C, Clemmons D R
Department of Medicine, University of North Carolina, Chapel Hill 27599, USA.
Endocrinology. 2000 Mar;141(3):1100-6. doi: 10.1210/endo.141.3.7386.
Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) has been shown to bind to extracellular matrix (ECM) with relatively high affinity, but the ECM components that mediate this interaction have not been identified. These studies show that radiolabeled IGFBP-5 specifically coprecipitates with two ECM proteins, thrombospondin-1 (TSP-1) and osteopontin (OPN). As TSP-1 binds avidly to heparin, as does IGFBP-5, the effect of glycosaminoglycans on the TSP-1/IGFBP-5 interaction was analyzed. Heparan and dermatan sulfate inhibited binding, whereas heparin increased binding. Chondroitin sulfate A and B had no effect. In contrast, both heparin and heparan sulfate significantly inhibited the OPN-IGFBP-5 interaction and chondroitin sulfate A, B, and C had no effect. To determine the region of IGFBP-5 that was involved in each interaction, synthetic peptides that spanned several regions of IGFBP-5 were tested for their capacity to competitively inhibit coprecipitation. A peptide that contained the amino acids between positions 201 and 218 resulted in 76% and 86% inhibition of binding to TSP-1 and OPN, respectively. Three other synthetic peptides that spanned regions ofIGFBP-5 with several charged residues had no effect. IGFBP-5 mutants that contained substitutions for basic residues in the 201-218 region were tested for their ability to bind to TSP-1 or OPN. A mutant with substitutions for amino acids at positions R201 and K202 and a mutant with substitutions for K211, R214, K217, and R218 had the greatest reduction in binding to TSP-1. Mutants containing substitutions for R214 alone and the combined K217A, R218A mutant had the greatest reductions in OPN binding. When the smooth muscle cell growth response to these components was assessed, IGF-I plus IGFBP-5 or the combination of TSP-1 or OPN with IGF-I potentiated the IGF-I effect. The addition of IGFBP-5 to these combinations resulted in further significant growth stimulation. Both OPN and TSP-1 specifically bind to IGFBP-5 with high affinity. These interactions may be important for concentrating intact IGFBP-5 in extracellular matrix and for modulating the cooperative interaction between the IGF-I receptor and integrin receptor signaling pathways.
胰岛素样生长因子（IGF）结合蛋白5（IGFBP-5）已被证明能以相对较高的亲和力与细胞外基质（ECM）结合，但介导这种相互作用的ECM成分尚未确定。这些研究表明，放射性标记的IGFBP-5能与两种ECM蛋白，即血小板反应蛋白1（TSP-1）和骨桥蛋白（OPN）特异性共沉淀。由于TSP-1与IGFBP-5一样能 avidly 结合肝素，因此分析了糖胺聚糖对TSP-1/IGFBP-5相互作用的影响。硫酸乙酰肝素和硫酸皮肤素抑制结合，而肝素增加结合。硫酸软骨素A和B没有影响。相比之下，肝素和硫酸乙酰肝素都显著抑制OPN-IGFBP-5相互作用，而硫酸软骨素A、B和C没有影响。为了确定IGFBP-5参与每种相互作用的区域，测试了跨越IGFBP-5几个区域的合成肽竞争性抑制共沉淀的能力。一个包含201至218位氨基酸的肽分别导致与TSP-1和OPN结合的抑制率为76%和86%。另外三个跨越IGFBP-5具有多个带电荷残基区域的合成肽没有影响。测试了在201-218区域含有碱性残基替代物的IGFBP-5突变体与TSP-1或OPN结合的能力。在R201和K202位置氨基酸被替代的突变体以及在K211、R214、K217和R218位置氨基酸被替代的突变体与TSP-1结合的减少最为显著。仅R214被替代的突变体以及K217A、R218A联合突变体与OPN结合的减少最为显著。当评估平滑肌细胞对这些成分的生长反应时，IGF-I加IGFBP-5或TSP-1或OPN与IGF-I的组合增强了IGF-I的作用。向这些组合中添加IGFBP-5导致进一步显著的生长刺激。OPN和TSP-1都以高亲和力特异性结合IGFBP-5。这些相互作用对于在细胞外基质中浓缩完整的IGFBP-5以及调节IGF-I受体和整合素受体信号通路之间的协同相互作用可能很重要。
