Parker A, Rees C, Clarke J, Busby W H, Clemmons D R
Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Mol Biol Cell. 1998 Sep;9(9):2383-92. doi: 10.1091/mbc.9.9.2383.
Insulin-like growth factor-binding protein-5 (IGFBP-5) has been shown to bind to fibroblast extracellular matrix (ECM). Extracellular matrix binding of IGFBP-5 leads to a decrease in its affinity for insulin-like growth factor-I (IGF-I), which allows IGF-I to better equilibrate with IGF receptors. When the amount of IGFBP-5 that is bound to ECM is increased by exogenous addition, IGF-I's effect on fibroblast growth is enhanced. In this study we identified the specific basic residues in IGFBP-5 that mediate its binding to porcine smooth-muscle cell (pSMC) ECM. An IGFBP-5 mutant containing alterations of basic residues at positions 211, 214, 217, and 218 had the greatest reduction in ECM binding, although three other mutants, R214A, R207A/K211N, and K202A/R206N/R207A, also had major decreases. In contrast, three other mutants, R201A/K202N/R206N/R208A, and K217N/R218A and K211N, had only minimal reductions in ECM binding. This suggested that residues R207 and R214 were the most important for binding, whereas alterations in K211 and R218, which align near them, had minimal effects. To determine the effect of a reduction in ECM binding on the cellular replication response to IGF-I, pSMCs were transfected with the mutant cDNAs that encoded the forms of IGFBPs with the greatest changes in ECM binding. The ECM content of IGFBP-5 from cultures expressing the K211N, R214A, R217A/R218A, and K202A/R206N/R207A mutants was reduced by 79.6 and 71.7%, respectively, compared with cells expressing the wild-type protein. In contrast, abundance of the R201A/K202N/R206N/R208A mutant was reduced by only 14%. Cells expressing the two mutants with reduced ECM binding had decreased DNA synthesis responses to IGF-I, but the cells expressing the R201A/K202N/R206N/R208A mutant responded well to IGF-I. The findings suggest that specific basic amino acids at positions 207 and 214 mediate the binding of IGFBP-5 to pSMC/ECM. Smooth-muscle cells that constitutively express the mutants that bind weakly to ECM are less responsive to IGF-I, suggesting that ECM binding of IGFBP-5 is an important variable that determines cellular responsiveness.
胰岛素样生长因子结合蛋白5（IGFBP - 5）已被证明能与成纤维细胞外基质（ECM）结合。IGFBP - 5与细胞外基质的结合导致其对胰岛素样生长因子 - I（IGF - I）的亲和力降低，这使得IGF - I能更好地与IGF受体达到平衡。当通过外源添加增加与ECM结合的IGFBP - 5的量时，IGF - I对成纤维细胞生长的作用增强。在本研究中，我们确定了IGFBP - 5中介导其与猪平滑肌细胞（pSMC）ECM结合的特定碱性残基。一个在第211、214、217和218位碱性残基发生改变的IGFBP - 5突变体与ECM的结合减少最为显著，尽管其他三个突变体R214A、R207A/K211N和K202A/R206N/R207A也有大幅减少。相比之下，其他三个突变体R201A/K202N/R206N/R208A、K217N/R218A和K211N与ECM的结合仅有极小程度的减少。这表明残基R207和R214对结合最为重要，而与之相邻的K211和R218的改变影响极小。为了确定ECM结合减少对细胞对IGF - I复制反应的影响，用编码ECM结合变化最大的IGFBP形式的突变体cDNA转染pSMC。与表达野生型蛋白的细胞相比，表达K211N、R214A、R217A/R218A和K202A/R206N/R207A突变体的培养物中IGFBP - 5的ECM含量分别降低了79.6%和71.7%。相比之下，R201A/K202N/R206N/R208A突变体的丰度仅降低了14%。表达两种ECM结合减少的突变体的细胞对IGF - I的DNA合成反应降低，但表达R201A/K202N/R206N/R208A突变体的细胞对IGF - I反应良好。这些发现表明，第207和214位的特定碱性氨基酸介导了IGFBP - 5与pSMC/ECM的结合。组成性表达与ECM弱结合的突变体的平滑肌细胞对IGF - I的反应较弱，这表明IGFBP - 5与ECM的结合是决定细胞反应性的一个重要变量。
