McCusker R H, Camacho-Hubner C, Bayne M L, Cascieri M A, Clemmons D R
Department of Medicine, University of North Carolina, Chapel Hill 27599.
J Cell Physiol. 1990 Aug;144(2):244-53. doi: 10.1002/jcp.1041440210.
The cell surface of human fibroblasts contains not only type I IGF receptors but at least two forms of IGFBPs. Studies were undertaken to analyze the mechanisms by which these IGFBPs alter IGF-I-cell surface interactions. Human fetal fibroblasts (GM10) and a human glioblastoma cell line (1690) were chosen for analysis. During assays to quantify [125I]-IGF-I binding, both cell lines were shown to release IGFBPs into the binding assay buffer. Under equilibrium conditions, [125I]-IGF-I preferentially associates with IGFBPs in the assay buffer (up to 40% of the [125I]-IGF-I added) since they have a higher affinity than type I IGF receptors or IGFBPs associated with the cell surface. Likewise the addition of increasing concentrations of unlabeled IGF-I results in preferential competition for binding to assay buffer IGFBPs. This results in a repartitioning of the [125I]-IGF-I that is bound to assay buffer IGFBPs onto cell surface binding sites. The degree of repartitioning is quantitatively related to the amount of [125I]-IGF-I bound to released IGFBPs. When cultures are exposed to cycloheximide before the binding assay, both the amount of IGFBPs that are released into the assay buffer and the amount of [125I]-IGF-I that is repartitioned are decreased. In contrast when [Gln3, Ala4, Tyr15, Leu16]-IGF-I ([QAYL]-IGF-I, an IGF analog that has unaltered affinity for type I IGF receptors) is iodinated and tested, the competition curve with unlabeled IGF-I shows no repartitioning effect. This form of IGF can be used to quantify type I receptor number independent of the presence of IGFBPs. IGF-I and the [QAYL]-IGF-I compete equally with the [125I]-[QAYL]-IGF-I for binding to cell surfaces, whereas unlabeled [QAYL]-IGF-I is greater than 25-fold less potent compared to IGF-I in competing with [125I]-IGF-I for cell surface binding. Specific binding of [125I]-[QAYL]-IGF-I to GM10 and 1690 cell surfaces is less than 20% of [125I]-IGF-I binding. These findings suggest that IGFBPs that are present on human fibroblast surfaces represent a large portion of the IGF binding sites. We conclude that the amount of IGFBPs released into assay buffer is a major determinant of the repartitioning of [125I]-IGF-I to cell surface binding sites and that both cell surface and assay buffer IGFBPs modulate type I IGF receptor binding.
人成纤维细胞的细胞表面不仅含有I型胰岛素样生长因子（IGF）受体，还至少含有两种形式的IGF结合蛋白（IGFBP）。开展了多项研究以分析这些IGFBP改变IGF-I与细胞表面相互作用的机制。选择人胎儿成纤维细胞（GM10）和人胶质母细胞瘤细胞系（1690）进行分析。在定量[125I]-IGF-I结合的实验中，两种细胞系均被证明会将IGFBP释放到结合实验缓冲液中。在平衡条件下，[125I]-IGF-I优先与实验缓冲液中的IGFBP结合（高达所添加[125I]-IGF-I的40%），因为它们比I型IGF受体或与细胞表面相关的IGFBP具有更高的亲和力。同样，添加浓度不断增加的未标记IGF-I会导致与实验缓冲液中IGFBP结合的竞争加剧。这导致与实验缓冲液中IGFBP结合的[125I]-IGF-I重新分配到细胞表面结合位点。重新分配的程度与结合到释放的IGFBP上的[125I]-IGF-I的量呈定量关系。当在结合实验前将培养物暴露于环己酰亚胺时，释放到实验缓冲液中的IGFBP的量以及重新分配的[125I]-IGF-I的量均会减少。相反，当[谷氨酰胺3、丙氨酸4、酪氨酸15、亮氨酸16]-IGF-I（[QAYL]-IGF-I，一种对I型IGF受体亲和力未改变的IGF类似物）被碘化并进行测试时，与未标记IGF-I的竞争曲线未显示重新分配效应。这种形式的IGF可用于独立于IGFBP的存在来定量I型受体数量。IGF-I和[QAYL]-IGF-I与[125I]-[QAYL]-IGF-I竞争结合细胞表面的能力相同，而未标记的[QAYL]-IGF-I在与[125I]-IGF-I竞争细胞表面结合时的效力比IGF-I低25倍以上。[125I]-[QAYL]-IGF-I与GM10和1690细胞表面的特异性结合不到[125I]-IGF-I结合的20%。这些发现表明，人成纤维细胞表面存在的IGFBP代表了大部分IGF结合位点。我们得出结论，释放到实验缓冲液中的IGFBP的量是[125I]-IGF-I重新分配到细胞表面结合位点的主要决定因素，并且细胞表面和实验缓冲液中的IGFBP均调节I型IGF受体的结合。
