MECHANISM OF ACTION OF INSULIN-LIKE GROWTH FACTORS

胰岛素样生长因子的作用机制

• 批准号: 3751999
• 负责人: S P NISSLEY
• 金额: --
• 依托单位: DIVISION OF CANCER BIOLOGY AND DIAGNOSIS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3751999
• 关键词: human tissue; immunoprecipitation; insulinlike growth factor; lysosomes; protein transport; tissue /cell culture; transfection; western blottings

A rapid, sensitive, dot-blot assay for insulin-like growth factor-II (IGF-II) based on chemiluminescence methodology has been further evaluated for possible interference by IGF binding proteins (IGFBPs). Six different IGFBPs were tested in the dot blot assay at concentrations up to 60-fold the concentration of IGF-II. The IGF-II signal was not diminished. By contrast, in a conventional radioimmunoassay using the same monoclonal antibody to IGF-II, the binding proteins produced significant interference, causing 23% to 87% competition of binding of 125I-IGF-II to the antibody. Thus the IGF-II dot blot assay is unique in allowing direct measurement of IGF-II in conditioned medium from cells in culture without interference by IGFBPs. Addition of IGF-I to MG-63 human osteosarcoma cells resulted in rapid activation of extracellular signal regulated kinase 2 (ERK2) as assessed by ion exchange HPLC and ERK kinase antibodies. ERK1 appeared to be already activated in serum-starved MG-63 cells and not to be further activated following addition of IGF-I. Thus ERK2 activation may be part of the signaling pathway for the IGF-I receptor. Further evaluation of fibroblasts from two patients with deletion of the distal long arm of chromosome 15 showed evidence for decreased expression of IGF-I receptor mRNA in one of the patients compared to age-matched controls. Evaluation of receptor function in a bioassay which measured the incorporation of [3H]thymidine into DNA in the presence of a full range of IGF-I concentrations showed no difference in the ED50 for IGF-I between the patient and control fibroblasts. We conclude from these findings and our earlier results that although there is evidence for decreased IGF-I receptor expression in fibroblasts from patients with chromosome 15q deletion syndrome, receptor function as assessed by cellular response to IGF-I is not impaired.

胰岛素样生长因子-II快速、灵敏的斑点印迹检测方法 基于化学发光方法学的(IGF-II)已进一步 评估IGF结合蛋白(IGFBPs)可能的干扰。 6种不同的IGFBPs在斑点印迹分析中进行了浓度测定。 高达IGF-II浓度的60倍。IGF-II信号不是 被削弱了。相比之下，在传统的放射免疫分析中使用 同样的IGF-II单抗，产生的结合蛋白 显著干扰，导致23%至87%的绑定竞争 125I-IGF-II与抗体结合。因此IGF-II斑点印迹分析是独一无二的 允许直接测量条件培养液中的IGF-II 在不受IGFBPs干扰的情况下培养细胞。 在MG-63人骨肉瘤细胞中加入IGF-I可迅速 细胞外信号调节蛋白2(ERK2)的激活 采用离子交换高效液相色谱法和ERK激酶抗体。ERK1似乎是 已经在血清匮乏的MG-63细胞中激活，不会进一步激活 在添加IGF-I后激活。因此，ERK2的激活可能是 IGF-I受体的信号通路。 对两例缺失该基因的患者成纤维细胞的进一步评价 15号染色体的远端长臂显示出减少的证据 IGF-I受体mRNA在其中一例患者中的表达 年龄匹配的对照组。生物测定中受体功能的评价 它测量了[~3H]胸腺嘧啶核苷在DNA中的掺入 存在全范围的IGF-I浓度显示没有差别 在患者和对照成纤维细胞之间的IGF-I的ED50。我们 从这些发现和我们早先的结果得出结论，尽管有 是成纤维细胞IGF-I受体表达降低的证据 染色体15q缺失综合征患者，受体功能为 通过细胞对IGF-I的反应评估没有受损。