Mechanism of Action of Insulin-like Growth Factors

胰岛素样生长因子的作用机制

• 批准号: 6433337
• 负责人: PETER NISSLEY
• 金额: --
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 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/6433337
• 关键词: biological signal transduction; genetic library; growth factor receptors; human subject; insulinlike growth factor; phosphorylation; protein structure function; receptor binding; tissue /cell culture

In addition to being important for normal fetal and postnatal growth, there is increasing evidence that insulin-like growth factors I and II (IGF-I, IGF-II) also support the growth of certain cancers. Biologic responses to IGF-I and IGF-II are signaled by the IGF-I receptor. Therefore, we are focusing our research effort on understanding signaling by the IGF-I receptor. We have used the yeast two-hybrid system to identify new binding partners for the IGF-I receptor. Two binding partners which we identified in a screen of a human fetal brain library were two isoforms of 14-3-3 protein and SOCS-2 (supressor of cytokine signaling). To explore a functional role for 14-3-3 in IGF-I receptor signaling we overexpressed IGF-I receptors that were mutated in the binding sites for 14-3-3 as defined in the yeast two-hybrid system. Deletion of the COOH tail of the receptor beyond residue 1310 resulted in a marked increase in the number of large colonies in a soft agar assay. When this mutation was combined with a S1283A mutation the number of large colonies decreased significantly. These results suggest that 14-3-3 may be involved in a transformation pathway emanating from the COOH tail of the receptor. To explore a functional role for SOCS-2 in IGF-I receptor signaling, we developed a tetracycline inducible system for SOCS-2 expression in MG-63 human osteosarcoma cells. We found that when SOCS-2 expression was induced, IGF-I stimulated DNA synthesis was inhibited by 50%, suggesting that SOCS-2 may also be a negative regulator of IGF-I receptor signaling. We have also performed a yeast two-hybrid screen of a MG-63 human osteosarcoma cell library using the IGF-I receptor cytoplasmic domain as bait. In addition to eight proteins which were previously identified as binding partners for the IGF-I receptor, seven new interactors were found. Baserga and his colleagues reported that an embryo fibroblast cell line derived from an IGF-I receptor knockout mouse could not be transformed by a variety of oncoproteins including SV40 large T antigen as assessed by anchorage independent growth. To confirm this potentially important finding we developed mouse embryo fibroblast lines from wild type (3) and receptor knockout (3) embryos and attempted to transform these cell lines with SV40 large T antigen. In agreement with Baserga's findings, the three receptor negative cell lines initially could not be transformed by SV40 large T antigen as measured by formation of large colonies in soft agar. However, with continued passage of clones from one of the receptor negative transfectants, all three clones formed large colonies in soft agar. Thus the requirement for the IGF-I receptor to be present in order to transform cells with SV40 large T antigen is not absolute.

除了对正常胎儿和出生后的生长很重要外，越来越多的证据表明胰岛素样生长因子I和II（IGF-I，IGF-II）也支持某些癌症的生长。 对IGF-I和IGF-II的生物学应答由IGF-I受体发出信号。因此，我们的研究重点是了解IGF-I受体的信号传导。我们已经使用酵母双杂交系统，以确定新的结合伙伴的IGF-I受体。我们在人胎脑文库的筛选中鉴定的两个结合配偶体是14 - 3 - 3蛋白和SOCS-2（细胞因子信号传导抑制剂）的两种同种型。 为了探索14 - 3 - 3在IGF-I受体信号传导中的功能作用，我们过表达IGF-I受体，所述IGF-I受体在酵母双杂交系统中定义的14 - 3 - 3的结合位点中突变。 在软琼脂测定中，缺失超过残基1310的受体的COOH尾导致大菌落数量显著增加。 当这种突变与S1283A突变相结合时，大菌落的数量显著减少。 这些结果表明，14 - 3 - 3可能参与从受体的COOH尾发出的转化途径。 为了探索SOCS-2在IGF-I受体信号传导中的功能作用，我们开发了一种四环素诱导的MG-63人骨肉瘤细胞中SOCS-2表达系统。我们发现，当SOCS-2表达被诱导时，IGF-I刺激的DNA合成被抑制50%，这表明SOCS-2也可能是IGF-I受体信号传导的负调节剂。我们还进行了酵母双杂交筛选MG-63人骨肉瘤细胞库使用IGF-I受体胞质结构域作为诱饵。 Baserga和他的同事们报道了一种来自IGF-I受体敲除小鼠的胚胎成纤维细胞系不能被多种癌蛋白转化，包括通过锚定非依赖性生长评估的SV40大T抗原。为了证实这一潜在的重要发现，我们从野生型（3）和受体敲除（3）胚胎中开发了小鼠胚胎成纤维细胞系，并试图用SV 40大T抗原转化这些细胞系。 与Baserga的发现一致，三种受体阴性细胞系最初不能被SV40大T抗原转化，如通过在软琼脂中形成大集落所测量的。然而，随着来自受体阴性转染子之一的克隆的继续传代，所有三个克隆在软琼脂中形成大的集落。 因此，为了用SV40大T抗原转化细胞而存在IGF-I受体的要求不是绝对的。