MECHANISM OF ACTION OF INSULIN-LIKE GROWTH FACTORS

胰岛素样生长因子的作用机制

• 批准号: 6290738
• 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/6290738
• 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 and the development of reagents that block IGF-I receptor function.We have used the yeast two-hybrid system to identify new binding partners for the IGF-I receptor. Last year, we reported that SOCS-2 (suppressor of cytokine signaling) was identified as a binding partner for the IGF-I receptor in a two-hybrid screen of a human fetal brain library. To determine whether or not other members of the SOCS family interact with the IGF-I receptor, SOCS-3 cDNA was cloned by utilizing the polymerase chain reaction. In the yeast two- hybrid system, SOCS-3 interacts with the autophosphorylated IGF-I receptor but not with the kinase negative receptor construct. In contrast, in in vitro experiments utilizing GST-SOCS-3 and IGF-I receptor from fibroblasts, and in transient transfection experiments in 293 human embryonic kidney fibroblasts, SOCS-3 binds to both the basal and activated receptor. SOCS-3 is weakly phosphorylated on tyrosine residues in an IGF-I dependent fashion and is more heavily phosphorylated on serine residues with or without stimulation of 293 cells with IGF-I. SOCS-3 is ubiquitinated in 293 cells and immunostaining shows that FLAG-SOCS-3 is localized in both the cytoplasm and nucleus. In addition , we have evidence that SOCS-3 can exist as a dimer in transfected 293 cells and self-associates in a yeast two-hybrid assay. These new findings expand the possibilities for the function of SOCS-3 in the cell. We have transfected IGF-I receptor negative fibroblasts with two truncated IGF-I receptor constructs, one consisting of the transmembrane and cytoplasmic domain of the IGF-I receptor and the second consisting of the transmembrane domain of oncogenic erbB-2 (neu) and the cytoplasmic domain of the IGF-I receptor. Clones from vector only transfection showed no anchorage independent growth whereas the truncated wild type IGF-I receptor clones and the truncated neu/IGF-I receptor clones showed many large colonies in the soft agar assay. In contrast, the proliferation of the two receptor clones in medium containing low serum was no different than the vector only clones. These results demonstrate a discordance between proliferation and anchorage-independent growth and provide support for the existence of a distinct transformation pathway emanating from the IGF-I receptor. - IGF-I receptor, SOCS-3, erbB- 2(neu),

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受体的信号传导和开发阻断IGF-I受体功能的试剂。我们使用酵母双杂交系统鉴定了IGF-I受体的新结合伴侣。去年，我们报道了SOCS-2（细胞因子信号转导抑制因子）被确定为IGF-I受体的结合伴侣在一个人胎脑文库的双杂交筛选。为了确定SOCS家族的其他成员是否与IGF-I受体相互作用，通过利用聚合酶链反应克隆SOCS-3 cDNA。在酵母双杂交系统中，SOCS-3与自磷酸化IGF-I受体相互作用，但不与激酶阴性受体构建体相互作用。相反，在利用来自成纤维细胞的GST-SOCS-3和IGF-I受体的体外实验中，以及在293人胚肾成纤维细胞中的瞬时转染实验中，SOCS-3结合到基础和活化受体两者。SOCS-3以IGF-I依赖的方式在酪氨酸残基上弱磷酸化，并且在用IGF-I刺激或不刺激293细胞的情况下在丝氨酸残基上更严重地磷酸化。SOCS-3在293细胞中被泛素化，免疫染色显示FLAG-SOCS-3定位于细胞质和细胞核中。此外，我们有证据表明，SOCS-3可以作为二聚体存在于转染的293细胞和酵母双杂交试验中的自我协会。这些新发现扩大了SOCS-3在细胞中功能的可能性。我们用两种截短的IGF-I受体构建体转染IGF-I受体阴性成纤维细胞，一种由IGF-I受体的跨膜和胞质结构域组成，第二种由致癌erbB-2（neu）的跨膜结构域和IGF-I受体的胞质结构域组成。来自仅载体转染的克隆没有显示出锚定非依赖性生长，而截短的野生型IGF-I受体克隆和截短的neu/IGF-I受体克隆在软琼脂测定中显示出许多大的集落。相比之下，两种受体克隆在含有低血清的培养基中的增殖与仅载体的克隆没有不同。这些结果表明，增殖和锚定非依赖性生长之间的不一致性，并提供支持的存在一个独特的转化途径，从IGF-I受体。- IGF-I受体，SOCS-3，erbB- 2（neu），