Identification and clinical use of the modifiers of small G proteins heir target proteins

我们鉴定了一些RAS下游分子，制备了它们的抗体和cDNA突变体，并对它们的功能进行了分析。(1)Ral下游分子的鉴定及其性质。我们鉴定POB1是一种新的RalBP1结合蛋白。POB1与Grb2结合，酪氨酸磷酸化，与EGF受体结合，与EGF形成复合体。POB1的N-末端区域具有EH结构域。Epsin和Eps15与POB1的EH结构域结合。这两种蛋白都与AP-2和笼状蛋白形成了一个复合体，并被认为对受体介导的内吞作用的调节很重要。(2)抗体的产生。免疫印迹法检测结果显示，在颌下腺、大脑、小脑、胸腺、心、肺、脾、肾上腺和睾丸中均有RAL的表达。大脑、前额叶突触小泡丰富。POB1和Epsin也存在于各种组织和细胞中。(3)功能分析。我们产生了Ral、RalBP1、POB1和Epsin的cDNA突变，并检测了它们与内吞作用的关系。Ral、RalBP1和POB1调节胰岛素和EGF受体的内吞作用，但不调节转铁蛋白的内吞作用，而Epsin参与这三种激动剂的受体介导的内吞作用。因此，Ral/RalBP1/POB1信号复合体将信号从胰岛素和EGF等配体传递到Eps15和Epsin，从而诱导配体依赖性的内吞作用，但不参与转铁蛋白受体等结构性内吞作用。POB1、Epsin和Eps15对于分析内吞作用的调节是有用的。由于有几种人类癌症具有参与内吞作用的分子突变，因此有可能通过筛选癌症中所述的Ral下游分子来建立一种新的基因诊断系统。

We identified some downstream molecules of Ras, generated their antibodies and cDNA mutants, and analyzed their functions. (1) Identification of downstream molecules of Ral and their characterization. We identified POB1 as a novel RalBP1-binding protein. POB1 bound to Grb2, was tyrosine-phosphorylated, and formed a complex with EGF receptor in response to EGF. The N-terminal region of POB1 has an EH domain. Epsin and Eps15 bound to the EH domain of POB1. Both proteins formed a complex with AP-2 and clathrin and was known to be important for the regulation of receptor-mediated endocytosis. (2) Generation of antibodies. Western blotting with the anti-Ral antibody demonstrated that Ral was present in submandibular gland, cerebrum, cerebellum, thymus, heart, lung, spleen, adrenal glands, and testis. m cerebrum, Ral was enriched in synaptic vesicles. POB1 and Epsin were also present in various tissues and cells. (3) Functional analyses. We generated cDNA mutations of Ral, RalBP1, POB1, and Epsin, and examined their involvement of endocytosis. Ral, RalBP1, and POB1 regulated the endocytosis of the receptors for insulin and EGF but not for transferrin, while Epsin was involved in the receptor-mediated endocytosis of these three agonists. Therefore, the signaling complex of Ral/RalBP1/POB1 transmitted the signal from a ligand such as insulin and EGF to Eps15 and Epsin, thereby induces the ligand-dependent endocytosis, but was not involved in the constitutive endocytosis such as the transferrin receptor.We believe that the antibodies and cDNA mutants of Ral. POB1, Epsin, and Eps15 are useful for the analyses of the regulation of endocytosis. Since there are several human cancers which have mutations of molecules involved in endocytosis, it is possible to make a novel system of gene diagnosis by screening the downstream molecules of Ral described here in cancers.