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

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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.