The long-term goal is to employ a multidisciplinary approach to gain a thorough understanding of the molecular mechanisms by which Rin1 regulates both insulin receptor (IR)-mediated trafficking and signaling. Elucidation of the mechanisms by which Rin1 regulates IR signaling and trafficking may serve as a model for tyrosine receptor kinases and other receptor signal transducing proteins. Each of these aims is significant because the mechanistic and physiologic functions of the Rin1 molecules are unknown, as are the mechanisms used to regulate Rin1 protein function. Thus, we have a unique opportunity to determine whether such effects are sufficient to alter the downstream effect of the IR signaling pathways, a question of considerable interest to the physiology of the insulin-signaling field. Therefore, the strength of this project is its ability to reveal new signaling regulatory mechanisms that are directly relevant to an important physiologic process and which may be defective in some cases of human disease. Accordingly, our objectives are to: 1) Determine how Rin1 is coupled to the activated insulin receptor. We will elucidate the specificity of this Rin1-insulin receptor interaction via mutagenesis and biochemical binding assays. 2) Determine how Rin1 is linked to the endocytic trafficking of the activated insulin receptor. We will address the mechanism of Rin1 function during internalization of insulin receptor through the activation of Rab5 GTPase. 3) Determine how Rin1 is integrated to the insulin receptor signaling transduction pathways. We postulate that Rin1 is required for insulin receptor signaling, which may favor one or more signal transduction pathways. 4) Determine the role of Rin1 in early endosome fusion. We will reconstitute and characterize Rin1-dependent endosome fusion by using a novel in vitro endosome fusion assay. Delineating the cellular and molecular biology of Rin1 will expand our understanding of the role of receptor internalization and membrane trafficking in signal transduction and will also help to define therapeutic targets for selectively modulating signal transduction via tyrosine kinase receptors. Diabetes affects an estimated 20 million Americans, making this disease one of the leading mortality risk factors due its association with greatly increased risk of heart disease, hypertension and cancer. This proposal is directly relevant to the goal of establishing molecular factors that lead to affecting insulin receptor signaling and action. It addresses whether functional modification of insulin signaling pathways, one that is predominant in metabolic tissues --impaired glucose metabolism-- and another being a growth factor-like pathway --cell growth ad proliferation-- lead to diabetes by impaired insulin receptor trafficking and signaling, a key question in the field. In this project, we have a unique opportunity to address this question by elucidating the molecular mechanism by which Ras interference 1 regulates insulin receptor-signaling and intracellular trafficking and may also serve as a model for tyrosine receptor kinases. The key feature of this project is its ability to reveal new signaling regulatory mechanisms that are relevant to an important physiologic process, such as diabetes.
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