Dynamin Function by Tyrosine Phoshorylation

描述(申请人提供)：重要的细胞反应，包括细胞 新陈代谢、增殖和分化都是由特定的 细胞外配体与其质膜锚定的相互作用 感受器。因此，配体与受体的可及性是一个重要的因素 生物调节中的一个方面。细胞表面受体的表达是 至少部分地由囊泡贩运所支配，通过调节 受体内化。内化(也称为内吞作用)是一种 重要的调节机制，这涉及到受体 再敏化，下调，以及最近在信号转导方面。 G蛋白偶联受体和受体酪氨酸的内吞作用 蛋白酪氨酸激酶(RTK)依赖于包被蛋白的内陷和分裂 囊泡从质膜进入胞浆。坑道的收缩 质膜上的囊泡分裂需要GTP酶的活性 动力素。最近的证据表明，蛋白质酪氨酸磷酸化是 也是激动剂调节的细胞表面受体内化所必需的， 尽管该蛋白酪氨酸磷酸化的位点仍未确定。 动力蛋白最近被证明直接与非受体相互作用。 酪氨酸激酶c-Src是一种酪氨酸磷酸化蛋白。因此，我们 酪氨酸激酶活性可能调节受体内吞作用的假说 通过作用于对受体内化特别重要的分子 动力剂。本提案引入了一种新的监管机制，以 动力蛋白在受体介导的内吞作用过程中的作用。中环 假说是激动剂调节的动力蛋白酪氨酸磷酸化 控制其在受体内化和信号转导中的作用。实验 将专注于确定涉及到的分子决定因素 受体调节的动力蛋白酪氨酸磷酸化及其作用的研究 动力蛋白酪氨酸磷酸化对其酶活性的影响。具体的 目的是：(1)证实β2肾上腺素能受体调节的酪氨酸 内源性动力蛋白I的磷酸化及其鉴定 残留；(2)确定β-2肾上腺素能的体内机制(S) 受体调节动力蛋白的酪氨酸磷酸化；以及(3)表达和 纯化重组动力素蛋白用于体外检测 确定Dynamin酪氨酸磷酸化在ITS中的生理作用 酶活性和受体介导的内吞作用。

DESCRIPTION (provided by applicant): Vital cellular responses including cell metabolism, proliferation and differentiation are all regulated by specific interactions between extracellular ligands and their plasma membrane-anchored receptors. Accessibility of ligand to receptor, is therefore, an important facet in biological regulation. Receptor expression on the cell surface is dictated, at least in part, by vesicle trafficking via the regulation of receptor internalization. Internalization (also termed endocytosis) is an important regulatory mechanism, which is implicated in receptor resensitization, downregulation, and more recently in signal transduction. Endocytosis of G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) is dependent on the invagination and fission of clathrin coated vesicles from the plasma membrane into the cytosol. Constriction of the pits and fission of vesicles from the plasma membrane requires the GTPase activity of dynamin. Recent evidence suggests that protein tyrosine phosphorylation is also required for agonist-regulated internalization of cell surface receptors, although the locus of this protein tyrosine phosphorylation remains undefined. Dynamin has recently been shown to directly interact with the non-receptor tyrosine kinase c-Src and to be a tyrosine phospho-protein. Therefore, we hypothesize that tyrosine kinase activity may regulate endocytosis of receptors by acting on molecules important for receptor internalization especially dynamin. The present proposal introduces a novel regulatory mechanism for dynamin's role in the process of receptor-mediated endocytosis. The central hypothesis is that agonist-regulated tyrosine phosphorylation of dynamin controls its function in receptor internalization and signaling. Experiments will focus on identifying the molecular determinants involved in receptor-regulated tyrosine phosphorylation of dynamin and elucidating the role of tyrosine phosporylation of dynamin on its enzymatic activity. The specific aims are: (1) To demonstrate the beta2 adrenergic receptor-regulated tyrosine phosphorylation of endogenous dynamin I and to identify phosphorylated residues; (2) To determine the in vivo mechanism(s) by which beta2 adrenergic receptors regulate tyrosine phosphorylation of dynamin; and (3) To express and purify recombinant dynamin proteins for use in vitro assays aimed at determining the physiologic role for tyrosine phosphorylation of dynamin on its enzymatic activity and receptor-mediated endocytosis.