Dynamin Function by Tyrosine Phoshorylation

酪氨酸磷酸化的动力功能

• 批准号: 7194650
• 负责人: Yehia Daaka
• 金额: $ 14.44万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7194650
• 关键词: PC12 cells; beta adrenergic receptor; biological signal transduction; cell line; dynamin; enzyme induction /repression; enzyme inhibitors; genetic regulation; immunocytochemistry; intermolecular interaction; molecular site; phosphorylation; protein localization; protein purification; protein sequence; protein structure function; protein tyrosine kinase; receptor expression; receptor mediated endocytosis; recombinant proteins; site directed mutagenesis; stimulant /agonist; tyrosine

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.

描述（由申请人提供）：重要的细胞反应，包括细胞 新陈代谢、增殖和分化都受到特定的调控 细胞外配体与其质膜锚定之间的相互作用 受体。因此，配体与受体的可及性是一个重要的因素 生物调节方面。细胞表面的受体表达为 至少部分地由通过调节的囊泡运输决定 受体内化。内化（也称为内吞作用）是一种 重要的调控机制，与受体有关 重新敏化、下调以及最近的信号转导。 G 蛋白偶联受体 (GPCR) 和受体酪氨酸的内吞作用 激酶（RTK）依赖于网格蛋白包被的内陷和裂变 囊泡从质膜进入细胞质。坑的收缩 囊泡从质膜分裂需要 GTP 酶活性 动力。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。 Dynamin 最近被证明可以直接与非受体相互作用 酪氨酸激酶 c-Src 是一种酪氨酸磷酸蛋白。因此，我们 假设酪氨酸激酶活性可能调节受体的内吞作用 通过作用于对受体内化重要的分子，尤其是 动力。本提案引入了一种新的监管机制 动力在受体介导的内吞作用过程中的作用。中央 假设是激动剂调节的动力酪氨酸磷酸化 控制其在受体内化和信号传导中的功能。实验 将重点关注确定参与的分子决定因素 受体调节的动力酪氨酸磷酸化并阐明其作用 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）证明β2肾上腺素受体调节的酪氨酸 内源性动力蛋白 I 的磷酸化并鉴定磷酸化 残留物； (2) 确定β2肾上腺素能的体内机制 受体调节动力的酪氨酸磷酸化； (3) 表达和 纯化重组动力蛋白用于体外测定 确定动力酪氨酸磷酸化对其的生理作用 酶活性和受体介导的内吞作用。