Cytoskeletal Interactions of Human Dlg Protein

描述(申请人提供)：hDlg是人类的同源物 果蝇盘状大肿瘤抑制物。我们最近确定了一部小说 一种名为GAKlN(鸟苷酸激酶相关激动素)的蛋白质，与 HDlg和PSD-95的鸟苷酸激酶样结构域。根据序列同源性，Gakin是 马达蛋白运动蛋白超家族的成员。这项建议的目的是 是为了检验Gakin-hDlg相互作用奠定基础的假设 关于MAGUKS与基于微管的偶联的一般范例 细胞骨架，这种相互作用对 细胞内的运输途径。为了检验这一假设，我们将检验： (1)hDlg与Gakin相互作用的一般性和特异性。我们建议 利用体外诱变技术定位Gakin和hDlg结合位点 表达，BlAcore分析。我们将确定加金的距离 绑定和测试其他成员中是否存在类似的交互 马古克家族的人。总而言之，这些研究可能会阐明一部小说 MAGUK通过运动蛋白与微管的物理偶联机制 摩托公司。(2)hDIG-GA KIN复合体相关蛋白质的鉴定。 我们将使用大规模蛋白质纯化和微测序技术来 鉴定与hDlg-Gakin蛋白复合体相关的蛋白质。 对这些组件的识别对于完全理解是必不可少的 MAGUK的脚手架和运输功能。(3)功能 HDlg-Gakin蛋白复合体的特性。我们建议， HDlg的膜转运和靶向是由Gakin介导的。为了测试这一点 假设，我们将介绍hDlg和Gakin的特定突变体 功能障碍，不能与淋巴样细胞、上皮细胞和神经细胞结合 并检测它们对hDlg定位和受体聚集的影响。这些 研究旨在测试Maguk-Motor链接在 蛋白质和膜泡靶向其他MAGUKS。(4)小鼠模型 挖掘不足。测试hDlg的功能角色的先决条件是 缺乏内源性hDlg或表达突变体的细胞系的可用性 形式的hDlg缺乏其鸟苷酸激酶结构域(Gukiess Dig)。使用基因 靶向技术，我们将在小鼠的直系同源基因中产生一个无GUKER突变体 小鼠的hDlg(MDlg)。这个目标将使我们能够评估生理上的 MDlg零突变对人骨肉瘤生长增殖的影响 造血细胞和神经细胞类型以及mDlg是否发挥肿瘤抑制作用 正如在果蝇体内观察到的DLG一样。

DESCRIPTION (provided by applicant): hDlg is the human homologue of the Drosophila discs large tumor suppressor. We have recently identified a novel protein termed GAKlN (Guanylate kinase Associated Kinesin) that binds to the guanylate kinase-like domain of hDlg and PSD-95. By sequence homology, GAKIN is a member of the kinesin superfamily of motor proteins. The aim of this proposal is to test the hypothesis that the GAKIN-hDlg interaction lays the foundation for a general paradigm of the coupling of MAGUKS to the microtubule-based cytoskeleton, and that this interaction is functionally important for the intracellular trafficking pathways. To test this hypothesis, we will examine: (1) Generality and specificity of hDlg interaction with GAKIN. We propose to map the GAKIN and hDlg binding sites using in vitro mutagenesis, protein expression, and BlAcore assays. We will determine the proximity of GAKIN binding and test for the existence of similar interactions within other members of the MAGUK family. Together, these studies are likely to elucidate a novel mechanism for the physical coupling of MAGUKs to microtubules via kinesin motors. (2) Identification of proteins associated with the hDlg-GA KIN complex. We will use large-scale protein purification and microsequencing techniques to identify the proteins associated with the hDlg-GAKIN protein complex. Identification of these components will be essential for complete understanding of scaffolding and transport functions of MAGUKs. (3) Functional characterization of the hDlg-GAKIN protein complex. We propose that the membrane trafficking and targeting of hDlg is mediated by GAKIN. To test this hypothesis, we will introduce specific mutants of hDlg and GAKIN that are functionally disabled for binding into lymphoid, epithelial, and neuronal cells and examine their effects on hDlg localization and receptor clustering. These studies are designed to test for the function of MAGUK-Motor link in the protein and membrane vesicle targeting of other MAGUKS. (4) Murine models of Dig deficiency. A prerequisite for testing the functional role of hDlg is the availability of cell lines lacking the endogenous hDlg or expressing a mutant form of hDlg lacking its guanylate kinase domain (Gukiess DIg). Using gene targeting techniques, we will generate a Gukless mutant in the murine ortholog of hDlg (mDlg) in mice. This aim will allow us to assess the physiological consequences of mDlg null mutation on the growth and proliferation of hematopoietic and neural cell types and whether mDlg acts as a tumor suppressor as observed for Dlg in Drosophila.