Cytoskeletal Interactions of Human Dlg Protein

描述（由申请人提供）：hDlg是人源化抗体的人同源物。 果蝇盘大肿瘤抑制因子。我们最近发现了一本小说 称为GAKIN（鸟苷酸激酶相关驱动蛋白）的蛋白质，其结合至 hDlg和PSD-95的鸟苷酸激酶样结构域。通过序列同源性，GAKIN是 运动蛋白质中驱动蛋白超家族的一员。这项建议的目的是 是为了验证GAKIN-hDlg相互作用奠定了基础的假设， 对于MAGUKS与基于微管的 细胞骨架，这种相互作用是功能重要的， 细胞内运输途径。为了验证这一假设，我们将研究： (1)hDlg与GAKIN相互作用的一般性和特异性。我们建议 使用体外诱变，蛋白质 表达和BlAcore测定。我们将确定GAKIN的距离 约束和测试其他成员之间是否存在类似的相互作用 关于MAGUK家族总之，这些研究很可能阐明一部小说， MAGUKs通过驱动蛋白与微管物理偶联的机制 电动机. (2)与hDlg-GA KIN复合物相关的蛋白质的鉴定。 我们将使用大规模蛋白质纯化和微测序技术， 鉴定与hDlg-GAKIN蛋白复合物相关的蛋白质。 这些组件的识别将是必不可少的完整的理解 MAGUK的脚手架和运输功能。(3)功能 hDlg-GAKIN蛋白复合物的表征。我们建议 hDlg的膜运输和靶向由GAKIN介导。为了验证这一 假设，我们将引入hDlg和GAKIN的特定突变体， 在功能上不能结合到淋巴、上皮和神经元细胞中 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-电机连接在 蛋白质和膜囊泡靶向其他MAGUKS。(4)的鼠模型 挖缺陷。测试hDlg的功能作用的先决条件是 缺乏内源性hDlg或表达突变体的细胞系的可用性 缺乏鸟苷酸激酶结构域的hDlg形式（Gukiess DIg）。使用基因 靶向技术，我们将在小鼠直系同源物中产生一个Guless突变体， 的hDlg（mDlg）。这一目标将使我们能够评估生理 mDlg无效突变对生长和增殖的影响 造血和神经细胞类型以及mDlg是否作为肿瘤抑制因子 如在果蝇中观察到的Dlg。

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.