PHOSPHOINOSITIDE REGULATION OF PHAGOCYTOSIS

描述(摘自申请人的摘要)：关节炎，是否由于 细菌感染或自身免疫性疾病的特征是 关节间隙中有吞噬的白细胞。吞噬细胞能够 通过多个途径摄取致病微生物、凋亡细胞和免疫复合物 细胞表面受体。他们发现，抑制磷脂酰肌醇 用药物抑制FcgR-和FcgR的3-激酶活性 补体受体(CR3)介导的吞噬作用。操纵 WT表达对三磷酸磷脂酰肌醇(PIP3)含量的影响 和PIP3磷酸盐(SH2结构域)的催化失活等位基因 含有肌醇5‘磷酸)也调节吞噬作用。最后， 来自Ship基因敲除小鼠的巨噬细胞显示FcgR和FcgR增强 CR3介导的吞噬作用，暗示PIP3促进吞噬作用。这 研究提案将解决这些酶调节的机制 吞噬作用。在特定的目标1中，他们将确定SH2域是否 它的抗吞噬功能是必需的。它们将进行亚克隆 将MYC标记的等位基因导入高效表达载体(PSFFV) 巨噬细胞系中的水平。在具体目标2中，他们将决定是否 已知的SHIP相互作用蛋白，如Shc，参与SHIP 下调FCR和CR3介导的吞噬作用。他们将使用 免疫共沉淀法鉴定体内船体结合蛋白 结扎FcgR或CR3后。对于他们将采用的体外测试 亲和层析使用GST融合的SH2结构域。此外, 他们还将使用亲和层析来识别潜在的新技术 与船舶相互作用的蛋白质。在具体目标3中，他们将确定PIP3是否 CR3“内向外”信令、“自外向内”信令、 或者同时使用原代巨噬细胞和转基因细胞系。他们会学习 PIP3在CR3的“自内向外”信令中扮演的角色如下 检测：CR3的表面表达，激活依赖表位的存在， 受体聚集、细胞骨架结合和CR3介导的ICAM结合 或者纤维蛋白原。为了研究“由外而内”的信号，他们将使用细胞系 表达具有结构性活性的CR3并测定PI3K活性，PIP3 产生、在ICAM或纤维蛋白原上扩散、肌动蛋白组装和吞噬 在CR3结扎后。

DESCRIPTION (Taken from the applicant's abstract): Arthritis, whether due to bacterial infections or autoimmune diseases, is characterized by the presence of phagocytic leukocytes in the joint spaces. Phagocytes are capable of ingesting pathogenic microbes, apoptotic cells and immune complexes by multiple cell surface receptors. They have found that inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) activity using pharmacologic agents inhibits FcgR- and complement receptor (CR3)-mediated phagocytosis. Manipulation of phosphatidylinositol (3,4,5) trisphosphate (PIP3) content by expression of WT and catalytically-inactive alleles of a PIP3 phosphates (the SH2 domain containing inositol 5' phosphates, SHIP) also regulates phagocytosis. Finally, macrophages derived from SHIP knock-out mice display enhanced For FcgR and CR3-mediated phagocytosis, implicating PIP3 in promoting phagocytosis. This research proposal will address the mechanisms by which these enzymes regulate phagocytosis. In Specific Aim 1, they will determine whether the SH2 domain of SHIP is required for its anti-phagocytic function. They will subclone Myc-tagged alleles of SHIP into a vector (pSFFV) that gives high expression levels in macrophage cell lines. In Specific Aim 2, they will determine whether known SHIP interacting proteins, such as Shc, participate in SHIP down-modulation of FcR- and CR3-mediated phagocytosis. They will use co-immunoprecipitation assays to identify in vivo SHIP-binding proteins following ligation of FcgR or CR3. For in vitro assays they will employ affinity chromatography using a GST fusion of the SHIP SH2 domain. In addition, they will also use affinity chromatography to identify potentially novel SHIP-interacting proteins. In Specific Aim 3, they will determine whether PIP3 production is required for CR3 "inside-out" signaling, "outside-in" signaling, or both using primary macrophages and transfected cells lines. They will study the role that PIP3 plays in "inside-out" signaling of CR3 using the following assays: surface expression of CR3, presence of an activation-dependent epitope, receptor clustering, cytoskeletal association, and CR3mediated binding to ICAM or fibrinogen. To study "outside-in" signaling they will use cell lines that express constitutively active CR3 and measure PI 3-kinase activity, PIP3 production, spreading on ICAM or fibrinogen, actin assembly and phagocytosis following CR3 ligation.