PHOSPHOINOSITIDE REGULATION OF PHAGOCYTOSIS

描述（摘自申请人摘要）：关节炎，无论是由于 细菌感染或自身免疫性疾病，其特征在于存在 关节间隙中的吞噬性白细胞吞噬细胞能够 通过多种途径感染致病微生物、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 3-激酶（PI 3-激酶）活性抑制FcgR-和 补体受体（CR 3）介导的吞噬作用。操纵 通过WT表达的磷脂酰肌醇（3，4，5）三磷酸（PIP 3）含量 和PIP 3磷酸的催化失活等位基因（SH 2结构域 含有肌醇5'磷酸，SHIP）也调节吞噬作用。最后， 来源于SHIP敲除小鼠的巨噬细胞显示增强的FcgR和 CR 3介导的吞噬作用，暗示PIP 3促进吞噬作用。这 研究计划将解决这些酶调节的机制， 吞噬作用在具体目标1中，他们将确定是否存在SH 2结构域， SHIP是其抗吞噬功能所必需的。他们将亚克隆 将MYC标记的SHIP等位基因导入载体（pSFFV）中， 巨噬细胞系中的水平。在具体目标2中，他们将确定是否 已知的SHIP相互作用蛋白，如Shc，参与SHIP 下调FcR和CR 3介导的吞噬作用。他们将使用 免疫共沉淀试验，以鉴定体内SHIP结合蛋白 在FcgR或CR 3连接后。对于体外试验，他们将采用 使用SHIP SH 2结构域的GST融合物的亲和层析。此外，本发明还提供了一种方法， 他们还将使用亲和色谱法来鉴定潜在的新的 SHIP相互作用蛋白。在具体目标3中，他们将确定PIP 3是否 CR 3“由内而外”信令、“由外而外”信令， 或使用原代巨噬细胞和转染的细胞系。他们将研究 PIP 3在CR 3的“由内而外”信号传导中所起的作用如下 测定：CR 3的表面表达，活化依赖性表位的存在， 受体聚集、细胞骨架结合和CR 3介导的ICAM结合 或纤维蛋白原。为了研究“由外向内”的信号传导，他们将使用 表达组成型活性CR 3并测量PI 3-激酶活性，PIP 3 产生、在ICAM或纤维蛋白原上铺展、肌动蛋白组装和吞噬 在CR 3连接之后。

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.