PHAGOCYTOSIS IN PULMONARY ALVEOLAR MACROPHAGES

肺泡巨噬细胞的吞噬作用

基本信息

批准号：
6498918
负责人：
STEVEN M GREENBERG
金额：
$ 34.1万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-02-01 至 2005-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6498918
关键词：
Rodentias affinity chromatography alveolar macrophages antibody receptor biological signal transduction chimeric proteins cytoskeletal proteins enzyme substrate myosins phagocytosis phosphatidylinositol 3 kinase phosphorylation protein protein interaction protein purification protein structure function protein tyrosine kinase site directed mutagenesis tissue /cell culture

项目摘要

DESCRIPTION (Applicant's Abstract): Phagocytosis is a phylogenetically ancient response to particulate stimuli adapted by specialized cells of the immune system. Among the best characterized phagocytic receptors are those that recognize the Fc portion of IgG (Fc1Rs). Phagocytosis via FcyRs requires the recruitment and activation Syk, a 72 kDa tyrosine kinase expressed predominantly in hematopoetic cells. In this proposal we describe experiments to explore the molecular mechanisms of FcyR-mediated phagocytosis in a variety of macrophages, including transformed macrophage cell lines and primary pulmonary alveolar macrophages. We will test a two-compartment model of phagocytosis that assigns separate signal transduction requirements for actin assembly and pseudopod extension. In Specific Aim 1, we will test the hypothesis that a Syk-binding protein is required for phagocytosis. We will identify those tyrosine residues on Syk that are required for phagocytic signaling and purify potential Syk substrates that associate with these residues. These experiments will utilize site-directed mutagenesis, expression of Syk and various Syk mutants in several complementary model systems of phagocytosis, and protein purification using affinity chromatography. In Specific Aim 2, we will test the hypothesis that Crk, an SH2/SH3 domain-containing adaptor protein, is recruited to Syk and that Crk is required for phagocytosis. We will isolate phosphotyrosyl-containing Crk-binding proteins that interact with Crk-SH2 and determine if they correspond to known phosphotyrosyl-containing proteins; if not, we will characterize them further. We will test the hypothesis that CrkL and/or CrkII is required specifically for FcyR-mediated phagocytosis and not other forms of phagocytosis; if so, we will attempt to identify CrkL- or CrkllSH3-binding proteins that might participate in phagocytosis. To address the mechanism of P1 3-kinase-dependent pseudopod extension, we have studied myosin X (M10), a PH domain-containing unconventional myosin with high affinity for PIP3 that is expressed in murine macrophages. Truncation fragments of M1O inhibit phagocytosis of IgG-coated targets. In contrast, these mutants do not affect "phagocytic cup" formation, nor do they inhibit membrane ruffling. This suggests that M10 is required specifically for pseudopod extension and phagocytosis. We will critically test this hypothesis and determine which domains of M10 are required for phagocytosis.

描述（申请人的摘要）：吞噬作用是一种系统发育古老的对特殊免疫细胞适应的颗粒刺激的反应系统。最具特征的吞噬细胞受体是那些识别 IgG 的 Fc 部分 (Fc1R)。通过 FcyR 进行吞噬作用需要募集和激活 Syk，一种表达的 72 kDa 酪氨酸激酶主要存在于造血细胞中。在这个提案中，我们描述了实验探索FcyR介导的多种细胞吞噬作用的分子机制巨噬细胞，包括转化的巨噬细胞系和原代巨噬细胞系肺泡巨噬细胞。我们将测试一个两室模型吞噬作用为肌动蛋白分配单独的信号转导要求组装和伪足延伸。在具体目标 1 中，我们将测试假设吞噬作用需要 Syk 结合蛋白。我们将识别 Syk 上吞噬细胞所需的酪氨酸残基信号传导并纯化与这些相关的潜在 Syk 底物残留物。这些实验将利用定点突变、表达 Syk 和各种 Syk 突变体在几个互补模型系统中的研究吞噬作用和使用亲和层析的蛋白质纯化。在具体目标 2，我们将检验假设 Crk，SH2/SH3 包含结构域的接头蛋白，被招募到 Syk 并且需要 Crk 用于吞噬作用。我们将分离含磷酸酪氨酰的 Crk 结合与 Crk-SH2 相互作用的蛋白质，并确定它们是否对应于已知的含磷酸酪氨酰的蛋白质；如果没有，我们将进一步描述它们的特征。我们将检验以下假设：CrkL 和/或 CrkII 是专门针对 FcγR介导的吞噬作用而非其他形式的吞噬作用；如果是这样，我们将尝试鉴定可能参与的 CrkL 或 CrkllSH3 结合蛋白在吞噬作用中。解决 P1 3 激酶依赖性伪足的机制扩展，我们研究了肌球蛋白 X (M10)，一种包含 PH 结构域的对小鼠中表达的 PIP3 具有高亲和力的非常规肌球蛋白巨噬细胞。 M1O 的截断片段抑制 IgG 包被的吞噬作用目标。相反，这些突变体不影响“吞噬杯”的形成，它们也不抑制膜波纹。这表明需要 M10 专门用于伪足延伸和吞噬作用。我们将严格测试这个假设并确定 M10 的哪些域是必需的吞噬作用。