PHAGOCYTOSIS IN PULMONARY ALVEOLAR MACROPHAGES

肺泡巨噬细胞的吞噬作用

• 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
• 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

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）：吞噬作用是系统发育古老的 对由免疫的专业细胞适应的颗粒刺激的反应 系统。在最佳特征的吞噬受体中，有 识别IgG（FC1RS）的FC部分。通过FCYRS的吞噬作用需要 招募和激活SYK，一种72 kDa酪氨酸激酶 主要在造血细胞中。在此提案中，我们描述了实验 探索FCYR介导的吞噬作用的分子机制 巨噬细胞，包括转化的巨噬细胞系和主要 肺肺泡巨噬细胞。我们将测试一个两室模型 分配肌动蛋白的单独信号转导要求的吞噬作用 组件和伪足扩展。在特定目标1中，我们将测试 假设吞噬作用需要Syk结合蛋白。我们将 识别吞噬细胞所需的SYK上那些酪氨酸残基 信号传导并净化与这些关联的潜在SYK底物 残留物。这些实验将利用位置定向的诱变，表达 在几个互补模型系统中的Syk和各种SYK突变体的 使用亲和色谱法吞噬作用和蛋白质纯化。在 特定目标2，我们将测试CRK，SH2/SH3的假设 将含域的适配器蛋白招募到SYK，需要CRK 用于吞噬作用。我们将隔离含磷酸酪蛋白的CRK结合 与CRK-SH2相互作用并确定它们是否对应已知的蛋白质 含磷酸酪氨基的蛋白；如果没有，我们将进一步描述它们。 我们将检验以下假设： FCYR介导的吞噬作用，而不是其他形式的吞噬作用；如果是这样，我们会 尝试识别可能参与的CRKL-或CRKLLSH3结合蛋白 在吞噬作用中。解决p1 3-激酶依赖性假脚架的机理 扩展，我们研究了肌球蛋白X（M10），一个含pH域的肌球蛋白 非常规的肌球蛋白具有高亲和力对PIP3的高亲和力，以鼠表示 巨噬细胞。 M1O的截短片段抑制IgG涂层的吞噬作用 目标。相反，这些突变体不影响“吞噬杯”的形成， 它们也不会抑制膜荷叶边。这表明需要M10 专门用于假卵形扩展和吞噬作用。我们将进行批判性测试 该假设并确定M10的哪些域需要 吞噬作用。