INTEGRINS AND PMN MOTILITY

整合素和 PMN 运动性

• 批准号: 2887281
• 负责人: William S. Hendey
• 金额: $ 9.87万
• 依托单位: RUSH UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2887281
• 关键词: actin binding protein; biological signal transduction; calcineurin; calcium flux; cell adhesion; cell motility; human tissue; integrins; leukocyte disorder; neutrophil; nucleic acid sequence; paxillin; phosphorylation; polymerase chain reaction; protein kinase C; protein sequence; receptor; transfection; vitronectin

DESCRIPTION (Adapted from Investigator's Abstract): This application for a FIRST AWARD is based on the hypothesis that the motility of polymorphonuclear leukocyte (PMN) on the vitronectin (Vn) substrate is regulated by the changes in intracellular calcium and calmodulin-dependent phosphatase, Calcineurin, via a Vn (vitronectin) receptor, that is, alphavbeta3, the alphav being an isoform unique to PMNs. It further proposes that in addition to the beta3, beta2 integrin receptor is involved in the phosphorylation- and calcium-dependent intracellular signalling pathways, and both these receptors are critical to the attachment/detachment processes related to PMN motility on vitronectin substrate. The studies are proposed under three specific aims. In the first specfic aim, characterization of alphav of PMN will be carried out with the anticipation that it is unique to PMN, and it differs in its N-terminal segment from alphav subunits distributed in other tissues. The characterization will include first generating the cDNA, from isolated PMN s RNA, and then reverse transcribing them by the polymerase chain reaction (PCR). After isolation of the alphav cDNA, it will be cleaved into short fragments by restriction endonucleases for sequence analyses. In addition to the nucleotide sequencing, peptide sequencing of the N-terminus will be performed. The second specific aim relates to the elucidation of the role of beta2 integrin in PMN attachment with the background contention that beta2 receptor is required for beta3-mediated attachment, and both beta2 and beta3 are essential to PMN attachment on the vitronectin substrate. In these experiments, the cell and cell lines deficient in beta2 subunit (leukocyte adhesion deficiency patients) and beta3 subunit (HL-60 cell line) receptors will be utilized. Various transfection experiments are proposed in this section to ascertain whether the beta3-deficient cells revert to their normal behavior after transfection with beta3 cDNA. Also, by antibody (AP5) manipulation it will be determined that if PMNs from LAD patients with beta2 receptor deficiency can be forced to attach to the Vn substrate utilizing beta3 receptor. In the third specific aim the role of phosphokinase-C (PKC) and calcineurin (a phosphatase) on the beta2 phosphorylation and beta3-mediated PMN motility will be examined. This specific aim is based on the contention that the motility of the PMN is related to the balance between the phosphorylation by PKC and dephosphorylation or phosphatase activity of calcineurin. The experiments with specific inhibitors of PKC and calcineurin will be performed to ascertain the phosphorylation of beta2. They will be followed by experiments in which signalling events distal to the beta2 phosphorylation will assessed, and these include phosphorylation of paxillin and changes in the intracellular calcium. Finally, relationship between beta2 phosphorylation and beta3-mediated attachment or detachment will be assessed.

描述（根据调查员的摘要改编）：此应用 第一个奖项是基于以下假设 牙动蛋白（VN）底物上的多形核白细胞（PMN）为 受细胞内钙和钙调蛋白依赖性的变化调节 磷酸酶，钙调蛋白，通过VN（玻璃纤维）受体，即 Alphavbeta3，Alphav是PMN独有的同工型。 进一步 提出，除了beta3外，还涉及β2整合素受体 在磷酸化和钙依赖性细胞内信号中 途径，这两个受体对于附件/分离至关重要 与玻璃体蛋白底物上的PMN运动有关的过程。 研究是 提出的三个特定目标。 在第一个特定目标中， PMN的Alphav的表征将与预期进行 它是PMN独有的，它在N末端段不同于 Alphav亚基分布在其他组织中。 表征将 包括首先从孤立的PMN S RNA产生cDNA，然后反向 通过聚合酶链反应（PCR）转录它们。 隔离后 在alphav cDNA的中，它将通过限制将其切成短片段 核酸内切酶进行序列分析。 除核苷酸 将进行N端的测序，肽测序。 这 第二个特定目的与阐明beta2整合素的作用有关 在PMN附着在背景争论中，beta2受体为 beta3介导的附件所需，beta2和beta3均为 PMN附着在玻璃直蛋白底物上必不可少的。 在这些 实验，细胞和细胞系缺乏β2亚基（白细胞 粘附缺乏患者）和β3亚基（HL-60细胞系）受体 将被利用。 在此提出了各种转染实验 一节确定beta3缺陷型细胞是否还原为其 用β3cDNA转染后的正常行为。 另外，通过抗体（AP5） 操纵将确定，如果来自LAD beta2患者的PMN 受体缺乏症可以被迫连接到利用VN基板上 beta3受体。 在第三个特定目标中，磷酸激酶-C（PKC）的作用 beta2磷酸化和钙调蛋白（一种磷酸酶）和 将检查beta3介导的PMN运动。 这个具体目标是基于 关于PMN的运动与余额有关的论点 PKC和去磷酸化或磷酸酶的磷酸化之间 钙调蛋白的活性。 PKC的特定抑制剂的实验 并将进行钙调蛋白以确定β2的磷酸化。 随后将进行实验，其中信号事件远端 将评估β2磷酸化，其中包括磷酸化 帕克西林和细胞内钙的变化。 最后，关系 在β2磷酸化和β3介导的附件或分离之间 将被评估。