INTEGRINS AND PMN MOTILITY

说明(改编自调查人员摘要)：此申请适用于 第一个奖项是基于这样一种假设，即 玻璃连结蛋白(VN)底物上的中性粒细胞(PMN)是 受细胞内钙和钙调素依赖性变化的调节 磷酸酶，钙调神经磷酸酶，通过VN(Vitronectin)受体，即， Alphavbeta3，Alphav是PMN特有的异构体。它进一步 提出除β3外，还涉及β2整合素受体。 在磷酸化和钙依赖的细胞内信号转导中 这两个受体对附着/脱离都是至关重要的 与中性粒细胞在玻璃连结蛋白底物上运动有关的过程。这些研究是 在三个具体目标下提出。在第一个特定的目标中， 中性粒细胞αv的表征将在预期中进行 它是PMN独有的，它的N端片段不同于 αV亚基分布在其他组织中。这部剧的特点将是 包括首先从分离的中性粒细胞S核糖核酸中产生cDNA，然后逆转 通过聚合酶链式反应(PCR)转录它们。隔离后 在甲型肝炎病毒的cdna中，它将被限制为短片段。 用于序列分析的内切酶。除了核苷酸之外 将进行N-末端的测序、多肽测序。这个 第二个特定目的与阐明β2整合素的作用有关 在PMN附件中，背景争论是Beta2受体是 是Beta3介导的连接所必需的，并且Beta2和Beta3都是 对于PMN附着在玻璃体粘连蛋白底物上是必不可少的。在这些 实验中，缺乏β2亚单位(白细胞)的细胞和细胞系 黏附缺陷患者)和β3亚单位(HL-60细胞株)受体 将会被利用。在这篇文章中提出了各种不同的转基因实验 部分，以确定Beta3缺陷细胞是否恢复到 转Beta3基因后动物行为正常。此外，通过抗体(AP5) 操作将被确定是否来自患有β2的LAD患者的PMN 受体缺陷可以被强迫附着在VN底物上，利用 β3受体。在第三个特定目的中，磷酸激酶C(PKC)的作用 和钙调神经磷酸酶(一种磷酸酶)对β2的磷酸化和 将检测β3介导的中性粒细胞运动能力。这一具体目标是基于 中性粒细胞的运动与平衡有关的争论 在PKC的磷酸化和去磷酸化或磷酸酶之间 钙调神经磷酸酶的活性。蛋白激酶C特异性抑制剂的实验研究 并将进行钙调神经磷酸酶以确定Beta2的磷酸化。 在它们之后将进行实验，在这些实验中，远端的信号事件 将评估Beta2的磷酸化，其中包括磷酸化。 和细胞内钙的变化。最后，关系 β2磷酸化与β3介导的附着或脱离之间的关系 将会被评估。

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.