INTEGRINS AND PMN MOTILITY

描述（改编自研究者摘要）：本申请 第一个奖项是基于这样的假设，即运动的 玻连蛋白（Vn）底物上的多形核白细胞（PMN）是 受细胞内钙和钙调素依赖性的变化调节 磷酸酶，钙调磷酸酶，通过Vn（玻连蛋白）受体，即， α v β 3，所述α v是PMN特有的同种型。 它进一步 提出除了β 3，β 2整合素受体参与 在磷酸化和钙依赖的细胞内信号传导中， 这两种受体对附着/分离都至关重要。 与玻连蛋白底物上PMN运动相关的过程。 这些研究 提出了三个具体目标。 在第一个具体目标中， PMN的α v的表征将与预期一起进行 它是PMN所特有的，它的N-末端片段与 α v亚单位分布在其他组织中。 特征将 包括首先从分离的PMN s RNA产生cDNA，然后逆转录 通过聚合酶链式反应（PCR）转录它们。 分离后 在α v cDNA中，它将被限制性内切酶切割成短片段， 用于序列分析的核酸内切酶。 除了核苷酸 测序，将进行N-末端的肽测序。 的 第二个具体目的涉及阐明β 2整联蛋白的作用 在PMN附着中，背景争论是β 2受体是 β 3介导的附着所需，β 2和β 3都是 这是PMN附着在玻连蛋白基质上所必需的。 在这些 实验中，β 2亚基（白细胞）缺陷的细胞和细胞系 粘附缺陷患者）和β 3亚单位（HL-60细胞系）受体 将被利用。 本文提出了各种转染实验。 部分，以确定β 3缺陷细胞是否恢复到其 用β 3 cDNA转染后的正常行为。 此外，通过抗体（AP 5） 将确定是否来自具有β 2 受体缺陷可以被迫附着到Vn基板利用 β 3受体。 在第三个具体目标中，磷酸激酶-C（PKC） 和钙调神经磷酸酶（一种磷酸酶）对β 2磷酸化的影响， 将检查β 3介导的PMN运动性。 这一具体目标是基于 中性粒细胞的运动性与平衡有关的论点 PKC磷酸化和去磷酸化或磷酸酶 calcineurin的活性。 PKC特异性抑制剂的实验 和钙调神经磷酸酶来确定β 2的磷酸化。 他们将随后进行实验，在这些实验中， 将评估β 2磷酸化，这些包括磷酸化 桩蛋白和细胞内钙的变化。 最后，关系 beta2磷酸化和beta3介导的附着或脱离之间 将被评估。

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.