LEUKOCYTE ADHESION--REGULATION OF L-SELECTIN FUNCTION

中性粒细胞是先天免疫中的关键效应细胞，在 具有病理后果的炎症环境，如脓毒症， 再灌注损伤和哮喘。已经确定的是中性粒细胞- 表达的黏附分子L-选择素在 将这些细胞导向不同的炎症环境。细胞质 L-选择素的结构域调节受体结合活性、细胞骨架 相互作用和内蛋白降解过程(脱落)，所有这些都是 已知影响L-选择素黏附。然而，我们对 与L胞浆结构域相互作用的细胞内因子--选择素 它们的功能模式是最小的。获得这种知识是 重要的是，因为它可能提供新的治疗能力来调节L- 选择素黏附和操纵白细胞向血管内皮细胞的募集 发炎。 我们的长期目标是了解L-选择素的黏附能力 为治疗目的而调节的。此应用程序的目标是 确定细胞内钙调节蛋白的作用方式 钙调蛋白对L-选择素黏附的调节作用这一点的中心假设是 应用是钙调蛋白与胞浆的共同结合 L-选择素结构域在调节L-选择素黏附中起重要作用。这是 基于令人信服的初步数据，证明了 钙调素与L胞浆结构域的特异性相互作用 选择素。破坏这种相互作用会大大减少这种积累 在一种模拟内皮配体上的白细胞的检测系统中 生理血流状态。建议的理由是 研究表明，一旦知道了分子间相互作用是如何 L-选择素的胞浆结构域和钙调蛋白之间调节L-选择素 选择素黏附，这一事件或特定的下游影响可能是 以新的和创新的方法在药理学上进行操纵以改变 炎症部位中性粒细胞聚集。我们特别好 准备进行这些研究，考虑到我们在以下领域的专业知识 黏附受体结构/功能，我们已建立的检测系统，以及 我们的新奇试剂。这项工作将在研究环境中完成 非常有利于它的顺利完成。明尼苏达大学 包括许多成熟的和资金充足的调查人员，例如， 在免疫学中心和癌症中心发现的那些 PI是的成员。我们建议通过执行以下操作来检验我们的假设 遵循三个具体目标。 1.确定L胞质结构域中所需的基序--选择素 用于钙调素结合。根据初步调查结果，我们的工作 假设L-选择素的高度碱性氨基末端区域 胞质结构域支持钙调蛋白结合。我们的方法将 涉及人L-选择素细胞质的定点突变 结构域，如截断和扫描丙氨酸点突变，以 确定钙调蛋白结合的关键残基。 2.确定直接破坏联合的影响 钙调素与L-选择素对黏附的影响。我们的工作假设，再一次 初步研究表明，钙调蛋白对L-选择素有调节作用 黏附，部分是通过诱导L-选择素蛋白分解来实现的。我们的 方法将涉及确定直接扰乱联合- 钙调素与L-选择素对L-选择素黏附的影响 通过模拟生理性血液流动的体外试验进行检查 条件。 3.评估调节钙调蛋白结合的细胞内机制 致L-选择素。根据初步发现，我们的工作假设是 细胞内钙离子浓度和/或L-选择素磷酸化 调节钙调蛋白与L-选择素的结合。我们的方法将涉及 L-选择素磷酸化对钙调素的影响 结合和L-选择素黏附，并研究钙离子依赖性 钙调素/L-选择素的相互作用。 这项拟议的工作具有创新性，因为它将有助于阐明 炎症刺激与调控的分子机制 白细胞的黏附功能。此外，我们还拥有新的试剂来 术后残留的L-选择素跨膜片段的研究 内蛋白水解性脱落，据我们所知，这并没有进行 在任何其他实验室。我们期望拟议的研究将 确定钙调蛋白在调节L选择素中的作用方式 粘附力。这些结果将是重要的，因为预计它们将提供 对L-选择素黏附调控的新见解 在炎症性疾病的新型治疗干预中。

Neutrophils are critical effector cells in innate immunity as well as in inflammatory settings with pathological consequences, such as sepsis, reperfusion injury, and asthma. It is well established that the neutrophil- expressed, adhesion molecule L-selectin plays an important role in directing these cells to diverse inflammatory settings. The cytoplasmic domain of L-selectin regulates receptor binding activity, cytoskeletal interactions, and endoproteolytic processing (shedding), all of which are known to affect L-selectin adhesion. However, our knowledge of the intracellular factors that interact with the cytoplasmic domain of L-selectin and their mode of function is minimal. Obtaining this knowledge is important, because, it may provide new therapeutic abilities to modulate L- selectin adhesion and manipulate leukocyte recruitment to sites of inflammation. Our long-term goal is to understand how L-selectin adhesion can be modulated for therapeutic purposes. The objective of this application is to determine the means by which the intracellular Ca+2 regulatory protein calmodulin regulates L-selectin adhesion. The central hypothesis of this application is that the co-association of calmodulin with the cytoplasmic domain of L-selectin is important in regulating L-selectin adhesion. This is based on compelling preliminary data that demonstrates a direct and specific interaction between calmodulin and the cytoplasmic domain of L- selectin. Disrupting this interaction profoundly reduces the accumulation of leukocytes on endothelial ligands in an assay system that simulates physiological blood flow conditions. The rationale for the proposed research is that once it is known how the intermolecular interaction between the cytoplasmic domain of L-selectin and calmodulin regulates L- selectin adhesion, this event or specific downstream effects might be manipulated pharmacologically in new and innovate approaches to alter neutrophil accumulation at sites of inflammation. We are particularly well prepared to undertake these studies considering our expertise in the area of adhesion receptor structure/function, our established assay systems, and our novel reagents. This work will be completed in a research environment very conducive to its successful completion. The University of Minnesota contains numerous established and well-funded investigators, for example, as those found in the Center for Immunology and the Cancer Center, which the PI is a member of. We propose to test our hypothesis by performing the following three specific aims. 1. Define the motif in the cytoplasmic domain of L-selectin that is required for calmodulin binding. Based on preliminary findings, our working hypothesis is that the highly basic NH2-terminal region of L-selectin's cytoplasmic domain supports calmodulin binding. Our approach will involve site directed mutagenesis of the human L-selectin cytoplasmic domain, such as truncation and scanning alanine point mutations, to identify critical residues for calmodulin binding. 2. Determine the effects of directly disrupting the co-association of calmodulin with L-selectin on adhesion. Our working hypothesis, again based on preliminary studies, is that calmodulin regulates L-selectin adhesion, in part, through the induction of L-selectin proteolysis. Our approach will involve determining the effects of directly disrupting the co- association of calmodulin with L-selectin on L-selectin adhesion, examined by an in vitro assay that simulates physiologic blood flow conditions. 3. Evaluate the intracellular mechanisms that regulate calmodulin binding to L-selectin. Based on preliminary findings, our working hypothesis is that intracellular Ca+2 concentrations and/or L-selectin phosphorylation regulate calmodulin binding to L-selectin. Our approach will involve determining the effects of L-selectin phosphorylation on calmodulin binding and L-selectin adhesion, and investigating the Ca+2 dependence of calmodulin/L-selectin interactions. The proposed work is innovative, because, it will help elucidate important molecular mechanisms linking inflammatory stimulation to the modulation of leukocyte adhesive functions. In addition, we possess novel reagents to study the transmembrane fragment of L-selectin that remains after endoproteolytic shedding, which to our knowledge is not being performed in any other laboratory. It is our expectation that the proposed study will determine the mode of function of calmodulin in regulating L selectin adhesion. These results will be significant as they are expected to provide new insights into the regulation of L-selectin adhesion, which may result in novel therapeutic interventions for inflammatory diseases.