LEUKOCYTE ADHESION--REGULATION OF L-SELECTIN FUNCTION

白细胞粘附--L-选择素功能的调节

• 批准号: 6537493
• 负责人: BRUCE K WALCHECK
• 金额: $ 29.55万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6537493
• 关键词: calmodulin; cell adhesion; human subject; intermolecular interaction; leukocytes; phosphorylation; point mutation; protein binding; protein sequence; protein structure function; selectins; site directed mutagenesis; tissue /cell culture; transfection

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.

中性粒细胞是先天性免疫以及免疫系统中的关键效应细胞。 具有病理后果的炎症环境，例如脓毒症， 再灌注损伤和哮喘。很明显中性粒细胞- 表达，粘附分子L-选择素在 将这些细胞引导到不同的炎症环境中。细胞质 L-选择素结构域调节受体结合活性，细胞骨架 相互作用和内切蛋白水解加工（脱落），所有这些都是 已知影响L-选择素粘附。然而，我们对 与L-选择素胞质结构域相互作用的细胞内因子 并且它们的功能模式是最小的。获得这些知识是 重要的是，因为它可能提供新的治疗能力，以调节L- 选择素粘附和操纵白细胞募集到 炎症 我们的长期目标是了解L-选择素粘附是如何 调制用于治疗目的。本申请的目的是 确定细胞内Ca+2调节蛋白 钙调素调节L-选择素粘附。这个问题的核心假设是 应用是钙调蛋白与细胞质的共缔合， L-选择素的结构域在调节L-选择素粘附中是重要的。这是 基于令人信服的初步数据，这些数据表明， 钙调素和L-胞质结构域之间的特异性相互作用 选择素破坏这种相互作用会极大地减少积累 白细胞在内皮配体上的表达， 生理血流条件。建议的理由 研究表明，一旦知道了分子间的相互作用 L-选择素和钙调素的胞质结构域之间调节L- 选择素粘附，这一事件或特定的下游效应可能是 以新的和创新的方法来改变 炎症部位中性粒细胞聚集。我们特别好 考虑到我们在以下领域的专业知识，准备进行这些研究 粘附受体的结构/功能，我们建立的检测系统， 我们的新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 包含了许多成熟和资金充足的调查人员，例如， 与免疫学中心和癌症中心发现的一样， PI是成员。我们建议通过执行以下操作来测试我们的假设： 三个具体目标。 1.定义L-选择素胞质结构域中所需的基序 用于钙调素结合。根据初步调查结果，我们的工作 一种假设是，L-选择素的高碱性NH 2-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诱变 结构域，如截短和扫描丙氨酸点突变，以 鉴定钙调蛋白结合的关键残基。 2.确定直接破坏以下物质的联合作用的影响： 钙调素和L-选择素对粘附的影响。我们的工作假设， 根据初步研究，钙调素调节L-选择素 粘附，部分地，通过诱导L-选择素蛋白水解。我们 方法将涉及确定直接破坏共同体的影响， 钙调素与L-选择素的结合对L-选择素粘附的影响， 通过模拟生理血流的体外试验进行检查 条件 3.评估调节钙调素结合的细胞内机制 到L-选择素根据初步的发现，我们的工作假设是 细胞内Ca+2浓度和/或L-选择素磷酸化 调节钙调素与L-选择素的结合。我们的方法将包括 测定L-选择素磷酸化对钙调素的影响 结合和L-选择素粘附，并研究Ca+2依赖性， 钙调素/L-选择素相互作用。 拟议的工作是创新的，因为它将有助于阐明重要的 将炎症刺激与调节联系起来的分子机制 白细胞粘附功能。此外，我们还拥有新的试剂， 研究L-选择素的跨膜片段， 据我们所知， 在任何其他实验室。我们期望拟议的研究将 确定钙调素在调节L选择素中的作用方式 粘连这些结果将是重要的，因为他们预计将提供 对L-选择素粘附调节的新见解，这可能导致 用于炎症性疾病的新型治疗干预。