Antigen Binding, Crosslinking and Transmembrane Signaling

抗原结合、交联和跨膜信号传导

基本信息

批准号：
8204988
负责人：
DAVID A HOLOWKA
金额：
$ 38.65万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1985
资助国家：
美国
起止时间：
1985-08-01 至 2014-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8204988
关键词：
Address Affinity Alanine Allergic Antigens Binding Biochemical Biological Biological Process Bone Marrow Calcium Calcium Channel Cell Communication Cell Culture Techniques Cell Degranulation Cell membrane Cell physiology Cells Cellular biology Chemotaxis Cholera Toxin Complex Coupled Cytoplasmic Granules Disease Dissociation Endoplasmic Reticulum Endosomes Epithelial Epithelial Cells Evaluation Event Exhibits Exocytosis Family member Fluorescence Resonance Energy Transfer Funding Ganglioside GM1 Grant Health Hematopoietic Homeostasis Host Defense Hypersensitivity IL3 gene IL4 gene IgE IgE Receptors Image Immune Immune Cell Activation Immune response Immunologic Surveillance Immunology Inflammation Intervention Intestines Invaded Lead Leukocytes Life Ligands Lipids Measurement Mediating Mediator of activation protein Membrane Methods Microscopy Molecular Molecular Genetics Mucous Membrane Pathway interactions Pattern Phosphatidylinositol 4,5-Diphosphate Phosphatidylinositols Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Physiological Play Process Production Property Protein Isoforms Protein Kinase C Proteins Public Health Rattus Reagent Receptor Activation Recruitment Activity Recycling Regulation Relative (related person)Research Role STIM1 gene Secretory Vesicles Seminal Signal Transduction Site Small Interfering RNA Speed Surface Testing Therapeutic Time Tissues Tyrosine Phosphorylation Work antigen binding cell motility chemical release computerized data processing crosslink cytokine extracellular imaging modality insight mast cell molecular imaging monolayer novel pathogen phosphatidylinositol 4-phosphate prototype public health relevance receptor reconstitution research study response segregation sensor synaptotagmin tissue preparation trafficking

项目摘要

DESCRIPTION (provided by applicant): Proposed work will continue our long-term efforts to understand molecular mechanisms by which crosslinking of IgE receptors on mast cells triggers complex cellular signaling processes that lead to important functional responses in immune host defense. Central to mast cell and other leukocyte signaling responses is mobilization of intracellular Ca2+, and we will investigate this spatio-temporally orchestrated process and its functional roles in mast cell exocytosis, cytokine production, and dynamic interactions with epithelial tissue. Specific Aim 1 focuses on elucidating molecular mechanisms of IgE receptor-activated Ca2+ responses, including novel antigen-stimulated Ca2+ waves that precede store-operated Ca2+ influx and impart spatial direction to this signaling response. The mechanism for activation of store-operated Ca2+ influx via recently identified Orai1/CRACM1 calcium channels will be characterized using fluorescence resonance energy transfer imaging for measurements of molecular proximity between this channel and the calcium sensor, STIM1. We will utilize this method to characterize the mechanism for regulation of Ca2+ influx by phosphatidylinositol 4,5- bisphosphate (PIP2) that we recently identified. Our evidence that different family members of phosphatidylinositol 4-phosphate 5-kinases synthesize functionally separate pools of PIP2 involved in Ca2+ mobilization leads us to propose that these PIP2 pools are spatially segregated in the plasma membrane, and we will test this hypothesis using biochemical, molecular genetic and imaging approaches. We established the participation of Ca2+ mobilization in IgE-receptor stimulated trafficking from recycling endosomes, and in Aim 2 we will test the role of this process in stimulated cytokine secretion important for adaptive immune responses. We will also test our hypothesis that protein kinase C regulates the access of secretory granules to Ca2+/PIP2/synaptotagmin complexes at the plasma membrane. In Aim 3 we will characterize cellular mechanisms for mucosal mast cell motility, chemotaxis, and dynamic interactions with intestinal epithelial cells, both in cell culture and in live tissue. Mast cell motility and interactions within physiological tissue are regulated by changes in Ca2+ levels, and our proposed work will extend our general understanding of Ca2+ mobilization as a fundamental yet complex regulator of hematopoietic cellular homeostasis and response. PUBLIC HEALTH RELEVANCE: Mast cells play a central role in allergic immune responses, and their significant participation in innate as well adaptive immunology is increasingly appreciated. Many aspects of mast cell function depend on highly orchestrated changes in intracellular Ca2+ in response to stimulation through the IgE receptor and by other means. Our proposed research draws from a broad range of experimental approaches to investigate molecular mechanisms for Ca2+-dependent signaling in key responses, including degranulation to release chemical mediators in allergies, cytokine production to recruit other cells in inflammation, and dynamic interactions of mast cells with epithelial cells as a first line of defense to invading pathogens. Elucidation of mast cell mechanisms will provide new opportunities for intervention in therapeutic applications and a clearer understanding of hematopoietic cell biology that is seminal to regulation of immune responses in health and disease.

描述（由申请人提供）：拟议的工作将继续我们的长期努力，以理解肥大细胞上IgE受体的分子机制，从而触发复杂的细胞信号传导过程，从而导致免疫宿主防御中重要的功能响应。肥大细胞和其他白细胞信号反应的中心是动员细胞内Ca2+，我们将研究这种空间策划的过程及其在肥大细胞胞外增生，细胞因子产生以及与上皮组织的动态相互作用中的功能作用。具体目标1的重点是阐明IgE受体激活的Ca2+反应的分子机制，包括新型抗原刺激的Ca2+波，这些抗原刺激的Ca2+波在商店经营的Ca2+倒入之前，并将空间方向赋予了这种信号响应。使用荧光谐振能量传递成像来测量该通道和钙传感器钙传感器之间的分子接近度，将通过最近鉴定的ORAI1/CRACM1钙通道激活储存的Ca2+流入机制。我们将利用这种方法来表征我们最近确定的磷脂酰肌醇4,5-双磷酸（PIP2）调节Ca2+流入的机制。我们的证据表明，磷脂酰肌醇4-磷酸5-激酶的不同家族成员合成了参与CA2+动员的功能分开的PIP2池，这会使我们提出这些PIP2池在质膜中在空间上被隔离，我们将使用生物学，分子，分子属性和影像学方法测试此假设。我们确定了Ca2+动员在IgE受体中的参与，从回收内体刺激了运输，在AIM 2中，我们将测试该过程在刺激的细胞因子分泌中对适应性免疫反应很重要的作用。我们还将检验我们的假设，即蛋白激酶C调节分泌颗粒对质膜上Ca2+/pip2/Synaptotagmin复合物的访问。在AIM 3中，我们将表征在细胞培养和活组织中，粘膜肥大细胞运动，趋化性和与肠上皮细胞的动态相互作用的细胞机制。肥大细胞运动和生理组织中的相互作用受CA2+水平的变化调节，我们提出的工作将扩展我们对CA2+动员的一般理解，作为造血细胞稳态和反应的基本但复杂的调节剂和反应。公共卫生相关性：肥大细胞在过敏性免疫反应中起着核心作用，并且越来越多地赞赏它们对先天性和适应性免疫学的重要参与。肥大细胞功能的许多方面取决于通过IgE受体和其他方式响应刺激的细胞内Ca2+的高度修复的变化。我们提出的研究从广泛的实验方法中汲取了研究，以研究关键反应中Ca2+依赖性信号传导的分子机制，包括脱粒以释放过敏反应中的化学介质，细胞因子产生以募集炎症中的其他细胞，以及肥大细胞与上皮细胞与上皮细胞的动态相互作用，作为对侵袭性载体的第一线。阐明肥大细胞机制将为治疗应用干预提供新的机会，并对造血细胞生物学有更清晰的了解，这是对健康和疾病中免疫反应的指导。