Novel Mechanisms of Calcium Signaling in B lymphocytes

B 淋巴细胞钙信号传导的新机制

• 批准号: 7887578
• 负责人: BRUCE D FREEDMAN
• 金额: $ 39.9万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7887578
• 关键词: Adaptor Signaling Protein; Antibodies; Antigen Receptors; Autoimmune Process; B-Lymphocytes; BLNK gene; Binding; Calcium; Calcium Signaling; Cations; Cell membrane; Clinic; Coupling; Cytoplasm; Cytoskeleton; Data; Development; Disease; Distal; Endoplasmic Reticulum; Generations; ITPR1 gene; Immune; Immune System Diseases; Immune response; Immunologic Deficiency Syndromes; Inflammatory; Link; Lipase; Lymphocyte; Lymphocyte Function; Mediating; Membrane; Microinjections; Molecular; Pathway interactions; Phosphorylation; Phosphotransferases; Play; Point Mutation; Protein Tyrosine Kinase; Proteins; Regulation; Role; STIM1 gene; Second Messenger Systems; Signal Transduction; Structure; Suggestion; T-Cell Activation; TRPC3 ion channel; Testing; Tyrosine; Work; base; inhibitor/antagonist; insight; novel; public health relevance; receptor; second messenger; sensor

DESCRIPTION (provided by applicant): Calcium (Ca2+) is a multifunctional second messenger that regulates lymphocyte differentiation and function. The pathways that initiate Ca2+signaling following antigen receptor engagement in lymphocytes are well delineated. For example, antigen receptor engagement on B cells, activates tyrosine kinases Lyn/Syk and the consequent activation of PLC32 results in the generation of IP3, which activates channels (IP3 receptors) on the endoplasmic reticulum (ER) membrane. Depletion of IP3-sensitive ER Ca2+ stores triggers relocalization of STIM1, the ER Ca2+ sensor, into punctate structures in junctional ER adjacent to the plasma membrane. Orai1, the recently identified CRAC channel pore located in the plasma membrane, also aggregates into puncta following activation, and its subsequent interaction with STIM1 results in "store-operated" CRAC channel activation. While it has long been thought that IP3-mediated store depletion is both necessary and sufficient for maximal CRAC activation, recent data, including our own data, clearly challenge this notion. Regulation of CRAC activation downstream of IP3-mediated store release was observed in lyn-/-syk-/- B cells following ER Ca2+stores depletion. In support of this idea, our studies demonstrate that CRAC activation is abrogated by either Lyn/Syk inhibitors or neutralizing anti-Lyn and -Syk antibodies introduced into the cytoplasm of single B cells. Our subsequent observation that Orai1 and Syk physically interact led us to hypothesize that Syk is involved in Orai1 redistribution required for CRAC activation. Accordingly, this proposal focuses on the role of Lyn and Syk in the regulation of post-store coupling (Aim1). Another indication that CRAC activity could be independently regulated is provided by our preliminary data that PLC32 directly interacts with Orai1, suggesting. Our data suggest that physical association of PLC32 and Orai1 is critical for CRAC activation and that such interactions can be inhibited by tyrosine phosphorylated TFII-I, a known Btk target. Based on these data and an analogous role for TFII-I in regulating PLC31-dependent activation of TRPC3 Ca2+ channels, we hypothesize that PLC3-2 regulates CRAC channel mediated Ca2+ entry in a lipase-independent manner that is negatively regulated by Btk dependent phosphorylation of TFII-I (tested in Aim 2). Finally, our recent work with Dan Billadeau demonstrates that the adaptor protein WAVE2 regulates Ca2+ entry, but not Ca2+ release from stores following T cell activation. Our preliminary results demonstrate that WAVE2 plays an analogous role in B cells. Furthermore, WAVE2 associates with STIM1 in B cells and dissociates following activation, suggesting a role in orienting STIM1 for interactions with Orai1. We will further explore the mechanistic role of WAVE2 in BCR-induced CRAC activation in Aim 3. Tight regulation of Ca2+signaling is critical for lymphocyte effector function and its dysregulation contributes to immunodeficiency as well as inflammatory diseases. Our proposed studies will identify additional mechanisms by which Ca2+ signaling could go awry in disease states. Results from these studies will also provide insight into approaches to ameliorate immunodeficiency diseases and/or autoimmune and inflammatory conditions. PUBLIC HEALTH RELEVANCE: Calcium is a multifunctional second messenger that regulates nearly every aspect of lymphocyte differentiation and function and thereby the immune response. The primary objective of these studies is to understand novel mechanisms by which calcium entry into lymphocytes is regulated. Results from these studies will identify targets and strategies both positive and negative manipulation of the immune response to ameliorate immunodeficiency diseases and/or to suppress development of autoimmune and inflammatory disease.

描述（由申请人提供）：钙（Ca 2+）是一种多功能第二信使，可调节淋巴细胞分化和功能。在淋巴细胞中抗原受体参与后启动Ca 2+信号传导的途径被很好地描绘。例如，B细胞上的抗原受体接合激活酪氨酸激酶林恩/Syk，随后PLC 32的激活导致IP 3的产生，IP 3激活内质网（ER）膜上的通道（IP 3受体）。消耗IP 3敏感的ER Ca 2+商店触发重新定位的STIM 1，ER Ca 2+传感器，进入点状结构的交界ER邻近质膜。Orai 1是最近鉴定的位于质膜中的CRAC通道孔，在激活后也聚集成斑点，并且其随后与STIM 1的相互作用导致“储存操作的”CRAC通道激活。虽然长期以来人们一直认为IP 3介导的储存耗尽对于最大化CRAC激活是必要的和充分的，但最近的数据，包括我们自己的数据，显然挑战了这一概念。在ER Ca 2+储存耗尽后，在林恩-/-syk-/- B细胞中观察到IP 3介导的储存释放下游的CRAC活化的调节。为了支持这一观点，我们的研究表明，CRAC激活被引入单个B细胞的细胞质中的林恩/Syk抑制剂或中和抗林恩和抗Syk抗体消除。我们随后观察到Orai 1和Syk在物理上相互作用，这使我们假设Syk参与了CRAC激活所需的Orai 1再分布。因此，本建议侧重于林恩和Syk的作用，在商店后耦合（AIM 1）的调节。我们的初步数据提供了CRAC活性可以独立调节的另一个迹象，即PLC 32直接与Orai 1相互作用，表明。我们的数据表明，PLC 32和Orai 1的物理关联对于CRAC激活是至关重要的，并且这种相互作用可以被酪氨酸磷酸化的TFII-I（一种已知的Btk靶标）抑制。基于这些数据和TFII-I在调节TRPC 3 Ca 2+通道的PLC 31依赖性激活中的类似作用，我们假设PLC 3 -2以不依赖脂肪酶的方式调节CRAC通道介导的Ca 2+内流，该方式由TFII-I的Btk依赖性磷酸化负调节（在目标2中测试）。最后，我们最近与Dan Billadeau的研究表明，衔接蛋白WAVE 2调节T细胞激活后的Ca 2+进入，但不调节Ca 2+从储存中释放。我们的初步结果表明WAVE 2在B细胞中起类似的作用。此外，WAVE 2与B细胞中的STIM 1相关联，并在激活后解离，这表明在定向STIM 1与Orai 1相互作用中的作用。我们将进一步探索WAVE 2在BCR诱导的CRAC激活中的机制作用。Ca 2+信号传导的紧密调节对于淋巴细胞效应器功能是至关重要的，并且其失调有助于免疫缺陷以及炎性疾病。我们提出的研究将确定Ca 2+信号在疾病状态下可能出错的其他机制。这些研究的结果还将提供对改善免疫缺陷疾病和/或自身免疫性和炎症性疾病的方法的深入了解。 公共卫生关系：钙是一种多功能的第二信使，几乎调节淋巴细胞分化和功能的各个方面，从而调节免疫应答。这些研究的主要目的是了解钙进入淋巴细胞的新机制。这些研究的结果将确定靶点和策略，包括免疫应答的积极和消极操纵，以改善免疫缺陷疾病和/或抑制自身免疫性疾病和炎症性疾病的发展。