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: 8605495
• 负责人: BRUCE D FREEDMAN
• 金额: $ 39.6万
• 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 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8605495
• 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; 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.

描述（由申请人提供）：钙（CA2+）是一个多功能的第二使者，可调节淋巴细胞分化和功能。很好地划定了淋巴细胞抗原受体接合后启动Ca2+信号传导的途径。例如，B细胞上的抗原受体接合，激活酪氨酸激酶Lyn/Syk，随之而来的PLC32激活导致IP3的产生，该IP3激活内质网（ER）膜上的通道（IP3受体）。 IP3敏感的ER Ca2+的耗竭将ER Ca2+传感器的触发稳定性稳定在与质膜相邻的连接ER中的点状结构中。 Orai1是位于质膜中的最近鉴定出的CRAC通道孔，在激活后也聚集在Puncta中，并且其随后与STIM1的相互作用导致“储存” CRAC通道激活。虽然长期以来一直认为IP3介导的商店耗竭既需要且足以满足最大CRAC激活，但最近的数据（包括我们自己的数据）清楚地挑战了这一概念。在ER Ca2+存储耗竭后，在Lyn - / - Syk - / - B细胞中观察到IP3介导的商店释放下游的CRAC激活的调节。为了支持这一想法，我们的研究表明，LYN/SYK抑制剂或中和抗Lyn和-syk抗体所引入的CRAC激活被引入了单个B细胞的细胞质。随后，我们对Orai1和Syk进行物理相互作用的观察结果使我们假设SYK参与了CRAC激活所需的ORAI1重新分布。因此，该提案的重点是Lyn和Syk在店后耦合调节中的作用（AIM1）。另一个迹象表明，我们的初步数据可以独立调节CRAC活动，该数据是PLC32直接与Orai1相互作用的迹象。我们的数据表明，PLC32和ORAI1的物理关联对于CRAC激活至关重要，并且这种相互作用可以被酪氨酸磷酸化TFII-I（已知的BTK靶标TFII-I）抑制。基于这些数据和TFII-I在调节TRPC3 Ca2+通道的依赖性依赖性的PLC31依赖性激活中的类似作用，我们假设PLC3-2调节以脂肪酶独立的方式调节CRAC通道介导的Ca2+进入，这些方式受BTK依赖性的依赖性脂肪酶依赖性调节，而TFI-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-2则依赖2）。最后，我们最近与Dan Billadeau的工作表明，衔接蛋白Wave2调节Ca2+进入，但在T细胞激活后不从商店中释放Ca2+。我们的初步结果表明，波2在B细胞中起类似的作用。此外，Wave2与B细胞中的STIM1与激活后解离物相关联，这表明在定位STIM1中的作用中与Orai1相互作用。我们将进一步探讨WAVE2在AIM 3中WAVE2在BCR诱导的CRAC激活中的机理作用。CA2+信号的严格调节对于淋巴细胞效应子功能至关重要，其功能障碍有助于免疫缺陷以及炎症性疾病。我们提出的研究将确定CA2+信号在疾病状态下可能出现问题的其他机制。这些研究的结果还将洞悉减轻免疫缺陷疾病和/或自身免疫性和炎症状况的方法。

项目成果

Avian tibial dyschondroplasia. II. Biochemical changes.

禽胫骨软骨发育不良。

• 发表时间: 1985
• 期刊: The American journal of pathology
• 作者: Freedman,BD;Gay,CV;Leach,RM
• 通讯作者: Leach,RM

Adhesion- and degranulation-promoting adapter protein is required for efficient thymocyte development and selection.

有效胸腺细胞发育和选择需要促进粘附和脱颗粒的接头蛋白。

• DOI: 10.4049/jimmunol.176.11.6681
• 发表时间: 2006
• 期刊: Journal of immunology (Baltimore, Md. : 1950)
• 作者: Wu,JenniferN;Gheith,Shereen;Bezman,NatalieA;Liu,Qing-Hua;Fostel,LindseyV;Swanson,AndrewM;Freedman,BruceD;Koretzky,GaryA;Peterson,ErikJ
• 通讯作者: Peterson,ErikJ

Evidence for voltage modulation of IL-2 production in mitogen-stimulated human peripheral blood lymphocytes.

在有丝分裂原刺激的人外周血淋巴细胞中电压调节 IL-2 产生的证据。

• 发表时间: 1992
• 期刊: Journal of immunology (Baltimore, Md. : 1950)
• 作者: Freedman,BD;Price,MA;Deutsch,CJ
• 通讯作者: Deutsch,CJ