Novel regulators of store-operated Ca2+ entry in immune systems

描述（由申请人提供）：Ca2+通过CRAC （Ca2+释放激活Ca2+）通道内流对免疫细胞功能至关重要。通过受体的刺激消耗细胞内Ca2+储存并随后导致质膜上CRAC通道的开放。最近，Orai1 （CRACM1）和STIM1分别被鉴定为CRAC通道的孔隙组分和Ca2+储存耗尽和CRAC通道打开之间的中介。然而，关于CRAC通道调控的分子组成和机制仍有许多有待了解的地方。我们在耗尽储存条件下使用大规模亲和蛋白纯化来鉴定CRAC通道的调节因子。我们已经确定了CRACR2A （Ca2+释放激活的Ca2+通道调节剂2A）是使用这种方法的CRAC通道的新调节剂。具体目的是：(1)验证Orai1-CRACR2A相互作用对CRAC通道功能至关重要的假设。我们将在Orai1缺失的原代细胞中分析不能与CRACR2A结合的Orai1突变体的活性。(2)验证CRACR2A稳定Orai1-STIM1复合物的假设。我们的初步数据表明，CRACR2A可以直接与Orai1和STIM1相互作用。Orai1和STIM1的功能将使用CRACR2A敲除细胞进行检测。将确定Orai1、STIM1和CRACR2A相互作用的功能重要性。(3)验证CRACR2A对T细胞功能至关重要的假设。在Jurkat T细胞中敲低CRACR2A可减少Ca2+的进入。我们将有条件地靶向CRACR2A基因，以确定CRACR2A在储存操作的Ca2+进入外周T细胞中的作用。由于最近才发现了Orai1和STIM1，因此对CRAC通道的新调节器的实验是及时的。在短期内，所提出的实验应该为研究哺乳动物细胞，特别是免疫细胞中储存操作的Ca2+进入的调节机制提供新的分子探针。从长远来看，我们将测试开发药物来调节CRAC通道功能的可能性；从而激活免疫。公共卫生相关性：我们建议通过蛋白质组学分析、进一步的功能分析和基因操作来了解免疫细胞激活的关键步骤——钙通过质膜钙通道进入的机制。我们利用这些方法成功鉴定了一个新的分子，CRACR2A，我们将重点阐明该分子在免疫系统中的新功能。我们的研究有助于开发能够激活或抑制免疫功能的药物，以治疗免疫系统相关问题，如自身免疫性疾病或移植排斥反应。

DESCRIPTION (provided by applicant): Ca2+ influx though CRAC (Ca2+ release activated Ca2+) channels is critical for immune cell functions. Stimulation through receptors depletes intracellular Ca2+ stores and subsequently leads to opening of CRAC channels on the plasma membrane. Recently, Orai1 (CRACM1) and STIM1 were identified as a pore component of the CRAC channel and a mediator between Ca2+ store depletion and CRAC channel opening, respectively. However, much remains to be understood about the molecular composition and the mechanism of CRAC channel regulation. We have used large scale affinity protein purification under store depleted conditions to identify regulators of the CRAC channel. We have identified CRACR2A (Ca2+ release activated Ca2+ channel Regulator 2A) as a novel regulator of CRAC channels using this approach. The Specific Aims are: (1) To test the hypothesis that Orai1-CRACR2A interaction is critical for the CRAC channel function. The activities of Orai1 mutants incapable of binding to CRACR2A will be analyzed in Orai1-null primary cells. (2) To test the hypothesis that CRACR2A stabilizes the Orai1-STIM1 complex. Our preliminary data suggest that CRACR2A can directly interact with both Orai1 and STIM1. The functions of Orai1 and STIM1 will be examined using CRACR2A knock-down cells. The functional importance of reciprocal interaction of Orai1, STIM1, and CRACR2A will be determined. (3) To test the hypothesis that CRACR2A is critical for the T cell function. Knock-down of CRACR2A in Jurkat T cells decreased Ca2+ entry. We will conditionally target the CRACR2A gene to determine the role of CRACR2A in store-operated Ca2+ entry in peripheral T cells. The experiments on the novel regulator of the CRAC channel are timely because of the very recent discovery of Orai1 and STIM1. In the short term, the proposed experiments should provide new molecular probes for investigating the regulatory mechanism of store-operated Ca2+ entry in mammalian cells, particularly in immune cells. In the long term, we will test the possibility of development of drugs to modulate the function of the CRAC channel; thereby immune activation. Public Health Relevance: We propose to understand the mechanism of a critical step of immune cell activation, calcium entry via plasma membrane calcium channels through proteomic analysis, further functional analysis, and gene manipulation. We successfully indentified a novel molecule, CRACR2A using these approaches and we will focus to elucidate the novel function of this molecule in the immune system. Our study can benefit development of drugs that can activate or repress immune functions as therapy for immune system related problems such as auto-immune diseases or graft rejection.