Understanding Spatial Interactions Between STIM and Orai

了解 STIM 和 Orai 之间的空间相互作用

基本信息

批准号：
9401912
负责人：
Robert M Nwokonko
金额：
$ 3.05万
依托单位：
PENNSYLVANIA STATE UNIV HERSHEY MED CTR
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-01 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9401912
关键词：
A kinase anchoring protein Address Adenylate Cyclase Affect Amino Acids Binding Binding Proteins Biological Assay C-terminal CREB1 gene Calcium Cell Differentiation process Cell membrane Cell physiology Cells Characteristics Complex Coupling Cyclic AMP Cyclic AMP-Dependent Protein Kinases Diffusion Dimerization Endoplasmic Reticulum Eukaryotic Cell Event FOS gene Fluorescence Recovery After Photobleaching Fluorescence Resonance Energy Transfer Gene Expression Genes Genetic Transcription Goals Immune Immunoprecipitation Ion Channel Label Ligands Link Mammals Measures Mediating Membrane Membrane Lipids Metabolism Microscopy Modeling Molecular Conformation Mutation Nature Neurons Pancreas Pathway interactions Positioning Attribute Protein Isoforms Proteins RNA Splicing Regulation Reporter Reporting Research Resolution Rest Role STIM1 gene Sarcoplasmic Reticulum Serine Signal Pathway Signal Transduction Signaling Protein Structural Models Testing Three-Dimensional Image Transcript Variant base cell type crosslink dimer functional mimics image reconstruction novel prevent public health relevance scaffold sensor

项目摘要

Project Summary/Abstract: DESCRIPTION: Store-operated calcium entry (SOCE) is a ubiquitous signaling mechanism in eukaryotic cells crucial for mediating longer–term Ca2+ signals and restoring endoplasmic/sarcoplasmic reticulum Ca2+ after ligand induced depletion. The key operators in SOCE are the Ca2+ selective PM hexameric Orai channels (predominantly Orai1) and the ER/SR resident, dimeric transmembrane calcium sensor proteins, STIM1 and STIM2. STIM1 is activated when ER/SR luminal Ca2+ is depleted, inducing it to unfold and bind to Orai1 channels in the PM. Active Orai1 channels create discrete microdomains of high Ca2+ within ER-PM junctions that contain roughly 100-fold greater Ca2+ concentrations than resting cytoplasmic levels. New EM studies reveal Orai1 channels within ER-PM junctions are closely spaced (9-13 nm), roughly the distance spanned by the STIM-Orai Activating Regions (SOAR) of dimeric STIM1. Although clustering of Orai channels is critical for generating such Ca2+-saturated microdomains, the actual mechanism by which clustering occurs is not understood. My studies address how clustering of Orai1 channels locally controls SOCE-mediated Ca2+ signals in junctions by “concentrating” or spatially confining Ca2+ entry through cross-linking Orai1 channels mediated by the dimeric SOAR domains in STIM1. The studies use soluble SOAR-dimers containing the critical F394H mutation that prevents SOAR binding to the Orai1 channels. I will investigate how a newly discovered and widely expressed splice variant of the STIM2 isoform (STIM2.1), containing an 8 amino acid insert adjacent to the critical F394 STIM-Orai1 binding domain in SOAR, functionally mimics the SOAR1(F394H) mutation. My aims address a critical gap in our understanding of how clustering of Orai1 spatially controls Ca2+ signals and their downstream effectors. My overall hypothesis is that STIM2.1 functions as an important negative regulator of STIM1 that prevents clustering of Orai1 channels, and controls the spatial characteristics of Ca2+ entry signals. Using new SOAR2.1 concatemeric dimers as probes, my aims are: (1) to use super-resolution microscopy to create 3D-image reconstructions of YFP-SOAR2.1 concatemer interactions with Orai1-CFP, and fluorescence recovery after photobleaching (FRAP) to assess membrane diffusion rates and size of YFP-OAR2.1 concatemers bound to Orai1; (2) to use genetically encoded Ca2+ indicators to measure localized Ca2+ signals in clusters, and a FRET-based cyclic AMP sensor, Epac2, to functionally report downstream Ca2+-dependent adenylyl cyclase (AC8) closely associated with Orai1 channels. Overall, these studies will determine how SOAR2.1 may mediate important control Orai1 channel clustering by preventing Orai1 cross-linking, and will assess how STIM2.1 may regulate Orai1 spacing and local Ca2+ microdomains mediated through downstream AC8 activity. Such studies bring together new understanding of the interdependence of local Ca2+ and cyclic AMP signals of major importance in pancreatic, neuronal, and immune cell function.

项目摘要/摘要：描述：商店经营的钙进入（SOCE）是真核细胞中普遍存在的信号机制对于介导长期CA2+信号和恢复内质/肌质网Ca2+的至关重要配体诱导的耗竭。 SOCE中的主要操作员是Ca2+选择性PM Hexameric Orai通道（主要是Orai1）和ER/SR居民，二聚体跨膜钙传感器蛋白，Stim1和 Stim2。当ER/SR Luminal Ca2+耗尽时，激活Stim1，诱导其展开并与Orai1结合 PM中的频道。主动ORAI1通道在ER-PM连接中创建高Ca2+的离散微域与静息细胞质水平相比，其含有大约100倍的Ca2+浓度。新的研究 ER-PM连接内的ORAI1通道紧密间隔（9-13 nm），大约是跨越的距离二聚体刺激的刺激区域（SOAR）。尽管Orai频道的聚类对于生成这样的Ca2+饱和微区域，聚类发生的实际机制不是理解。我的研究探讨了Orai1通道的聚类如何在局部控制SOCE介导的Ca2+ 通过“集中”或空间限制Ca2+进入连接的ORAI1通道，在连接处的信号由STIM1中的二聚体Soar域介导。研究使用包含临界F394H突变阻止了与Orai1通道的飙升。我将调查一个新的含有8氨基酸的STIM2同工型的发现并广泛表达的剪接变体插入与临界F394 stim-orai1结合结构域相邻的，在功能上模仿 SOAR1（F394H）突变。我的目标解决了我们对Orai1聚集的理解的关键差距空间控制Ca2+信号及其下游效应。我的总体假设是STIM2.1功能作为阻止Orai1通道聚类并控制空间的重要负调节器 CA2+进入信号的特征。使用新的SOAR2.1串联二聚体作为问题，我的目的是：（1）使用超分辨率显微镜创建yfp-soar2.1 convatemer Itspetherions的3D图像重建使用Orai1-CFP，并在光漂白后荧光回收（FRAP）评估膜扩散速率 YFP-OAR2.1 confemers的大小与Orai1结合；（2）使用一般编码的Ca2+指示器测量簇中的局部Ca2+信号，以及基于FRET的循环AMP传感器EPAC2，以便在功能上报告与Orai1通道密切相关的下游Ca2+依赖性腺苷酸环化酶（AC8）。总体而言，这些研究将确定SOAR2.1如何通过防止 ORAI1交联，并将评估STIM2.1如何调节ORAI1间距和局部Ca2+微区域通过下游AC8活性介导。这样的研究汇集了对局部Ca2+和循环AMP信号的相互依赖性在胰腺，神经元和免疫细胞功能。