白三烯B实时成像 4介导的细胞迁移和BLT1与β- arrestin的相互作用
Summary
本文介绍的方法，以确定具体的配体的白细胞趋化反应和识别细胞表面的受体，并用活细胞成像技术的胞质蛋白质之间的相互作用。
Abstract
属于G蛋白偶联受体（GPCRs）的7个跨膜蛋白家族介导的细胞外信号传导到细胞内的反应。 GPCRs的控制多种生物学功能，如趋化，细胞内钙释放，基因调控配体依赖性通过异三聚体G蛋白的 1-2 。配体结合诱导了一系列构象变化，导致激活异三聚体G蛋白调节的第二信使，如环磷酸腺苷（cAMP）的，三磷酸肌醇（IP3）和二酰基甘油（DG）的水平。激活受体配体结合伴随还发起了一系列活动，以衰减信号通过脱敏，封存和/或内部化的受体。 GPCRs的脱敏过程中发生通过受体磷酸化G蛋白受体激酶（GRKs）和β- arrestins 3随后结合。 β- arrestins是胞浆蛋白转运到细胞膜G蛋白偶联受体激活后，磷酸化的受体结合（大多数情况下）通过促 进受体的内化4-6存在。
白三烯B 4（LTB 4）是一种促炎性脂质分子，从花生四烯酸途径介导其行动派生通过GPCRs的，LTB 4受体1（BLT1;高亲和力受体）和LTB 4受体2（BLT2;低亲和力受体）7-9。 LTB 4 BLT1通路已被证明是在一些炎症性疾病，包括哮喘，关节炎，动脉粥样硬化10-17的关键。当前的纸张描述发展到显示器的LTB 4诱导白细胞迁移和BLT1的相互作用与β- arrestin的，在使用活细胞显微成像技术18-19中的受体易位的方法。
骨髓C57BL / 6小鼠的树突状细胞分离和培养作为先前描述 20-21 。这些细胞在活细胞成像方法进行了测试，证明LTB 4诱导细胞迁移。人类BLT1用红色荧光蛋白（BLT1 – RFP）的标签，在C -末端与绿色荧光蛋白（β- ARR – GFP）标记的β- arrestin1了两个质粒转染大鼠嗜碱性Leukomia（RBL – 2H3）细胞株18日至19日 。这些蛋白质和本地化之间的相互作用的动力学进行了监测使用的活细胞视频显微镜。在当前纸张的方法描述了利用显微技术研究G蛋白偶联受体在活细胞的功能反应。还介绍了当前的纸张MetaMorph软件的使用，以量化的荧光强度，以确定受体的动力学和胞浆蛋白的相互作用。
Protocol
方法显微镜的说明活细胞成像实验用的TE – FM系统连接到尼康的Eclipse TE300倒置显微镜落射荧光。显微镜配备加热阶段。一个凉爽的总部管理单元数字B / W CCD（罗伯科学）相机和LAMDA 10-2光学过滤器更换（萨特仪器公司）是连接到显微镜。激发和发射波长控制滤光轮和兰巴10-2滤光轮控制器控制仪器有限公司，萨特曝光时间为500 ms应该足够，以查看RFP或GFP在活细胞…
Discussion
活细胞成像是一个强大的工具，表现出特定的蛋白质的功能和互动，因为他们在实时发生。在这个手稿中描述的方法，清楚地表明，LTB 4能诱导树突状细胞的快速迁移。这些方法不仅扩大LTB 4个功能多样的细胞类型方面，它们允许类似的方法被应用到其他各种趋化因子和测试他们在不同的白细胞分人口趋化剂的功效。在这个系统中的荧光成像，可以实时监测的信号事件。此外，这?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究是由美国国立卫生赠款AI – 52381，CA138623和肯塔基州的肺癌研究委员会和机构支持从格雷厄姆詹姆斯布朗癌症中心的支持。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Cell lines: | ||||
| Rat Basophilic Leukomia Cell line (RBL-2H3) or HEK293 cells. | ATCC | CRL-2256 | ||
| Media: | ||||
| Delbecco’s modified Eagle’s Medium (DMEM) | Invitrogen | 11995 | ||
| Phenol red free RPMI or DMEM | Invitrogen | 11835-030 | ||
| Fetal Bovine Serum | Invitrogen | 16000-044 | ||
| L-Glutamine (200 mM) | Invitrogen | 25030 | ||
| Penicillin-streptomycin (10000 U/mL) | Invitrogen | 15140 | ||
| Trypsin, 0.05% (1X) with EDTA 4Na, liquid | Invitrogen | 25300 | ||
| HEPES (1M) | Invitrogen | 15630 | ||
| Others: | ||||
| 35 mm sterile glass coverslip-bottomed Fluoro dishes (0.17 mm thick) (WillCo-dish) | WPI | FD35-100 | ||
| Sterile Gene Pulser Cuvette (0.4 cm electrode gap) (Bio-Rad) | Bio-Rad | 16552088 | ||
| Instruments/software: | ||||
| Gene Pulser II electroporater | Bio-Rad | |||
| TE-FM Epi-Fluorescence system attached to Nikon Inverted Microscope Eclipse TE300 | Nikon | |||
| Metamorph Software | Universal Imaging | |||
| Vertical Micro-pipette puller | Narishige International | |||
| Micro-Forge M-900 | Narishige International | |||
| Hadraulic Micromanipulator MO-188NE | Narishige International | |||
| Coarse Manual Manipulator, MN-188NE | Narishige International | |||
| cDNA constructs: | ||||
| cDNA of G-Protein coupled receptor tagged with red fluorescence protein at C-terminus (hBLT1-RFP) | Jala et al 2005 | |||
| cDNA of cytosolic protein tagged with GFP (β-arrestin1-GFP in present study). | Jala et al 2005 |
Referências
- Wess, J. G-protein-coupled receptors: molecular mechanisms involved in receptor activation and selectivity of G-protein recognition. FASEB J. 11, 346-354 (1997).
- Gether, U. Uncovering molecular mechanisms involved in activation of G protein-coupled receptors. Endocr Rev. 21, 90-113 (2000).
- Pierce, K. L., Premont, R. T., Lefkowitz, R. J. Seven-transmembrane receptors. Nat Rev Mol Cell Biol. 3, 639-650 (2002).
- Lefkowitz, R. J. G. protein-coupled receptors. III. New roles for receptor kinases and beta-arrestins in receptor signaling and desensitization. J Biol Chem. 273, 18677-18680 (1998).
- Shenoy, S. K., Lefkowitz, R. J. Multifaceted roles of beta-arrestins in the regulation of seven-membrane-spanning receptor trafficking and signalling. Biochem J. 375, 503-515 (2003).
- . Beta-arrest or. Nature. 383, 447-450 (1996).
- Serhan, C. N., Haeggstrom, J. Z., Leslie, C. C. Lipid mediator networks in cell signaling: update and impact of cytokines. Faseb J. 10, 1147-1158 (1996).
- Tager, A. M., Luster, A. D. BLT1 and BLT2: the leukotriene B(4) receptors. Prostaglandins Leukot Essent Fatty Acids. 69, 123-134 (2003).
- Toda, A., Yokomizo, T., Shimizu, T. Leukotriene B4 receptors. Prostaglandins Other Lipid Mediat. 68-69, 575-585 (2002).
- Haribabu, B. Targeted disruption of the leukotriene B(4) receptor in mice reveals its role in inflammation and platelet-activating factor-induced anaphylaxis. J Exp Med. 192, 433-438 (2000).
- Subbarao, K. Role of leukotriene B4 receptors in the development of atherosclerosis: potential mechanisms. Arterioscler Thromb Vasc Biol. 24, 369-375 (2004).
- Jala, V. R., Haribabu, B. Leukotrienes and atherosclerosis: new roles for old mediators. Trends Immunol. 25, 315-322 (2004).
- Heller, E. A. Inhibition of atherogenesis in BLT1-deficient mice reveals a role for LTB4 and BLT1 in smooth muscle cell recruitment. Circulation. 112, 578-586 (2005).
- Miyahara, N. Requirement for leukotriene B4 receptor 1 in allergen-induced airway hyperresponsiveness. Am J Respir Crit Care Med. 172, 161-167 (2005).
- Terawaki, K. Absence of leukotriene B4 receptor 1 confers resistance to airway hyperresponsiveness and Th2-type immune responses. J Immunol. 175, 4217-4225 (2005).
- Shao, W. H., Del Prete, A., Bock, C. B., Haribabu, B. Targeted disruption of leukotriene B4 receptors BLT1 and BLT2: a critical role for BLT1 in collagen-induced arthritis in mice. J Immunol. 176, 6254-6261 (2006).
- Kim, N. D., Chou, R. C., Seung, E., Tager, A. M., Luster, A. D. A unique requirement for the leukotriene B4 receptor BLT1 for neutrophil recruitment in inflammatory arthritis. J Exp Med. 203, 829-835 (2006).
- Jala, V. R., Shao, W. H., Haribabu, B. Phosphorylation-independent beta-arrestin translocation and internalization of leukotriene B4 receptors. J Biol Chem. 280, 4880-4887 (2005).
- Jala, V. R., Haribabu, B. Real-time analysis of G protein-coupled receptor signaling in live cells. Methods Mol Biol. 332, 159-165 (2006).
- Del Prete, A., A, . Regulation of dendritic cell migration and adaptive immune response by leukotriene B4 receptors: a role for LTB4 in up-regulation of CCR7 expression and function. Blood. 109, 626-631 (2007).
- Salogni, L. Activin A induces dendritic cell migration through the polarized release of CXC chemokine ligands 12 and 14. Blood. 113, 5848-5856 (2009).
- Boudreau, J., Koshy, S., Cummings, D., Wan, Y. Culture of myeloid dendritic cells from bone marrow precursors. J Vis Exp. , (2008).
Tags
Leukotriene B4Cell MigrationBLT1β-arrestinG-protein Coupled ReceptorsTransmembrane Protein FamilyExtracellular SignalsIntracellular ResponsesChemotaxisCalcium ReleaseGene RegulationLigand BindingConformational ChangesHeterotrimeric G-proteinsSecond MessengersCyclic Adenosine Monophosphate (cAMP)Inositol Triphosphate (IP3)Diacyl Glycerol (DG)DesensitizationSequestrationInternalizationG-protein Receptor Kinases (GRKs)Cytosolic ProteinsMembrane TranslocationPro-inflammatory Lipid MoleculeArachidonic Acid Pathway
Citar este artigo
Jala, V. R., Haribabu, B. Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with β-arrestin. J. Vis. Exp. (46), e2315, doi:10.3791/2315 (2010).