Heterotrimeric G Protein Signaling In Allergic Inflammation

过敏性炎症中的异三聚体 G 蛋白信号传导

基本信息

批准号：
7964378
负责人：
Kirk m Druey
金额：
$ 85.49万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7964378
关键词：
1-Phosphatidylinositol 3-Kinase Adenylate Cyclase Affect Allergens Allergic Anaphylaxis Antigen Receptors Antigens Area Asthma Attention Attenuated B-Lymphocytes Basophils Binding Biochemical Biological Models Breast Cancer Cell CCL17 gene CD4 Positive T Lymphocytes CREB1 gene Cancer Cell Growth Cell Degranulation Cell Line Cell Nucleus Cells Collaborations Complex Cyclic AMP DNA Binding Development Disease Enzymes Epithelial Cells Family Family member G(q) Alpha G-Protein-Coupled Receptors GTP-Binding Protein Regulators GTP-Binding Protein alpha Subunits, Gs GTP-Binding Proteins Gene Deletion Gene Expression Gene Targeting Generations Genetic Genetic Transcription Goals Growth Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Heterotrimeric GTP-Binding Proteins Human G(i) Alpha Proteins Idiopathic anaphylaxis IgE Immunity In Vitro Infection Inflammation Inflammatory Investigation Knowledge Laboratories Ligands Link Lung Lymphocyte Mammary Neoplasms Mediating Mediator of activation protein Metabolism Modeling Mus Mutation National Institute of Allergy and Infectious Disease Parasitic Diseases Pathway interactions Patients Phosphatidylinositols Phosphotransferases Physiological Process Production Proteins RGS Proteins Regulation Role Schistosoma Schistosoma mansoni Signal Pathway Signal Transduction Site Small Interfering RNA System T-Lymphocyte Testing Th2 Cells Therapeutic Agents Time Work base cell growth cell type chemokine chemokine receptor crosslink cytokine desensitization design domain mapping forskolin receptor granulocyte in vivo mast cell novel overexpression promoter protein expression receptor receptor coupling response transcription factor

项目摘要

Mast cells (MCs) and T lymphocytes are two cell types integral to development of an allergic response and asthma. The signature response of each of these cells, degranulation and cytokine production, respectively, is induced primarily by cross-linking of the receptor for antigen. In addition, both mast cells and T cells express numerous inflammation-generating receptors coupled to heterotrimeric G proteins (GPCRs). The purpose of this study is to understand mechanisms of intracellular G-protein-coupled signal transduction in these cells and subsequent pathways to inflammation. In particular, the project focuses on the control of G protein activity in inflammatory processes by a novel family of regulators of G protein signaling (RGS proteins), which inhibit function of G alpha-i and G alpha-q, but not G alpha-s, subunits by increasing their GTPase activity. G alpha subunits oscillate between GDP- (inactive) and GTP- (active) bound forms based on ligand occupancy of the associated receptor. The GTPase accelerating (GAP) activity of RGS proteins limits the time of interaction of active G-alpha and its effectors, resulting in desensitization of GCPR signaling. Despite a growing body of knowledge concerning the biochemical mechanisms of RGS action, little is known about the physiological role of these proteins in native mammalian systems. In the previous year's work, we identified an RGS protein, RGS13, which inhibits IgE-mediated mast cell degranulation and anaphylaxis in mice by counteracting activation of the critical downstream enzyme phosphoinositide-3 kinase (PI3 kinase). These results uncovered a new physiological function of RGS proteins with broad implications for cell growth, metabolism, and immunity: the direct inhibition of PI3 kinase. We hypothesized that abnormalities in RGS13 expression or function may exist in patients with idiopathic anaphylaxis or other disorders characterized by increased mast cell reactivity. Because we discovered during the course of this work that several RGS proteins regulated PI3 kinase, we investigated whether RGS family members homologous to RGS13 such as RGS16 behaved in the same way in different cell types. During 2009, we extended our findings by describing regulation of PI3 kinase by RGS16 in breast cancer cells. Because a substantial percentage of breast tumors have RGS16 mutations and reduced RGS16 protein expression, we investigated the link between regulation of PI3K activity by RGS16 and breast cancer cell growth. We found that RGS16 reduced growth of breast cancer cells by suppressing PI3 kinase-induced proliferation. We also mapped the domains on RGS16 and PI3 kinase (p85 subunit) required for direct interaction. These studies helped clarify the mechanism by which RGS proteins mitigate PI3 kinase activity. The next step will be to co-crystallize the RGS-PI3K complex to enable design of therapeutic agents that mimic the action of RGS proteins. Such compounds might be eventually tested for their ability to alleviate mast-cell mediated allergic disorders. Unexpectedly, RGS13 overexpression in an epithelial cell line inhibited cAMP generation induced by stimulation of a Gs-coupled receptor and by forskolin, a direct activator of adenylyl cyclase. The biochemical basis for this effect was investigated using downstream activators of this signaling pathway. We found that RGS13 acts in the nucleus where it binds the activated (phosphorylated) form of the transcription factor CREB, which is the target of the cAMP pathway. RGS13 overexpression inhibited CREB promoter occupancy in vivo and suppressed CREB-dependent gene expression, while siRNA-mediated knockdown of RGS13 expression had the opposite effect. RGS13-deficient B lymphocytes displayed increased CREB DNA binding and transcription of a CREB target gene, OCA-B. We are currently studying whether RGS13 deficiency affects cyclic AMP-induced IgE production by B cells, a CREB-dependent mechanism. Another major area of investigation in this project is the regulation of chemokine GPCR-mediated recruitment of inflammatory cells to sites of allergic inflammation. We found that RGS16 is expressed in activated Th1, Th2, and Th17 CD4+ lymphocytes. RGS16-deficient T cells migrate more to the Th2-specific chemokine CCL17 in vitro, and we found more Th2 cells in the lungs of allergen-challenged mice Rgs16-/- mice than in wild type counterparts. From these preliminary results, we conclude that RGS16 attenuates Th2 responses to Schistosoma antigens. We plan to confirm these results in a full model of S. mansoni infection in collaboration with Dr. Thomas Wynn (Laboratory of Parasitic Diseases, NIAID). Finally, a newer focus of this project is to identify the chemokine receptor/G protein/RGS protein axis utilized by mouse basophils in allergic inflammation. These cells have received considerable recent attention as crucial mediators of Th2 responses and anaphylaxis. This project is just underway.

肥大细胞 (MC) 和 T 淋巴细胞是过敏反应和哮喘发展中不可或缺的两种细胞类型。这些细胞各自的特征反应，即脱颗粒和细胞因子产生，主要是通过抗原受体的交联来诱导的。此外，肥大细胞和 T 细胞都表达大量与异三聚体 G 蛋白 (GPCR) 偶联的炎症生成受体。本研究的目的是了解这些细胞中细胞内 G 蛋白偶联信号转导的机制以及随后的炎症途径。特别是，该项目重点关注通过新型 G 蛋白信号传导调节因子（RGS 蛋白）家族控制炎症过程中的 G 蛋白活性，这些调节因子抑制 G α-i 和 G α-q 的功能，但不抑制 G α- s，亚基通过增加其 GTP 酶活性。 G α 亚基根据相关受体的配体占据情况在 GDP-（无活性）和 GTP-（活性）结合形式之间振荡。 RGS 蛋白的 GTP 酶加速 (GAP) 活性限制了活性 G-α 与其效应物相互作用的时间，导致 GCPR 信号转导脱敏。尽管关于 RGS 作用的生化机制的知识越来越多，但人们对这些蛋白质在天然哺乳动物系统中的生理作用知之甚少。在去年的工作中，我们发现了一种 RGS 蛋白 RGS13，它通过抵消关键下游酶磷酸肌醇 3 激酶（PI3 激酶）的激活来抑制 IgE 介导的肥大细胞脱粒和过敏反应。这些结果揭示了 RGS 蛋白的新生理功能，对细胞生长、代谢和免疫具有广泛影响：直接抑制 PI3 激酶。我们假设患有特发性过敏反应或其他以肥大细胞反应性增加为特征的疾病的患者中可能存在 RGS13 表达或功能异常。因为我们在这项工作过程中发现了几种 RGS 蛋白调节 PI3 激酶，所以我们研究了与 RGS13 同源的 RGS 家族成员（例如 RGS16）在不同细胞类型中是否以相同的方式表现。 2009 年，我们通过描述乳腺癌细胞中 RGS16 对 PI3 激酶的调节来扩展我们的发现。由于很大一部分乳腺肿瘤具有 RGS16 突变并且 RGS16 蛋白表达减少，因此我们研究了 RGS16 对 PI3K 活性的调节与乳腺癌细胞生长之间的联系。我们发现 RGS16 通过抑制 PI3 激酶诱导的增殖来减少乳腺癌细胞的生长。我们还绘制了直接相互作用所需的 RGS16 和 PI3 激酶（p85 亚基）上的结构域。这些研究有助于阐明 RGS 蛋白减轻 PI3 激酶活性的机制。下一步将是共结晶 RGS-PI3K 复合物，以设计模拟 RGS 蛋白作用的治疗剂。最终可能会测试此类化合物缓解肥大细胞介导的过敏性疾病的能力。出乎意料的是，上皮细胞系中的 RGS13 过表达抑制了 Gs 偶联受体刺激和腺苷酸环化酶直接激活剂毛喉素 (forskolin) 诱导的 cAMP 生成。使用该信号通路的下游激活剂研究了这种效应的生化基础。我们发现 RGS13 在细胞核中起作用，它与转录因子 CREB 的激活（磷酸化）形式结合，而 CREB 是 cAMP 途径的靶标。 RGS13过表达抑制体内CREB启动子占据并抑制CREB依赖性基因表达，而siRNA介导的RGS13表达敲低则具有相反的效果。 RGS13 缺陷的 B 淋巴细胞表现出 CREB DNA 结合和 CREB 靶基因 OCA-B 转录增加。我们目前正在研究 RGS13 缺陷是否会影响 B 细胞环 AMP 诱导的 IgE 产生，这是一种 CREB 依赖性机制。该项目的另一个主要研究领域是趋化因子 GPCR 介导的炎症细胞募集到过敏性炎症部位的调节。我们发现 RGS16 在活化的 Th1、Th2 和 Th17 CD4+ 淋巴细胞中表达。在体外，RGS16 缺陷的 T 细胞更多地迁移到 Th2 特异性趋化因子 CCL17，我们发现，与野生型小鼠相比，过敏原攻击的 Rgs16-/- 小鼠的肺部有更多的 Th2 细胞。从这些初步结果中，我们得出结论，RGS16 减弱了 Th2 对血吸虫抗原的反应。我们计划与 Thomas Wynn 博士（寄生虫病实验室，NIAID）合作，在曼氏链球菌感染的完整模型中确认这些结果。最后，该项目的一个新重点是确定小鼠嗜碱性粒细胞在过敏性炎症中使用的趋化因子受体/G 蛋白/RGS 蛋白轴。这些细胞作为 Th2 反应和过敏反应的关键介质，最近受到了相当多的关注。这个项目刚刚开始。