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

• 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
• 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

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（MCS）和T淋巴细胞是两种细胞类型，是过敏反应和哮喘发展的不可或缺的细胞类型。 这些细胞中每个细胞的签名反应分别脱粒和细胞因子的产生主要是通过抗原受体的交联引起的。 此外，肥大细胞和T细胞都表现出与异源三聚体G蛋白（GPCR）耦合的许多炎症生成受体。这项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，该项目的重点是通过新的G蛋白信号传导（RGS蛋白）家族来控制炎症过程中G蛋白活性，该家族通过增加GTPase活性来抑制G alpha-I和G alpha-Q的功能，但不抑制G alpha-S的功能，但不抑制GALPHA-S的功能。 Gα亚基在GDP-（非活动）和GTP-（活动）结合形式之间振荡，基于相关受体的配体占用率。 RGS蛋白的GTPase加速（GAP）活性限制了活性G-Alpha及其效应子的相互作用时间，从而导致GCPR信号脱敏。尽管关于RGS作用的生化机制的知识越来越多，但这些蛋白质在天然哺乳动物系统中的生理作用知之甚少。 在上一年的工作中，我们鉴定了RGS蛋白RGS13，该蛋白通过抵消关键下游磷酸肌醇-3激酶（PI3激酶）的激活来抑制小鼠IgE介导的肥大细胞脱粒和过敏反应。这些结果发现了RGS蛋白的新生理功能，对细胞生长，代谢和免疫力具有广泛的影响：PI3激酶的直接抑制作用。 我们假设在特发性过敏反应或其他以肥大细胞反应性升高的特征的患者中，RGS13表达或功能异常可能存在。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RGS家族成员是否与RGS13同源（例如RGS16）在不同的细胞类型中的表现相同。 在2009年期间，我们通过描述RGS16在乳腺癌细胞中对PI3激酶的调节来扩展发现。由于大量乳腺肿瘤具有RGS16突变并降低RGS16蛋白表达，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激酶诱导的增殖来降低乳腺癌细胞的生长。我们还映射了直接相互作用所需的RGS16和PI3激酶（p85亚基）的域。这些研究有助于阐明RGS蛋白减轻PI3激酶活性的机制。下一步将是将RGS-PI3K复合物共结合，以实现模仿RGS蛋白的作用的治疗剂。这些化合物最终可能会因减轻桅杆细胞介导的过敏疾病的能力而进行测试。 出乎意料的是，在上皮细胞系中的RGS13过表达抑制了刺激GS耦合受体和腺苷环酶的直接激活剂Forskolin引起的营地产生。使用该信号通路的下游激活剂研究了这种作用的生化基础。我们发现RGS13作用在其结合了转录因子CREB的活化（磷酸化）形式的核中，该因子是cAMP途径的靶标。 RGS13的过表达抑制了体内CREB启动子占用率，并抑制了CREB依赖性基因表达，而siRNA介导的RGS13表达的敲低表达具有相反的作用。 RGS13缺陷型B淋巴细胞显示出Creb靶基因OCA-B的CREB ​​DNA结合和转录增加。我们目前正在研究RGS13缺乏症是否影响循环AMP诱导的IgE生产B细胞，B细胞是CREB依赖性机制。 该项目的另一个主要研究领域是调节趋化因子GPCR介导的炎症细胞对过敏性炎症部位的募集。我们发现RGS16在活化的Th1，Th2和Th17 CD4+淋巴细胞中表达。 RGS16缺陷的T细胞在体外迁移到Th2特异性的趋化因子CCL17，并且在过敏原挑战者的小鼠肺中发现的Th2细胞比野生型对应物中的Th2细胞更多。从这些初步结果中，我们得出的结论是，RGS16减弱了TH2对血吸虫抗原的反应。我们计划通过与托马斯·怀恩（Thomas Wynn）博士合作（寄生疾病实验室，NIAID）的完整模型来确认这些结果。 最后，该项目的一个较新的重点是鉴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大的关注。这个项目刚刚开始。