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: 9354742
• 负责人: Kirk m Druey
• 金额: $ 16.94万
• 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/9354742
• 关键词: Adhesions; Allergens; Allergic; Allergic Disease; Allergic inflammation; Antigens; Area; Basophils; Binding; Biochemical; Blood Platelets; CXCL12 gene; Cells; Chemotactic Factors; Chemotaxis; Chronic; Collagen; Cytoplasmic Granules; Defect; Development; Effector Cell; Exhibits; Extrinsic asthma; Family; Family member; G Protein-Coupled Receptor Signaling; G(q) Alpha; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Protein Regulators; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Gene Deletion; Genes; Goals; Guanosine Diphosphate; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Helminths; Heterotrimeric GTP-Binding Proteins; Hypersensitivity; Immune; Immunologic Deficiency Syndromes; Infection; Inflammation; Inflammatory; Interleukin-4; Investigation; Knowledge; Laboratories; Leukocyte Trafficking; Leukocytes; Ligands; Light; Lung; Lymphocyte; Mediating; Modeling; Molecular; Mouse Strains; Mus; Mutation; Nose; Oranges; Papain; Pathway interactions; Patients; Peptide Hydrolases; Phenotype; Physiological; Platelet Activation; Process; Property; Proteins; Research; Rhinitis; Ribonucleases; Role; Signal Transduction; Site; Skin; Source; T-Lymphocyte; Thrombin; Time; Tissues; Universities; allergic response; base; chemokine; cytokine; desensitization; genetic approach; granulocyte; human subject; lymph nodes; macrophage; mast cell; neutrophil; novel; overexpression; receptor; receptor coupling; release of sequestered calcium ion into cytoplasm; research study; response; trafficking

Mast cells (MCs), granulocytes, and lymphocytes are integral to the development of an allergic response. Allergic inflammation may also be generated through activation of receptors coupled to heterotrimeric G proteins (GPCRs). The purpose of this study is to understand mechanisms of G protein-mediated signal transduction in immune cells, with a focus on GPCR-mediated trafficking of leukocytes to sites of allergic inflammation. GPCRs activate a core pathway of heterotrimeric G proteins, which bind guanosine triphosphate (GTP) in exchange for guanosine diphosphate (GDP). The GTP-bound form of the G protein alpha subunit induces downstream signaling cascades, including intracellular calcium flux responsible for MC/basophil degranulation. This project focuses on a family of regulators of G protein signaling (RGS proteins), which inhibit the function of G alpha-i and G alpha-q, but not G alpha-s, proteins 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, relatively little is known about the physiological role of these proteins in allergic inflammation. A major area of investigation is the recruitment of inflammatory cells to sites of inflammation. Chemokines are a major class of compounds acting on leukocyte GPCRs, which orchestrate immune cell trafficking, and RGS proteins including RGS5, RGS13, and RGS16 inhibit chemokine signaling by desensitizing GPCR signals. A second research area is the trafficking of mast cells and granulocytes during allergic responses. Many allergens contain intrinsic proteolytic activity and bind protease activated GPCRs. Although sensitization to protease allergens, such as papain, helminth infection, chronic allergic skin inflammation, and nasal rhinitis are associated with basophil recruitment to inflamed tissue or to draining lymph nodes (LNs), the precise role of basophils and mechanisms involved in their recruitment is incompletely understood. We are generating mouse strains containing mast cells or basophils hyper- or hyporesponsive to chemokines in order to study the contribution of these cells to various allergic responses. In FY16, we contributed key experiments to a collaborative study with Dr. Rosenberg (LAD/IIS) to characterize chemoattractant properties of an RNAse family member (mEar 11), which is expressed in macrophages and is upregulated by Th2 cytokines (IL-4/13). We demonstrated that mEar11 acts as a potent chemoattractant for macrophages. This property did not depend on mEar11 RNase activity. Further investigation of mEar11 in models of allergic disease may shed light on the function of these leukocytes at sites of tissue inflammation. In another collaborative project with Dr. Khasawneh (Western University), we demonstrated a critical negative regulatory function of RGS16 in platelet activation. Platelets from RGS16-deficient mice exhibited enhanced aggregration, granule secretion, and adhesion in response to GPCR ligands thrombin and CXCL12 as well as collagen. Patients with undefined immunodeficiencies and novel mutations in G proteins and/or RGS proteins are being characterized in collaborative studies with Drs. Orange and Su. A final area of investigation is the role of RGS5 in neutrophil trafficking. Using Rgs5-/- mice, we discovered in 2016 that neutrophils deficient in RGS5 do not traffic normally to sites of inflammation. Neutrophils isolated from Rgs5 gene deleted mice display enhanced chemotaxis to proinflammatory chemokines. Current studies are aimed at understanding the molecular mechanisms underlying this phenotype and determining whether the defects are leukocyte-intrinsic.

肥大细胞（MC）、粒细胞和淋巴细胞是过敏反应发展的组成部分。 过敏性炎症也可以通过激活与异源三聚体G蛋白（GPCR）偶联的受体而产生。本研究的目的是了解G蛋白介导的免疫细胞信号转导机制，重点是GPCR介导的白细胞运输到过敏性炎症部位。 GPCR激活异源三聚体G蛋白的核心途径，其结合三磷酸鸟苷（GTP）以交换二磷酸鸟苷（GDP）。G蛋白α亚基的GTP结合形式诱导下游信号级联，包括负责MC/嗜碱性粒细胞脱粒的细胞内钙流。该项目的重点是G蛋白信号传导调节因子（RGS蛋白）家族，其通过增加GT3活性抑制G α-i和G α-q蛋白的功能，但不抑制G α-s蛋白的功能。G α亚基基于相关受体的配体占有率在GDP-（非活性）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及其效应物相互作用的时间，导致GCPR信号转导的脱敏。尽管关于RGS作用的生化机制的知识越来越多，但关于这些蛋白质在过敏性炎症中的生理作用的知识相对较少。 研究的一个主要领域是炎症细胞向炎症部位的募集。趋化因子是作用于白细胞GPCR的主要类别的化合物，其协调免疫细胞运输，并且RGS蛋白包括RGS5、RGS13和RGS16通过使GPCR信号脱敏来抑制趋化因子信号传导。 第二个研究领域是过敏反应期间肥大细胞和粒细胞的运输。许多过敏原含有内在的蛋白水解活性并结合蛋白酶激活的GPCR。尽管对木瓜蛋白酶等蛋白酶过敏原、蠕虫感染、慢性过敏性皮肤炎症和鼻鼻炎的致敏与嗜碱性粒细胞向炎症组织或引流淋巴结（LN）的募集有关，但嗜碱性粒细胞的确切作用及其募集机制尚不完全清楚。我们正在产生含有肥大细胞或嗜碱性粒细胞的小鼠品系，这些细胞对趋化因子的反应性过高或过低，以研究这些细胞对各种过敏反应的贡献。 在2016财年，我们为与Rosenberg博士（LAD/IIS）的合作研究提供了关键实验，以表征RNAse家族成员（mEar 11）的化学引诱物特性，该家族成员在巨噬细胞中表达并受Th2细胞因子（IL-4/13）上调。我们证明了mEar11作为巨噬细胞的有效化学引诱物。该特性不依赖于mEar11 RNase活性。在过敏性疾病模型中对mEar11的进一步研究可能会揭示这些白细胞在组织炎症部位的功能。 在与Khasawneh博士（西方大学）的另一个合作项目中，我们证明了RGS16在血小板活化中的关键负调节功能。来自RGS16缺陷小鼠的血小板表现出响应于GPCR配体凝血酶和CXCL12以及胶原蛋白的增强的聚集、颗粒分泌和粘附。 在与橙子和苏博士的合作研究中，正在对G蛋白和/或RGS蛋白中具有未确定的免疫缺陷和新突变的患者进行表征。 研究的最后一个领域是RGS5在中性粒细胞运输中的作用。使用Rgs5-/-小鼠，我们在2016年发现，缺乏RGS5的中性粒细胞不能正常运输到炎症部位。 从Rgs5基因缺失的小鼠中分离的中性粒细胞显示出对促炎趋化因子的增强的趋化性。目前的研究旨在了解这种表型的分子机制，并确定缺陷是否是白细胞内在的。