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: 7592215
• 负责人: Kirk m Druey
• 金额: $ 42.83万
• 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/7592215
• 关键词: Adenylate Cyclase; Allergens; Allergic; Anaphylaxis; Antibodies; Antigen Receptors; Asthma; B-Lymphocytes; Binding; Biochemical; Biological Models; Bone Marrow; CD3 Antigens; CD8-Positive T-Lymphocytes; CREB1 gene; Cell Degranulation; Cell Line; Cell Nucleus; Cells; Cutaneous; Cyclic AMP; DNA Binding; Data; Development; Disease; Enzymes; Epithelial Cells; Exhibits; Family; 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; Guanosine Diphosphate; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Heterotrimeric GTP-Binding Proteins; Human; Human G(i) Alpha Proteins; Idiopathic anaphylaxis; IgE; Inflammation; Inflammatory; Knowledge; Ligands; Lung; Lymphocyte; Lymphoid Tissue; Mediating; Modeling; Molecular Analysis; Mus; Pathway interactions; Patients; Phosphatidylinositols; Phosphorylation; Phosphotransferases; Physiological; Process; Production; Protein Overexpression; Proteins; Purpose; RGS Proteins; Role; Route; Signal Pathway; Signal Transduction; Signaling Protein; Small Interfering RNA; Structure of germinal center of lymph node; System; T-Cell Receptor; T-Lymphocyte; Time; base; cell motility; cell type; chemokine; crosslink; cytokine; desensitization; forskolin receptor; granulocyte; in vivo; mast cell; migration; mouse Gdi2 protein; novel; promoter; receptor; receptor coupling; reconstitution; release of sequestered calcium ion into cytoplasm; 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. RGS13, a GAP for Gi and Gq, but not Gs, was found to be expressed in murine and human mast cells (MCs) and B lymphocytes. Rgs13-deficient mice were generated to determine the function of RGS13 in these cell types. Allergic (MC-dependent) physiological responses were evaluated in these mice. Surprisingly, both cutaneous and systemic anaphylaxis induced by cross-linking of MC antigen receptors with IgE antibody were markedly increased in Rgs13-deficient mice. This abnormality was found to be caused by accentuated IgE-evoked degranulation of RGS13-deficient cultured bone marrow-derived mast cells (BMMCs), and reconstitution of these BMMCs with RGS13 inhibited degranulation. In addition, the GAP activity of RGS13 was not required for this effect. Rather, subsequent detailed molecular analysis revealed that Rgs13 regulates IgE-mediated allergic responses by interacting with the p85 regulatory subunit of the enzyme phosphoinositide-3-kinase (PI3K), which is a critical component of the allergen-induced signaling route leading to MC degranulation. These results have uncovered a new physiological function of RGS13 in mast cells. We hypothesize that abnormalities in RGS13 expression or function may exist in patients with idiopathic anaphylaxis or other disorders accompanied by increased mast cell reactivity. 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. An RGS highly homologous to RGS13, RGS16, is highly expressed in mouse and human activated T lymphocytes. We are studying the function of RGS16 in T cell migration and activation using Rgs16-deficient mice, which we have recently generated. Rgs16 was found to be upregulated by T-cell-receptor stimulation of both murine CD4 and CD8 cells. Preliminary data suggest high RGS16 expression in germinal center T lymphocytes isolated from mouse lymphoid tissue. Activation and proliferation induced by T cell receptor stimulation as well as chemokine-mediated migration of RGS16-deficient human T cells are being analyzed using siRNA. Jurkat T cells stably transfected with RGS16 siRNA exhibited increased activation in response to CD3 stimulation, as determined by calcium flux, NFAT activity, and intracellular signaling protein phosphorylation (ERK, ZAP70). The biochemical basis for these abnormalities are currently being investigated.

肥大细胞（MC）和T淋巴细胞是过敏反应和哮喘发展不可或缺的两种细胞类型。 这些细胞中的每一种的特征性应答，即脱粒和细胞因子产生，分别主要由抗原受体的交联诱导。 此外，肥大细胞和T细胞都表达许多与异源三聚体G蛋白（GPCR）偶联的炎症产生受体。本研究的目的是了解这些细胞的细胞内G蛋白偶联信号转导机制和随后的炎症途径。特别是，该项目的重点是通过G蛋白信号传导（RGS蛋白）调节剂的新家族控制炎症过程中的G蛋白活性，该家族通过增加其GT3活性抑制G α-i和G α-q亚基的功能，但不抑制G α-s亚基的功能。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。尽管有关RGS作用的生化机制的知识越来越多，但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。 发现RGS13（Gi和Gq的GAP，但不包括Gs）在鼠和人肥大细胞（MC）和B淋巴细胞中表达。产生RGS13缺陷小鼠以确定RGS13在这些细胞类型中的功能。在这些小鼠中评价过敏性（MC依赖性）生理反应。令人惊讶的是，在Rgs13缺陷小鼠中，由MC抗原受体与IgE抗体交联诱导的皮肤和全身过敏反应均显著增加。 发现这种异常是由缺乏RGS13的培养骨髓源性肥大细胞（BMMC）的IgE诱发的脱颗粒加剧引起的，而这些BMMC与RGS13的重建抑制了脱颗粒。 此外，RGS13的GAP活性不是这种效应所必需的。 相反，随后的详细分子分析表明，Rgs13调节IgE介导的过敏反应，通过与酶磷酸肌醇-3-激酶（PI3K），这是一个关键组成部分的过敏原诱导的信号转导途径，导致MC脱粒的p85调节亚基相互作用。这些结果揭示了RGS13在肥大细胞中的新的生理功能。 我们假设RGS13表达或功能异常可能存在于特发性过敏反应或其他伴有肥大细胞反应性增加的疾病患者中。 出乎意料的是，RGS13在上皮细胞系中的过表达抑制了由GS偶联受体和毛喉素（一种腺苷酸环化酶的直接激活剂）刺激诱导的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，在那里它结合转录因子CREB的活化（磷酸化）形式，CREB是cAMP途径的靶点。RGS13过表达抑制CREB启动子在体内的占用和抑制CREB依赖的基因表达，而siRNA介导的RGS13表达的敲低具有相反的效果。RGS13缺陷型B淋巴细胞显示CREB DNA结合和CREB靶基因OCA-B转录增加。 与RGS13高度同源的RGS（RGS16）在小鼠和人活化T淋巴细胞中高度表达。我们正在研究RGS16在T细胞迁移和活化中的功能，使用我们最近产生的RGS16缺陷小鼠。 发现Rgs16通过鼠CD4和CD8细胞的T细胞受体刺激而上调。初步数据表明RGS16在从小鼠淋巴组织分离的生发中心T淋巴细胞中高表达。使用siRNA分析由T细胞受体刺激诱导的活化和增殖以及趋化因子介导的RGS16缺陷型人T细胞的迁移。如通过钙通量、NFAT活性和细胞内信号蛋白磷酸化（ERK，ZAP 70）所确定的，用RGS16 siRNA稳定转染的Jurkat T细胞表现出响应于CD3刺激的增加的活化。 目前正在研究这些异常的生化基础。