Heterotrimeric G Protein Signaling In Allergic Inflammation

肥大细胞(MC)和T淋巴细胞是过敏反应和哮喘发生过程中不可或缺的两种细胞类型。这些细胞的标志性反应，脱颗粒和细胞因子的产生，主要是由抗原受体的交联性诱导的。此外，肥大细胞和T细胞都表达许多与异三聚体G蛋白(GPCRs)偶联的致炎受体。这项研究的目的是了解这些细胞内G蛋白偶联信号转导的机制以及随后的炎症途径。特别是，该项目专注于通过一系列新的G蛋白信号调节蛋白(RGS蛋白)来控制炎症过程中的G蛋白活性，这些蛋白通过提高Gα-I和Gα-Q亚单位的GTP酶活性来抑制Gα-I和Gα-Q亚单位的功能，而不是抑制Gα-S亚单位的功能。Gα亚基根据相关受体的配基占有率在GDP结合形式(不活跃)和GTP结合形式(活跃)之间振荡。RGS蛋白的GTP酶加速(GAP)活性限制了活性G-α与其效应分子相互作用的时间，导致GCPR信号失敏。尽管关于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。 RGS13是GI和GQ的间隙，但不表达Gs，在小鼠和人肥大细胞(MC)和B淋巴细胞中有表达。建立RGS13基因缺陷小鼠，以确定RGS13在这些细胞类型中的功能。对这些小鼠的过敏(MC依赖)生理反应进行评估。令人惊讶的是，在RGS13缺陷小鼠中，由MC抗原受体与IgE抗体交联所引起的皮肤和全身过敏反应显著增加。这种异常是由于RGS13缺陷的培养的骨髓肥大细胞(BMMCs)的IgE诱导的脱颗粒加重所致，而用RGS13重组这些BMMCs抑制了脱颗粒。此外，RGS13的GAP活性不是这一效应所必需的。相反，随后的详细分子分析揭示，Rgs13通过与磷脂酰肌醇-3-激酶(PI3K)的P85调节亚基相互作用来调节IgE介导的过敏反应，PI3K是过敏原诱导的导致MC脱颗粒的信号通路的关键组成部分。这些结果揭示了RGS13在肥大细胞中的一种新的生理功能。我们假设RGS13的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强的疾病患者中。 出乎意料的是，RGS13在上皮细胞系中的过表达抑制了由Gs偶联受体和腺苷环化酶直接激活剂Forsklin诱导的cAMP生成。使用该信号通路的下游激活剂研究了这一效应的生化基础。我们发现RGS13作用于细胞核，在那里它与转录因子CREB的激活(磷酸化)形式结合，CREB是cAMP途径的靶标。RGS13过表达抑制了CREB启动子在体内的占位，抑制了CREB依赖的基因表达，而siRNA介导的RGS13表达下调具有相反的作用。RGS13缺陷的B淋巴细胞表现出CREB DNA结合增加和CREB靶基因OCA-B转录增加。 与RGS13高度同源的RGS RGS16在小鼠和人活化的T淋巴细胞中高表达。我们正在利用我们最近培育的RGS16缺陷小鼠来研究RGS16在T细胞迁移和激活中的作用。RGS16被发现通过T细胞受体刺激小鼠的CD4和CD8细胞而上调。初步数据表明，从小鼠淋巴组织中分离的生发中心T淋巴细胞高表达Rgs16。利用siRNA分析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T细胞的迁移。稳定表达Rgs16 siRNA的Jurkat T细胞在CD3刺激下表现出更高的活性，通过钙离子通量、NFAT活性和细胞内信号蛋白磷酸化(ERK、ZAP70)来确定。这些异常的生化基础目前正在调查中。

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.