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RGS 13（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RGS 13缺陷小鼠以确定RGS 13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，由MC抗原受体与IgE抗体交联诱导的皮肤和全身过敏反应在RGS 13缺陷小鼠中显著增加。 这种异常是由于培养的骨髓来源的肥大细胞（BMMCs）的增强IgE诱发的脱粒引起的。用RGS 13重建这些BMMC抑制脱粒。 此外，RGS 13差距活性不是这种效应所必需的。 相反，随后的详细分子分析显示，RGS 13通过与磷酸肌醇-3-激酶（PI 3 K）的p85调节亚基相互作用来调节IgE介导的过敏反应，PI 3 K是导致MC脱粒的变应原诱导的信号传导途径的关键组分。这些结果揭示了RGS 13在肥大细胞中的新的生理功能。 我们假设RGS 13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，RGS 13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RGS 13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。RGS 13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RGS 13表达的敲低具有相反的效果。RGS 13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RGS 13高度同源的RGS，RGS 16，在小鼠和人活化的T淋巴细胞中高度表达。我们正在使用RGS 16缺陷小鼠研究RGS 16在T细胞迁移和活化中的功能。 这些小鼠的类扁桃体器官含有与野生型小鼠相当数量的淋巴细胞。 RGS 16的表达上调的T细胞受体刺激的小鼠CD 4和CD 8细胞。 RGS 16敲除小鼠的脾B和T细胞在暴露于趋化因子CCL 21、CXCL 12和CXCL 13后在体外迁移更多。 目前的研究涉及检查T依赖性抗原免疫后的免疫应答。

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 caused by enhanced IgE-evoked degranulation of cultured bone marrow-derived mast cells (BMMCs). 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. Lymphoid organs of these mice contain comparable numbers of lymphocytes as their wild-type counterparts. RGS16 expression was upregulated by T-cell-receptor stimulation of both murine CD4 and CD8 cells. Splenic B and T cells from RGS16 knockout mice migrated more in vitro after exposure to the chemokines CCL21, CXCL12, and CXCL13. Current studies involve examination of the immune responses after immunization with T-dependent antigens.