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。 在前一年的工作中，我们确定了RGS蛋白，RGS13，它抑制IgE介导的肥大细胞脱粒和过敏反应在小鼠中通过抵消激活的关键下游酶磷酸肌醇-3激酶（PI3激酶）。这些结果揭示了RGS蛋白的一种新的生理功能，对细胞生长、代谢和免疫具有广泛的意义：直接抑制PI3激酶。 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以肥大细胞反应性增加为特征的疾病患者中。因为我们在这项工作的过程中发现，几种RGS蛋白调节PI3激酶，我们研究了与RGS13同源的RGS家族成员如RGS16在不同细胞类型中是否以相同的方式表现。 在2009年期间，我们通过描述RGS16在乳腺癌细胞中对PI3激酶的调节来扩展我们的发现。由于相当大比例的乳腺肿瘤具有RGS16突变和RGS16蛋白表达降低，我们研究了RGS16调节PI3K活性与乳腺癌细胞生长之间的联系。我们发现RGS16通过抑制PI3激酶诱导的增殖来减少乳腺癌细胞的生长。我们还绘制了RGS16和PI3激酶（p85亚基）直接相互作用所需的结构域。这些研究有助于阐明RGS蛋白减轻PI3激酶活性的机制。下一步将是使RGS-PI3K复合物共结晶，以设计模拟RGS蛋白作用的治疗剂。这些化合物最终可能会被测试其缓解肥大细胞介导的过敏性疾病的能力。 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。我们目前正在研究RGS13缺乏是否会影响B细胞产生环腺苷酸诱导的IgE，这是一种CREB依赖性机制。 该项目的另一个主要研究领域是趋化因子GPCR介导的炎症细胞向过敏性炎症部位募集的调节。我们发现RGS16在活化的Th1、Th2和Th17 CD4+淋巴细胞中表达。在体外，RGS16缺陷型T细胞更多地迁移到Th2特异性趋化因子CCL17，我们发现在过敏原攻击小鼠Rgs16-/-小鼠的肺中比野生型小鼠有更多的Th2细胞。从这些初步结果，我们得出结论，RGS16减弱Th2血吸虫抗原的反应。我们计划在一个完整的S模型中证实这些结果。与托马斯韦恩博士（寄生虫病实验室，NIAID）合作研究曼氏感染。 最后，本项目的一个新的重点是确定趋化因子受体/G蛋白/RGS蛋白轴利用小鼠嗜碱性粒细胞在过敏性炎症。这些细胞作为Th2应答和过敏反应的重要介质最近受到了相当大的关注。这个项目正在进行中。

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. 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.