Heterotrimeric G Protein Signaling In Allergic Inflammation

肥大细胞(MC)、粒细胞和淋巴细胞是过敏反应发生过程中不可或缺的部分。过敏性炎症也可能通过激活与异三聚体G蛋白(GPCRs)偶联的受体而产生。本研究的目的是了解免疫细胞中G蛋白介导的信号转导机制，重点是G蛋白介导的白细胞向变态反应性炎症部位的转运。 GPCRs激活异源三聚体G蛋白的核心途径，与鸟苷三磷酸(GTP)结合，以换取鸟苷二磷酸(GDP)。G蛋白α亚单位的GTP结合形式诱导下游信号级联反应，包括负责MC/嗜碱性粒细胞脱颗粒的细胞内钙离子通量。该项目关注于G蛋白信号转导调节蛋白家族(RGS蛋白)，它们通过增加GTP酶的活性来抑制Gα-I和Gα-Q蛋白的功能，但不抑制Gα-S蛋白的功能。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，但对这些蛋白质在过敏性炎症中的生理作用知之甚少。 一个主要的研究领域是炎症细胞在炎症部位的募集。趋化因子是作用于白细胞GPCRs的一类主要化合物，它协调免疫细胞的运输，RGS蛋白包括RGS5、RGS13和RGS16通过脱敏GPCR信号来抑制趋化因子信号。 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。许多变应原具有固有的蛋白分解活性，并与蛋白水解酶激活的GPCRs结合。尽管对木瓜酶、蠕虫感染、慢性过敏性皮肤炎和鼻炎等蛋白酶变应原的致敏作用与嗜碱性粒细胞聚集到炎症组织或引流淋巴结(LNS)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。我们正在培育含有肥大细胞或嗜碱性粒细胞对趋化因子高或低反应的小鼠品系，以研究这些细胞对各种过敏反应的贡献。 在2016财年，我们与Rosenberg博士(LAD/IIS)合作进行了一项关键实验，以表征RNase家族成员(MEAR 11)的趋化特性，该成员在巨噬细胞中表达，并受到Th2细胞因子(IL-4/13)的上调。我们证明了mEar11对巨噬细胞具有很强的趋化作用。此属性不依赖于mEar11核糖核酸酶活性。在过敏性疾病模型中进一步研究mEar11可能有助于了解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，我们展示了RGS16在血小板激活中的关键负调节功能。Rgs16基因缺陷小鼠的血小板对GPCR配体凝血酶和CXCL12以及胶原的反应表现出增强的聚集、颗粒分泌和粘附性。 在与奥兰治博士和苏博士的合作研究中，患有不明免疫缺陷和G蛋白和/或RGS蛋白新突变的患者正在进行研究。 最后一个调查领域是RGS5在中性粒细胞贩运中的作用。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基因缺失小鼠分离的中性粒细胞对促炎趋化因子具有增强的趋化作用。目前的研究旨在了解这种表型背后的分子机制，并确定这种缺陷是否是白细胞固有的。

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.