H1R Signaling and Immune Deviation in EAE

EAE 中的 H1R 信号传导和免疫偏差

• 批准号: 7852561
• 负责人: CORY TEUSCHER
• 金额: $ 34.26万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-19 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7852561
• 关键词: Adjuvant; Affect; Alleles; Amines; Amino Acids; Arrestins; Autoimmune Diseases; Autoimmune Process; Bordetella pertussis; CD4 Positive T Lymphocytes; Candidate Disease Gene; Cell surface; Cells; Central Nervous System Diseases; Chimeric Proteins; Complement; Data; Development; Disease; Disease Resistance; Disease susceptibility; Encephalomyelitis; Endoplasmic Reticulum; Enhancers; Exhibits; Experimental Autoimmune Encephalomyelitis; Exposure to; G Protein-Coupled Receptor Signaling; G-Protein Signaling Pathway; G-Protein-Coupled Receptors; GTP-Binding Proteins; Gene Cluster; Gene Transfer Techniques; Genes; Genetic; Genetic Polymorphism; Histamine; Histamine H1 Receptors; Hypersensitivity; Immune; Immune response; In Vitro; Inbred Strains Mice; Inflammation Mediators; Inflammatory; Interferons; Interleukin-17; Knowledge; Lead; Ligand Binding; Link; Linkage Disequilibrium; Maps; Mediating; Mitogen-Activated Protein Kinases; Modeling; Molecular Chaperones; Multiple Sclerosis; Mus; Mutation; Pathway interactions; Peptides; Pertussis Toxin; Phenotype; Play; Predisposition; Process; Production; Receptor Activation; Receptor Signaling; Regulation; Resistance; Role; SJL/J Mouse; Signal Pathway; Signal Transduction; Surface; T-Lymphocyte; Transfection; Ursidae Family; central nervous system demyelinating disorder; cytokine; gene cloning; human MAPK14 protein; immune function; in vivo; loss of function; mouse model; mutant; novel; novel therapeutics; oligodendrocyte-myelin glycoprotein; overexpression; protein activation; protein folding; public health relevance; receptor expression; research study; restoration; trafficking

DESCRIPTION (provided by applicant): Histamine is a potent mediator of inflammation and regulator of innate and adaptive immune responses, and autoimmune diseases such as experimental allergic encephalomyelitis (EAE), the principal autoimmune model of multiple sclerosis (MS). EAE is an inflammatory demyelinating disease of the central nervous system with a critical genetic component. We identified Bphs as a susceptibility locus for EAE and positional candidate gene cloning demonstrated Bphs to be the histamine H1 receptor (Hrh1/H1R). As in many autoimmune diseases, CD4+ T cells play a key role in MS and EAE through production of cytokines such as interferon-g and interleukin-17. T cell-specific expression of the susceptible (BphsS/H1RS) allele allowed for EAE development on a H1RKO background, while expression of the resistant (BphsR/H1RR) allele in the same fashion did not. H1R alleles differ by three amino acids in the third intracellular loop (H1RS: Pro263Val312Pro330; H1RR: Leu263Met312Ser330). This domain is associated with signal transduction, but may also be important in protein folding and trafficking, like the intracellular domains of other G-protein coupled receptors (GPCRs). In transfection studies, H1RR surface expression was substantially lower than H1RS, with H1RR exhibiting intracellular retention in the endoplasmic reticulum (ER). In Aim 1 we will dissect the mechanism(s) responsible for the ER-retention of the H1RR allele and whether pharmacological chaperones can restore H1RR surface expression and affect disease in vivo. Pharmacological chaperones restore surface expression and function of polymorphic GPCRs in vitro, and knowledge gained from the proposed in vivo experiments may have broad implications for GPCR-diseases. We also have evidence of a functional, complementary mechanism that can overcome the ER retention process in BphsR mice. Compilation of Hrh1 sequence data and Bphs data from ~100 inbred mouse strains, revealed strains that were phenotypically BphsS, but bear the H1RR allele. Preliminary mapping studies linked the locus to Hrh1 and because it corrects BphsR, we refer to this gene as Bphs-enhancer (Bphse). SJL/J mice exhibit another Hrh1-linked phenotype, spontaneous histamine sensitivity (Shs). We hypothesize that Bphse and Shs reside within a functional linkage disequilibrium (LD)-domain linked to Hrh1 and may contain genes related to H1R folding, trafficking, or signaling. In Aim 2 we will use a forward genetics approach to positionally clone and identify Bphse and Shs. H1R signaling through p38 mitogen activated protein kinase (MAPK) is important in EAE susceptibility. For many GPCRs sustained MAPK activation involves non-G-protein signaling through b-arrestins. Since we found no difference in G-protein activation of allelic H1R-Ga fusion proteins, this suggested that H1R signaling may also involve a novel non-G-protein b-arrestin pathway. Thus in Aim 3 we will examine whether non-G-protein signaling downstream of the H1R is required for EAE development. Non-G-protein signaling by GPCRs is an emerging paradigm and new pharmacologics are being developed to selectively target these pathways. PUBLIC HEALTH RELEVANCE: The function of G-protein coupled receptors (GPCRs) such as the histamine H1 receptor (H1R) can be regulated by their subcellular localization and the signaling pathways they elicit. Proper folding and cell surface expression of GPCRs is required for ligand binding and signaling. Mutations that lead to improper folding and/or intracellular trafficking comprise the largest class of GPCR mutations that result in disease. Our finding that H1R alleles controlling susceptibility to autoimmune disease exhibit differential cell surface expression and altered intracellular trafficking, with the resistant allele being retained within the endoplasmic reticulum, was the first demonstration that polymorphisms influencing GPCR trafficking and cell surface expression can regulate immune functions. Understanding the mechanisms leading to differences in the trafficking and cell surface expression of the H1R alleles, and how they can be manipulated in vivo using this naturally occurring mouse model, will undoubtedly aid in the development of new therapeutic strategies for diseases in which gain- or loss-of-function mutants leading to GPCR misfolding and/or improper intracellular trafficking are implicated.

描述（由申请人提供）：组胺是炎症的有效介质，是先天性和适应性免疫反应以及自身免疫性疾病的调节剂，例如实验性过敏性脑脊髓炎（EAE），多发性硬化症（MS）的主要自身免疫模型。 EAE 是一种中枢神经系统炎症性脱髓鞘疾病，具有重要的遗传因素。我们确定 Bphs 是 EAE 的易感位点，并且位置候选基因克隆证明 Bphs 是组胺 H1 受体 (Hrh1/H1R)。与许多自身免疫性疾病一样，CD4+ T 细胞通过产生干扰素-g 和白介素-17 等细胞因子，在 MS 和 EAE 中发挥关键作用。易感 (BphsS/H1RS) 等位基因的 T 细胞特异性表达允许在 H1RKO 背景下发生 EAE，而以相同方式表达抗性 (BphsR/H1RR) 等位基因则不允许。 H1R 等位基因在第三个细胞内环中存在三个氨基酸差异（H1RS：Pro263Val312Pro330；H1RR：Leu263Met312Ser330）。该结构域与信号转导相关，但在蛋白质折叠和运输中也可能很重要，就像其他 G 蛋白偶联受体 (GPCR) 的胞内结构域一样。在转染研究中，H1RR 表面表达显着低于 H1RS，H1RR 在内质网 (ER) 中表现出细胞内滞留。在目标 1 中，我们将剖析负责 H1RR 等位基因 ER 保留的机制，以及药理学伴侣是否可以恢复 H1RR 表面表达并影响体内疾病。药理学伴侣可在体外恢复多态性 GPCR 的表面表达和功能，并且从拟议的体内实验中获得的知识可能对 GPCR 疾病具有广泛的影响。我们还有证据表明存在一种功能性互补机制，可以克服 BphsR 小鼠的 ER 滞留过程。对约 100 个近交小鼠品系的 Hrh1 序列数据和 Bphs 数据进行汇编，发现这些品系具有 BphsS 表型，但带有 H1RR 等位基因。初步定位研究将该基因座与 Hrh1 联系起来，并且由于它纠正了 BphsR，我们将该基因称为 Bphs 增强子 (Bphse)。 SJL/J 小鼠表现出另一种 Hrh1 相关表型，即自发组胺敏感性 (Shs)。我们假设 Bphse 和 Shs 位于与 Hrh1 连接的功能连锁不平衡 (LD) 结构域内，并且可能包含与 H1R 折叠、运输或信号传导相关的基因。在目标 2 中，我们将使用正向遗传学方法来定位克隆和鉴定 Bphse 和 Shs。通过 p38 丝裂原激活蛋白激酶 (MAPK) 的 H1R 信号传导对于 EAE 易感性非常重要。对于许多 GPCR 来说，持续的 MAPK 激活涉及通过 β-抑制蛋白的非 G 蛋白信号传导。由于我们发现等位基因 H1R-Ga 融合蛋白的 G 蛋白激活没有差异，这表明 H1R 信号传导可能还涉及一种新的非 G 蛋白 b-抑制蛋白途径。因此，在目标 3 中，我们将检查 H1R 下游的非 G 蛋白信号传导是否是 EAE 发展所必需的。 GPCR 的非 G 蛋白信号传导是一种新兴范例，并且正在开发新的药理学来选择性地针对这些途径。 公共卫生相关性：组胺 H1 受体 (H1R) 等 G 蛋白偶联受体 (GPCR) 的功能可通过其亚细胞定位及其引发的信号通路进行调节。 GPCR 的正确折叠和细胞表面表达是配体结合和信号转导所必需的。导致不当折叠和/或细胞内运输的突变构成了导致疾病的最大一类 GPCR 突变。我们发现控制自身免疫性疾病易感性的 H1R 等位基因表现出差异的细胞表面表达和改变的细胞内运输，而抗性等位基因保留在内质网内，这是影响 GPCR 运输和细胞表面表达的多态性可以调节免疫功能的首次证明。了解导致 H1R 等位基因运输和细胞表面表达差异的机制，以及如何使用这种自然发生的小鼠模型在体内操纵它们，无疑将有助于开发新的治疗策略，用于治疗涉及导致 GPCR 错误折叠和/或不当细胞内运输的功能获得或丧失突变体的疾病。