Adapter molecules on C3a receptor signaling in mast cells

肥大细胞中 C3a 受体信号传导的衔接分子

• 批准号: 7929958
• 负责人: Hydar Ali
• 金额: $ 35.81万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-14 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7929958
• 关键词: Affinity; Allergens; Allergic; Anaphylatoxins; Arrestins; Artificial skin; Basophilic leukemia; Binding; Biochemical; Bone Marrow; CD34 gene; Cell Culture Techniques; Cell Degranulation; Cell Line; Cell physiology; Cell surface; Cells; Complement; Complement 3a; Complement 5a; Cutaneous; Disease; G-Protein-Coupled Receptors; GTP-Binding Proteins; Human; Hypersensitivity; IgE Receptors; Inflammation; Inflammatory; Investigation; Ligands; Lung; Lung Inflammation; Mediating; Mediator of activation protein; Modeling; Mus; Neuropeptides; Play; Proteins; Proteomics; Receptor Signaling; Regulation; Research; Role; Signal Pathway; Signal Transduction; Tertiary Protein Structure; Testing; Tissues; Vascular Permeabilities; adapter protein; base; chemokine; in vivo; mast cell; mutant; novel therapeutic intervention; receptor; receptor expression; receptor function; reconstitution; response; transcription factor

It is well documented that aggregation of high affinity IgE receptors (FceRI) on mast cells mediates allergic and hypersensitivity diseases. Not surprisingly, this receptor has been the subject of intense research investigation. Mast cells also express cell surface G protein coupled receptors (GPCRs) for a number of inflammatory ligands such as complement components C3a, C5a, neuropeptides, and chemokines, which modulate allergic and inflammatory diseases. Surprisingly, however, regulation of GPCR function in mast cells remains largely unexplored. This probably reflects the difficulty in isolating sufficient numbers of tissue mast cells for biochemical and functional studies. Furthermore, murine bone marrow-derived mast cells (BMMCs), which have been extensively used for studies on FceRI signaling, do not respond to many of the GPCR ligands. Our studies have focused on C3a receptor signaling in mast cells because of its demonstrated importance in allergic and inflammatory diseases. We found that although BMMCs express C3aR at low levels, they are weakly responsive to C3a. We therefore used two human mast cell lines (HMC-1 and LAD2) as well as primary human mast cell cultures derived from CD34+ cells. We found that these mast cells endogenously express C3a receptors (C3aR) and are highly responsive to the anaphylatoxin for signaling and mediator release. Basophilic leukemia RBL-2H3 cells express FceRI but are unresponsive to C3a. Accordingly, we found that RBL-2H3 cells stably expressing human C3aR respond to C3a for mediator release via signaling pathways that appears to be identical to that in human mast cells. Our preliminary studies revealed new roles of adapter molecules on the regulation of C3aR signaling in mast cells, which are G protein independent. C3aR is unique among GPCRs expressed in mast cells in that its carboxyl terminus possesses a class I PSD-95/Dlg/Zo1 (PDZ) motif but its role in receptor regulation is unknown. Using a newly developed proteomic array containing most of the known class I PDZ domains, we have shown that only a few of these domains associate with carboxyl terminus of C3aR (Ct-C3aR). Based on our preliminary studies, we hypothesize that interaction of C3aR with PDZ domain proteins provides a signaling platform for C3a-induced mast cell activation. In aim #1, we will delineate the role of PDZ domain adapter proteins on the regulation of C3aR function in mast cells. In aim #2, we will test the hypothesis that a second adapter molecule, b-arrestin also associates with C3aR but it modulates C3aR-PDZ domain interaction to inhibit mast cell signaling. In aim #3, we will modulate inflammation in vivo by targeting C3aR-PDZ protein interaction in mast cells. Collectively, these studies will generate significant new information on how C3aR signaling modulates mast cell function and may offer novel therapeutic approaches for the treatment of allergic and inflammatory diseases.

有充分证据表明，肥大细胞上高亲和力IgE受体（FceRI）的聚集介导了 过敏性和超敏性疾病。毫不奇怪，这种受体一直是深入研究的对象 调查肥大细胞还表达细胞表面G蛋白偶联受体（GPCR），用于多种免疫调节。 炎性配体如补体成分C3 a、C5 a、神经肽和趋化因子， 调节过敏性和炎症性疾病。然而，令人惊讶的是，肥大细胞中GPCR功能的调节 大部分尚未开发。这可能反映了分离足够数量的组织肥大细胞的困难 用于生物化学和功能研究的细胞。此外，鼠骨髓来源的肥大细胞（BMMC）， 其已广泛用于FceRI信号传导的研究，但不应答许多GPCR配体。 我们的研究集中在肥大细胞中的C3 a受体信号传导，因为它在肥大细胞中的重要性已被证明。 过敏性和炎症性疾病。我们发现，尽管BMMCs表达C3 aR的水平较低， 对C3 a反应弱。因此，我们使用了两种人肥大细胞系（HMC-1和LAD 2）以及原代肥大细胞。 来源于CD 34+细胞的人肥大细胞培养物。我们发现这些肥大细胞内源性表达C3 a， 受体（C3 aR），并高度响应过敏毒素的信号和介质释放。嗜碱性 白血病RBL-2 H3细胞表达FceRI，但对C3 a无应答。因此，我们发现RBL-2 H3细胞 稳定表达的人C3 aR通过信号通路响应C3 a释放介质， 与人肥大细胞中的相同。我们的初步研究揭示了衔接子分子在细胞凋亡中的新作用。 调节肥大细胞中的C3 aR信号传导，其是G蛋白非依赖性的。C3 aR在GPCR中是独特的 在肥大细胞中表达，因为其羧基末端具有I类PSD-95/Dlg/Zo 1（PDZ）基序，但其 在受体调节中的作用尚不清楚。使用一种新开发的蛋白质组阵列， I类PDZ结构域，我们已经表明，只有少数这些结构域与羧基末端的 C3aR（Ct-C3aR）。基于我们的初步研究，我们假设C3 aR与PDZ的相互作用 结构域蛋白为C3 a诱导的肥大细胞活化提供了信号平台。在目标#1中，我们将 描述PDZ结构域衔接蛋白对肥大细胞中C3 aR功能调节的作用。在目标#2中， 我们将检验第二个衔接分子b-抑制蛋白也与C3 aR相关，但它 调节C3 aR-PDZ结构域相互作用以抑制肥大细胞信号传导。 在目标#3中，我们将调整 通过靶向肥大细胞中的C3 aR-PDZ蛋白相互作用在体内抑制炎症。这些研究将 产生关于C3 aR信号传导如何调节肥大细胞功能的重要新信息，并可能提供新的 用于治疗过敏性和炎性疾病的治疗方法。