Adapter molecules on C3a receptor signaling in mast cells

已有文献表明，肥大细胞上高亲和力IgE受体(FceRI)的聚集 过敏和过敏性疾病。毫不奇怪，这种受体一直是密集研究的主题。 调查。肥大细胞也表达细胞表面G蛋白偶联受体(GPCRs) 炎症配体，如补体成分C3a、C5a、神经肽和趋化因子，它们 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 在很大程度上仍未被探索。这可能反映了分离足够数量的组织肥大细胞的困难。 用于生化和功能研究的细胞。此外，小鼠骨髓来源的肥大细胞(BMMCs)， 已被广泛应用于FceRI信号转导研究的GPCRs不能与许多GPCRs反应。 我们的研究主要集中在肥大细胞中的C3a受体信号转导，因为它在 过敏性和炎症性疾病。我们发现，尽管BMMC低水平表达C3aR，但它们 对C3a反应弱。因此，我们使用了两个人类肥大细胞系(HMC-1和LAD2)以及原代 CD34+细胞来源的人肥大细胞培养。我们发现这些肥大细胞内源性表达C3a 受体(C3aR)和对过敏性毒素高度反应的信号和介质释放。嗜碱性 白血病RBL-2H3细胞表达FceRI，但对C3a无反应。相应地，我们发现RBL-2H3细胞 稳定表达的人C3aR对C3a的反应是通过似乎是 与人类肥大细胞中的相同。我们的初步研究揭示了接头分子在 肥大细胞中C3aR信号的调节，肥大细胞不依赖于G蛋白。C3aR在GPC中是独一无二的 在肥大细胞中表达的原因是其羧基末端具有I类PSD-95/DLG/Zo1(PDZ)基序，但其 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，该阵列包含大多数已知的 I类PDZ结构域，我们已经证明这些结构域中只有几个与 C3aR(Ct-C3aR)。基于我们的初步研究，我们假设C3aR与PDZ的相互作用 结构域蛋白为C3a诱导的肥大细胞活化提供了信号平台。在目标1中，我们将 阐明PDZ结构域适配蛋白在肥大细胞C3aR功能调节中的作用。在目标2中， 我们将检验第二个接头分子b-arrestin也与C3aR相关的假设，但它 调节C3aR-PDZ结构域的相互作用以抑制肥大细胞信号转导。在目标3中，我们将调整 体内炎症通过靶向肥大细胞中C3aR-PDZ蛋白的相互作用。总的来说，这些研究将 产生关于C3aR信号如何调节肥大细胞功能的重要新信息，并可能提供新的 治疗过敏性和炎症性疾病的治疗方法。

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.