Roles of novel MRGPRX2/MrgprB2 signaling in mast cells on host defense and Inflammation

摘要： 除了高亲和力 IgE 受体 (FcεRI) 外，人结缔组织肥大细胞 (MCTC) 还表达 最近描述的 G 蛋白偶联受体 (GPCR)，称为 Mas 相关 GPCR-X2 (MRGPRX2)。我们的 实验室第一个证明宿主防御抗菌肽（HDP）激活人类肥大细胞 通过 MRGPRX2，这可能有助于先天免疫。新出现的证据表明 MRGPRX2- 介导的局部肥大细胞激活通过招募中性粒细胞清除细菌感染并促进 控制再感染的适应性免疫。然而，宿主防御失调和过度生成 作为宿主防御肽的一部分，LL-37 有助于红斑痤疮的发病机制。 MrgprB2 是鼠标 人类 MRGPRX2 的对应物和我们的初步数据表明 LL-37 介导的实验 与野生型小鼠相比，MrgprB2-/- 小鼠的红斑痤疮显着减少。 MRGPRX2 还服务 作为一种新型 GPCR，适用于 P 物质、血激肽-1 和垂体腺苷酸环化酶等神经肽 激活肽（PACAP）。此外，MRGPRX2 是一种“非典型阿片受体”，并且有一些副作用 阿片类药物的作用可能是通过该受体通过肥大细胞脱颗粒介导的。然而，分子 其下游信号传导的激活和调节所涉及的机制仍然很大程度上未知。 除了 G 蛋白外，许多 GPCR 激动剂还通过招募接头蛋白来发出信号 称为β-抑制蛋白。该通路不仅有助于GPCR脱敏，而且提供了一个平台 用于多种不依赖于 G 蛋白的信号传导。偏向的 GPCR 激动剂优先激活通路 由G蛋白（G蛋白偏向）或β-抑制蛋白（β-抑制蛋白偏向）介导。激活两者的激动剂 途径被称为平衡激动剂。最近，我们做出了令人惊讶的观察，虽然复合 48/80 激活 G 蛋白和 β-arrestin（平衡激动剂），血管生成宿主防御肽激活 仅 G 蛋白，但不包括 β-arrestin（G 蛋白偏向激动剂）。根据这些发现，我们假设 平衡和 G 蛋白偏向 MRGPRX2 激动剂通过不同机制激活肥大细胞 促进体内不同的生物反应。因为 MrgprB2 是人类的小鼠对应物 MRGPRX2，我们将在许多体内研究中纳入该受体。在目标#1中，我们将确定哪个 已知的肥大细胞促分泌剂（HDP、神经肽、阿片类药物和 FDA 批准的假过敏药物） 药物）作为 MRGPRX2 的平衡和 G 蛋白偏向激动剂。我们还将确定结构 受体上促进其与 G 蛋白和 β-抑制蛋白偶联的决定因素。在目标#2中，我们将 通过靶向 MRGPRX2/MrgprB2 的平衡/G 蛋白调节宿主防御、假性过敏和红斑痤疮 体内肥大细胞中的偏向信号传导。在目标#3中，我们将确定 β-arrestin1 和 β-arrestin2 在 MRGPRX2 和 MrgprB2 介导的体外信号传导和体内生物反应。完成本研究 将提供调节 MRGPRX2 介导的宿主防御和炎症疾病的新方法。

Summary: In addition to the high affinity IgE receptor (FcεRI), human connective tissue mast cells (MCTC) express a recently described G protein coupled receptor (GPCR), known as Mas-related GPCR-X2 (MRGPRX2). Our lab was the first to demonstrate that host defense antimicrobial peptides (HDPs) activate human mast cells via MRGPRX2, which likely contributes to innate immunity. Emerging evidence suggests that MRGPRX2- mediated local mast cell activation clears bacterial infection via the recruitment of neutrophils and promotes adaptive immunity to control reinfection. However, dysregulation of host defense and excessive generation of the host defense peptide, LL-37 contributes to the pathogenesis of rosacea. MrgprB2 is the mouse counterpart of human MRGPRX2 and our preliminary data demonstrated that LL-37-mediated experimental rosacea is significantly reduced in MrgprB2-/- mice when compared to wild-type mice. MRGPRX2 also serves as a novel GPCR for neuropeptides such as substance P, hemokinin-1 and pituitary adenylate cyclase- activating peptide (PACAP). Furthermore, MRGPRX2 is an “atypical opioid receptor” and some of the side effects opioids are likely mediated via mast cell degranulation through this receptor. However, the molecular mechanisms involved in the activation and regulation of its downstream signaling remains largely unknown. In addition to G proteins, many GPCR agonists also signal via the recruitment of adapter proteins known as β-arrestins. This pathway not only contributes to GPCR desensitization but also provides a platform for a variety of G protein-independent signaling. Biased GPCR agonists preferentially activate pathways mediated by G proteins (G protein-biased) or β-arrestins (β-arrestin-biased). Agonists that activate both pathways are known as balanced agonists. Recently, we made the surprising observation that while compound 48/80 activates both G protein and β-arrestin (balanced agonist) an angiogenic host defense peptide activates only G protein but not β-arrestin (G protein-biased agonist). Based on these findings, we hypothesize that balanced and G protein-biased MRGPRX2 agonists activate mast cells via different mechanisms to promote distinct biological responses in vivo. Because MrgprB2 is the mouse counterpart of the human MRGPRX2, we will incorporate this receptor for many of our in vivo studies. In aim #1, we will determine which of the known mast cell secretagogues (HDPs, neuropeptides, opioids and FDA-approved pseudo-allergic drugs) act as balanced and G protein-biased agonists for MRGPRX2. We will also identify the structural determinants on the receptor that facilitate their coupling to G protein and β-arrestins. In aim #2, we will modulate host defense, pseudo-allergy and rosacea by targeting MRGPRX2/MrgprB2’s balanced/G protein biased signaling in mast cells in vivo. In aim #3, we will determine the roles of β-arrestin1 and β-arrestin2 on MRGPRX2 and MrgprB2-mediated signaling in vitro and biological responses in vivo. Completion of this study will provide novel approaches to modulate MRGPRX2-mediated host defense and inflammatory diseases.