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

总结： 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（MRGPRX 2）。我们 该实验室首次证明宿主防御抗菌肽（HDPs）激活人类肥大细胞 通过MRGPRX 2，这可能有助于先天免疫。新出现的证据表明，MRGPRX 2- 介导的局部肥大细胞活化通过募集中性粒细胞清除细菌感染， 获得性免疫来控制再感染然而，宿主防御的失调和过度生成 LL-37是宿主防御肽中的一种，参与了酒渣鼻的发病机制。MrgprB 2是小鼠 人MRGPRX 2的对应物，我们的初步数据表明，LL-37介导的实验 当与野生型小鼠相比时，MrgprB 2-/-小鼠中的红斑痤疮显著减少。MRGPRX 2还提供 作为神经肽如P物质、血激肽-1和垂体腺苷酸环化酶的新型GPCR， 激活肽（PACAP）。此外，MRGPRX 2是一种“非典型阿片受体”， 阿片类药物的作用可能是通过该受体通过肥大细胞脱粒介导的。然而，分子 参与其下游信号传导的激活和调节的机制在很大程度上仍然未知。 除了G蛋白，许多GPCR激动剂也通过募集衔接蛋白来传递信号 称为β-抑制蛋白。这条通路不仅有助于GPCR脱敏， 用于多种G蛋白非依赖性信号传导。偏性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。激动剂激活两者 这些途径被称为平衡激动剂。最近，我们做了一个令人惊讶的观察， 48/80激活G蛋白和β-抑制蛋白（平衡激动剂），一种血管生成宿主防御肽激活 只有G蛋白，而没有β-arrestin（G蛋白偏向激动剂）。基于这些发现，我们假设， 平衡的和G蛋白偏向的MRGPRX 2激动剂通过不同的机制激活肥大细胞， 促进体内不同的生物反应。因为MrgprB 2是人类的老鼠对应物， MRGPRX 2，我们将在我们的许多体内研究中纳入这种受体。在目标#1中，我们将确定 已知的肥大细胞促分泌素（HDPs、神经肽、阿片类药物和FDA批准的假过敏性 药物）作为MRGPRX 2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簇。在目标#2中，我们将 通过靶向MRGPRX 2/MrgprB 2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置信号传导。在目标#3中，我们将确定β-arrestin 1和β-arrestin 2在 体外MRGPRX 2和MrgprB 2介导的信号传导和体内生物学应答。完成本研究 将提供新的方法来调节MRGPRX 2介导的宿主防御和炎症性疾病。

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.