Novel Roles of G-Protein-Coupled Receptor Kinase-2 (GRK2) on MrgprB2-mediated Mast Cell Response

G 蛋白偶联受体激酶 2 (GRK2) 对 MrgprB2 介导的肥大细胞反应的新作用

• 批准号: 10151986
• 负责人: Monica Thapaliya
• 金额: $ 4.6万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10151986
• 关键词: Novel; Roles; Protein; Coupled; Receptor

ABSTRACT/PROJECT SUMMARY Human Mas-related G protein coupled receptor (GPCR)-X2 (MRGPRX2) and its mouse orthologue MrgprB2 are predominantly expressed in connective tissue type mast cells and contribute to drug-induced pseudoallergy, non-histaminergic itch and neurogenic inflammation. Emerging evidence suggests that MRGPRX2/B2 is activated by a wide spectrum of cationic ligands but the molecular mechanisms involved in its regulation are largely unknown. Classical GPCRs are regulated by a process of desensitization via phosphorylation by GPCR kinases (GRKs) to prevent the detrimental effects of sustained signaling. In particular, GRK2, the most widely studied member of this family of kinases, has the ability to phosphorylate both GPCRs and non-receptor substrates and interact with a diverse repertoire of protein partners in addition to its function in receptor desensitization. It was recently demonstrated that GRK2 positively regulates FcεRI and negatively regulates anaphylatoxin C3a receptor (C3aR) signaling in mast cells. However, the possibility that GRK2 modulates MRGPRX2/B2 signaling in mast cells has yet to be determined. My expectation was that GRK2 would serve to desensitize MRGPRX2 responses in mast cells such that its overexpression would attenuate signaling and that silencing its expression would enhance the response. However, my preliminary data demonstrated the opposite suggesting that as for FceRI, GRK2 contributes to MRGPRX2/B2 signaling in mast cells. Based on my unexpected findings, I propose to test the novel hypothesis that GRK2 promotes MrgprB2-mediated mast cell signaling in vitro and contributes to pseudoallergy, non-histaminergic itch, neurogenic inflammation and in vivo. Because global GRK2 knockout mice are embryonic lethal, I have generated mice with mast cell-specific deletion of GRK2. Studies in Aim 1 will determine the effects of GRK2-deletion on mast cell degranulation and cytokine/chemokine generation in response to MrgprB2 ligands implicated in pseudoallergy (Ciprofloxacin, Icatibant), non-histaminergic itch (Proadrenomedullin N- terminal 20 peptide, fragment 9-20 (PAMP9-20) and neurogenic inflammation (Substance P, (SP)).The hypothesis that GRK2 mediates its effect on MrgprB2-mediated responses via the modulation of Syk, phospholipase Cg, protein kinase B (Akt) and NF-kB signaling pathways will be tested. MrgprB2-mediated mast cell degranulation (early response) is responsible for pseudoallergy and non-histaminergic itch whereas neurogenic inflammation depends on the cytokine/chemokine generation (late response). Studies in Aim 2 will test the hypothesis that mast cell-specific deletion of GRK2 modulate MrgprB2-mediated pseudoallergy, non-histaminergic itch and neurogenic inflammation in vivo. A comprehensive understanding of the mechanism via which GRK2 regulates MRGPRX2/B2 function in mast cells may lead to novel approaches for modulating pseudoallergy, non-histaminergic itch and neurogenic inflammation.

摘要/项目摘要 人Mas相关G蛋白偶联受体-X2(MRGPRX2)及其小鼠同源基因MRgprB2 主要在结缔组织类型的肥大细胞中表达，并有助于药物诱导 假性过敏、非组胺能瘙痒和神经源性炎症。新出现的证据表明 MRGPRX2/B2被广泛的阳离子配体激活，但涉及的分子机制包括 它的监管在很大程度上是未知的。经典的GPCR受脱敏过程的调节，通过 被GPCRK(GRKs)磷酸化，以防止持续信号的有害影响。在……里面 特别是，GRK2，这一家族中研究最广泛的成员，具有磷酸化的能力 GPCRs和非受体底物，并与不同的蛋白质伙伴相互作用 它在受体脱敏中的作用。最近的研究表明，GRK2正向调节FcεRI 负向调节肥大细胞中的过敏性毒素C3a受体(C3aR)信号。然而，这种可能性 GRK2是否调节肥大细胞中的MRGPRX2/B2信号尚不确定。我的期望是 GRK2将用于使肥大细胞中MRGPRX2反应脱敏，从而其过表达将 减弱信号，沉默其表达将增强反应。不过，我的初选 数据表明，对于FceRI，GRK2参与了MRGPRX2/B2信号转导 在肥大细胞中。基于我意想不到的发现，我提议测试GRK2促进的新假设 MRgprB2介导的肥大细胞信号转导与假变态反应、非组胺能 瘙痒、神经源性炎症和活体内。因为全球GRK2基因敲除小鼠是胚胎致死的，所以我 已经产生了肥大细胞特异性GRK2缺失的小鼠。目标1中的研究将确定 MRgprB2对肥大细胞脱颗粒和细胞因子/趋化因子产生的影响 与假性过敏(环丙沙星，icatibant)、非组胺能瘙痒(肾上腺髓质素原N- 末端20肽，片段9-20(PAMP9-20)与神经源性炎症(P物质，(SP)) 假设GRK2通过调节Syk来调节其对MRgprB2介导的反应的影响， 将测试磷脂酶CG、蛋白激酶B(Akt)和核因子-kB信号通路。MrgprB2介导的 肥大细胞脱颗粒(早期反应)是假性过敏和非组胺能瘙痒的原因，而 神经源性炎症依赖于细胞因子/趋化因子的产生(晚期反应)。AIM 2中的研究 将测试肥大细胞特异性的GRK2缺失调节MRgprB2介导的假过敏的假设， 体内非组胺能瘙痒和神经源性炎症。全面了解 GRK2调节肥大细胞MRGPRX2/B2功能的机制可能导致新的途径 用于调节假性过敏、非组胺能瘙痒和神经源性炎症。