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/项目总结 人Mass相关G蛋白偶联受体（GPCR）-X2（MRGPRX 2）及其小鼠直向同源物MrgprB 2 主要在结缔组织型肥大细胞中表达， 假性变态反应、非组胺能瘙痒和神经源性炎症。新出现的证据表明 MRGPRX 2/B2被广泛的阳离子配体激活，但涉及MRGPRX 2/B2的分子机制是不确定的。 其规则在很大程度上是未知。经典的GPCR受脱敏过程的调节， 通过GPCR激酶（GRKs）的磷酸化来防止持续信号传导的有害作用。在 特别地，GRK 2是该激酶家族中研究最广泛的成员，具有磷酸化 GPCR和非受体底物，并与多种蛋白质伴侣相互作用， 它在受体脱敏中的作用。最近研究表明GRK 2正调控FcεRI 并负调节肥大细胞中的过敏毒素C3 a受体（C3 aR）信号传导。然而， GRK 2调节肥大细胞中的MRGPRX 2/B2信号传导尚未确定。我的期望是 GRK 2将用于使肥大细胞中的MRGPRX 2应答脱敏，使得其过表达将 减弱信号传导，并且沉默其表达将增强应答。然而，我的初步 数据显示相反的情况，表明对于FceRI，GRK 2有助于MRGPRX 2/B2信号传导 在肥大细胞中。基于我意想不到的发现，我建议测试GRK 2促进 体外MrgprB 2介导的肥大细胞信号传导并导致假性过敏，非组胺能 瘙痒、神经源性炎症和体内。因为GRK 2基因敲除小鼠是胚胎致死的， 已经产生了肥大细胞特异性缺失GRK 2的小鼠。目标1中的研究将确定 GRK 2缺失对肥大细胞脱粒和响应于MrgprB 2的细胞因子/趋化因子产生的影响 与假性变态反应有关的配体（环丙沙星、艾替班特）、非组胺能瘙痒（肾上腺髓质素前体N- 末端20肽，片段9-20（PAMP 9 -20）和神经源性炎症（P物质，（SP））。 假设GRK 2通过调节Syk介导其对MrgprB 2介导的应答的作用， 将测试磷脂酶Cg、蛋白激酶B（Akt）和NF-κ B信号传导途径。MrgprB 2介导 肥大细胞脱粒（早期反应）是假性变态反应和非组胺能性瘙痒的原因， 神经源性炎症取决于细胞因子/趋化因子的产生（迟发反应）。研究目的2 将检验GRK 2的肥大细胞特异性缺失调节MrgprB 2介导的假变态反应的假设， 体内非组胺能瘙痒和神经源性炎症。全面了解 GRK 2调节肥大细胞中MRGPRX 2/B2功能的机制可能会导致新的方法 用于调节假性变态反应、非组胺能瘙痒和神经源性炎症。