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

基本信息

批准号：
10725107
负责人：
Monica Thapaliya
金额：
$ 2.63万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2022-12-22
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10725107
关键词：
Affinity Allergens Allergic Disease Allergic Reaction Anaphylatoxins Antigens Attenuated Biological Assay Blood Vessels CCL3 gene Cell Degranulation Ciprofloxacin Complement 3a Connective Tissue Data Disease Embryo Family member G protein coupled receptor kinase G-Protein-Coupled Receptors GTP-Binding Proteins Generations Histamine Host Defense Human IgE IgE Receptors Immunity In Vitro Inflammation Inflammatory Inflammatory Response Knockout Mice Knowledge Ligands LoxP-flanked allele Mediating Mediator Membrane Proteins Modality Molecular Mus N-terminal NF-kappa B NFKB Signaling Pathway Neurogenic Inflammation Neuropeptides Pathologic Pathway interactions Peptides Pharmaceutical Preparations Phospholipase Phosphorylation Phosphotransferases Play Process Production Proteins Proto-Oncogene Proteins c-akt Pruritus Reaction Receptor Signaling Regulation Role Route Signal Transduction Substance P Testing Therapeutic Tryptase Tyrosine Phosphorylation United States Food and Drug Administration antimicrobial beta-n-acetylhexosaminidase chemokine cytokine desensitization expectation icatibant in vivo mast cell novel novel strategies overexpression prevent receptor receptor function release of sequestered calcium ion into cytoplasm response

项目摘要

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/项目摘要与MAS MAS相关的G蛋白偶联受体（GPCR）-X2（MRGPRX2）及其小鼠直系同源物MRGPRB2 主要在结缔组织类型肥大细胞中表达，并有助于药物诱导伪铝，非兴奋性瘙痒和神经源性注射。新兴证据表明 MRGPRX2/B2被广泛的阳离子配体激活，但涉及的分子机制它的调节在很大程度上未知。经典的GPCR受到脱敏过程的调节 GPCR激酶（GRK）磷酸化以防止持续信号的有害作用。在特别是该家族的激酶家族中最广泛研究的成员GRK2具有磷酸化的能力 GPCR和非受体底物都与蛋白质伴侣的潜水员库相互作用它在受体脱敏中的功能。最近证明GRK2积极调节FcεRI 并对肥大细胞中的过敏毒素C3A受体（C3AR）信号进行负调节。但是，可能性 GRK2调节肥大细胞中的MRGPRX2/B2信号传导尚未确定。我的期望是 GRK2将使肥大细胞中的mrgprx2响应脱敏，以使其过表达将减弱信号传导和沉默的表达将增强响应。但是，我的初步数据证明了相反的表明，与FCERI有关，GRK2有助于MRGPRX2/B2信号传导在肥大细胞中。根据我意外的发现，我建议测试GRK2促进的新颖假设 MRGPRB2介导的肥大细胞信号在体外，并有助于伪选择性，非高速素药能瘙痒，神经源性感染和体内。因为全球GRK2淘汰小鼠是胚胎致死的，所以我已经产生了具有GRK2的肥大细胞特异性缺失的小鼠。 AIM 1的研究将确定对MRGPRB2的肥大细胞脱粒和细胞因子/趋化因子产生的GRK2删除伪铝（环丙沙星，iCatibant），非基胺能经中实施的配体末端20肽，片段9-20（PAMP9-20）和神经源注射（物质P，SP）。 GRK2通过调节SYK的调节，介导了其对MRGPRB2介导的反应的影响将测试磷脂酶CG，蛋白激酶B（AKT）和NF-KB信号通路。 MRGPRB2介导肥大细胞脱粒（早期响应）负责伪曲和非抗二敏能瘙痒神经源性炎症取决于细胞因子/趋化因子产生（晚期反应）。 AIM 2的研究将检验以下假设：GRK2的肥大细胞特异性缺失调节MRGPRB2介导的伪符号，在体内的非希斯明能瘙痒和神经源注射。对 GRK2调节肥大细胞中MRGPRX2/B2功能的机制可能导致新方法用于调节假性，非基因氨酸瘙痒和神经源性注射。