Functional Analysis of Mrgpr Family in Itch Sensation

描述(由申请人提供)：我们研究的目标是了解组胺非依赖性瘙痒的细胞和分子机制。背根神经节(DRG)的初级感觉神经元通过皮肤中的外周轴突检测瘙痒刺激，并通过其中枢轴突将信号发送到脊髓，从而在瘙痒的产生中发挥重要作用。最具特点的瘙痒媒介是组胺。然而，抗组胺药物在大多数瘙痒情况下都无效，这表明参与了组胺非依赖性途径。理解组胺非依赖性瘙痒的主要障碍是缺乏标记DRG中瘙痒敏感神经元的分子标记物，以及由组胺以外的瘙痒刺激直接激活的细胞表面受体。最近，我们发现了一种G蛋白偶联受体MRGPrs家族，它特异性地表达在一小部分DRG神经元中，作为抗疟疾药物氯喹的受体发挥作用，并且是氯喹引起的瘙痒所必需的。除氯喹外，表达mRgprs和mrgpr的DRG神经元对其他几种致痒化合物也有反应，提示mRgprs是直接感受这些化合物的新型瘙痒受体，表达这些受体的神经元转导瘙痒信号。在这项研究中，我们将采用分子、遗传学、行为学和电生理学的方法来分析瘙痒中MRGPR的功能和表达MRGPR的DRG神经元的特性。我们的初步数据表明，MRGPrs介导了蛋白酶激活受体2(PAR2)的多肽激动剂SLIGRL引起的瘙痒。这一令人惊讶的结果挑战了传统的观念，即PAR2在DRG中作为瘙痒受体介导蛋白酶诱导的瘙痒。目的I验证在瘙痒信号转导中，MRGPrs作为PAR2裂解N末端的受体的假说。我们最近发现了一种小分子化合物，它可以在异种细胞中通过包括氯喹和SLIGRL在内的几种致痒化合物特异性地抑制人和小鼠MRGPR的激活。在AIM II中，我们将确定该拮抗剂的治疗是否可以阻断氯喹和SLIGRL诱导的小鼠神经和行为反应。我们的细胞分析表明，表达mrgpr的神经元是瘙痒敏感神经元。在目标III中，我们将使用我们新建立的转基因小鼠系，在其中用GFP-Cre标记表达MRGPR的DRG神经元，以研究这些神经元在外周和脊髓中的电生理特性和轴突投射。对mrgprs和mrgpr表达神经元的功能分析将有助于深入了解瘙痒的关键机制，并为新的瘙痒疗法的开发打开大门。

DESCRIPTION (provided by applicant): The goal of our research is to understand the cellular and molecular mechanisms of histamine-independent itch. Primary sensory neurons in dorsal root ganglia (DRG) play an essential role in generating itch by detecting itch stimuli through their peripheral axons in the skin and sending the signals to the spinal cord via their central axons. The best characterized itch mediator is histamine. However, anti-histamine drugs are ineffective in most itch conditions suggesting the involvement of histamine- independent pathways. The major hurdle in understanding histamine-independent itch is the dearth of molecular markers that label itch-sensitive neurons in DRG and cell surface receptors directly activated by itch stimuli other than histamine. Recently, we have shown that Mrgprs, a family of G protein-coupled receptors specifically expressed in a small subset of DRG neurons, function as receptors for the anti- malaria drug chloroquine and are required for chloroquine-induced itch. Besides chloroquine, Mrgprs and Mrgpr-expressing DRG neurons also respond to several other itch-inducing compounds suggesting that Mrgprs are novel itch receptors by directly sensing these compounds and that neurons expressing these receptors transduce itch signals. In this proposal, we will take molecular, genetic, behavioral, and electrophysiological approaches to dissect the functions of Mrgprs and properties of Mrgpr-expressing DRG neurons in itch. Our preliminary data show that Mrgprs mediate itch induced by SLIGRL, a peptide agonist for protease-activated receptor 2 (PAR2). This surprising result challenges the traditional notion that PAR2 functions as the itch receptor in DRG to mediate protease-induced itch. Aim I is to test the hypothesis that Mrgprs function as receptors for the cleaved N-terminus of PAR2 generated by proteases in itch signaling. We have recently identified a small molecule compound that can specifically inhibit human and mouse Mrgpr activation by several itch-inducing compounds including chloroquine and SLIGRL in heterologous cells. In Aim II, we will determine whether treatment with the antagonist can block chloroquine- and SLIGRL-induced neuronal and behavioral responses in mice. Our cellular analyses suggest that Mrgpr-expressing neurons are itch-sensitive neurons. In Aim III, we will use our newly generated transgenic mouse lines in which Mrgpr-expressing DRG neurons are labeled by GFP-Cre to study the electrophysiological properties and axonal projections of these neurons in the periphery and the spinal cord. Functional analysis of Mrgprs and Mrgpr-expressing neurons will provide insight into key mechanisms of itch as well as open the door for the development of novel itch therapeutics.