The Structure and Function of the CRTH2 PDG2 Receptor

描述(由申请人提供)： 前列腺素D2(PGD2)是一种主要的环氧合酶代谢物，其释放被认为与哮喘的炎症和气道高反应性增加有关。PGD2似乎部分通过最近克隆的名为CRTH2的GPCR发挥作用。CRTH2的表达模式与该受体在变态反应性疾病发病机制中的重要作用相一致。这一建议的主要假设是，免疫炎症反应中关键的PGD2诱导的变化至少部分是由CRTH2诱导的反应介导的。我们进一步认为，PGD2代谢产物包括PGJ2和15d PGJ2的部分作用是通过CRTH2受体介导的。为了验证这一假设，我们在特定目标1中提出了定义小鼠CRTH2受体的药理学。我们将在细胞培养中表达CRTH2受体，并确定其配体结合和信号转导特性。在特定的目标2中，我们将通过定点突变来鉴定对配体结合和信号转导至关重要的氨基酸残基。以建模和分子动力学模拟为指导，我们将在CRTH2受体中引入点突变，并在细胞培养系统中表达突变受体。在具体目标3中，我们将探索PGD2是否通过CRTH2受体调节哮喘小鼠模型的炎症效应和/或支气管收缩。我们将在CRTH2基因敲除小鼠中分析卵清蛋白(OVA)过敏性致敏作用，这是一种TH2型免疫模型，并将其与DP缺失动物进行比较。我们进一步建议将CRTH2缺失的小鼠与DP缺失的小鼠杂交，以测试两种PGD2受体缺失的效果。这些研究的完成将有助于阐明PGD2在过敏性呼吸道疾病细胞迁移和渗透中的生理学作用，并将为了解该受体在过敏性炎症小鼠模型中的作用提供洞察力。

DESCRIPTION (provided by applicant): Prostaglandin D2 (PGD2) is a major cyclooxygenase metabolite, and its release has been hypothesized to contribute to the inflammation and increased airway hyperreactivity observed in asthma. PGD2 appears to act in part through a recently cloned GPCR designated CRTH2. CRTH2 mRNA expression pattern is consistent with an important role for this receptor in the etiology of allergic disease. The principal hypothesis of this proposal is that critical PGD2 evoked changes in the immune inflammatory response are mediated, at least in part, by CRTH2-evoked responses. We further propose that some of the effects of PGD2 metabolites including PGJ2 and 15d PGJ2 are mediated via the CRTH2 receptor. To test this hypothesis we propose in Specific Aim 1 to define the pharmacology of the mouse CRTH2 receptor. We will express the CRTH2 receptor in cell culture and determine its ligand binding and signal transduction properties. In Specific Aim 2 we will identify amino acid residues critical for ligand binding and signal transduction by site directed mutagenesis. Using modeling and molecular dynamics simulations as a guide, we will introduce point mutations into the CRTH2 receptor, and express mutant receptors in cell culture systems. In Specific Aim 3 we will explore whether PGD2 modulates inflammatory effects and/or bronchoconstriction in mouse models of asthma via the CRTH2 receptor. We will analyze ovalbumin (OVA) allergic sensitization, a TH2 type immune model, in CRTH2 knockout mice and compare this with DP null animals. We further propose to cross the CRTH2 null mice with DP null mice to test the effect of loss of both PGD2 receptors. Completion of these studies should aid in the elucidation of the physiologic role of PGD2 in cellular migration and infiltration in allergic airway disease and will provide insight as to the role of this receptor in mouse models of allergic inflammation.