STRUCTURE & FUNCTION RELATIONSHIP: PROSTANOID RECEPTORS

描述(申请人提供)：该项目的长期目标是了解前列腺素受体如何调节人体生理病理血管和血液动力学过程，包括止血、血栓形成和炎症。前列腺素包括血栓素A2(TXA2)、前列环素12(PGI2)和前列腺素D2(PGD2)、前列腺素E2(PGE2)和前列腺素F2(PGF2)。TXA2是一种强有力的血小板聚集刺激因子和平滑肌收缩因子。PGI2的作用本质上与TXA2相反。PGE2作为一种血管扩张剂或缩血管药，在亚型受体的介导下发挥着不同或相反的功能。TXA2和PGI2也是由它们的受体介导的。所有的受体都属于G蛋白偶联受体家族，有七个跨膜结构域，并与不同的信号偶联。血栓素A2受体(TP)、前列环素受体(IP)和四种亚型前列腺素E_2受体(EP)的结构和功能关系尚不清楚，几乎没有关于前列腺素如何识别它们的受体和通过G蛋白特异地传递信号的结构信息。TXA2、PGI2和PGE2受体介导的不同作用使我们假设，这些受体在其细胞外配体识别位点和细胞内G蛋白偶联位点具有不同的3D结构。最近，我们已经部分鉴定了TP和IP受体胞外区和部分胞内G蛋白偶联区的配体结合口袋。对TP、IP和典型的EP受体的特异性配体识别和G蛋白偶联位置的进一步表征应揭示它们的差异。以下特定目标将利用重组受体突变体、合成肽、圆二色谱和二维核磁共振光谱来鉴定组成TP受体激动剂识别口袋的片段和关键残基，以便与拮抗剂识别口袋进行比较；2.确定IP受体胞外结构域的溶液构象，并确定组成配体识别口袋的片段和关键残基；3.确定人EP3受体胞外结构域的溶液构象，并鉴定组成其特异性配体识别口袋的残基，以与TP和IP受体的溶液构象进行比较；4.确定组成胞内结构域的片段的溶液构象。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand how prostanoid receptors mediate human physiopathological vascular and hemodynamic processes, including hemostasis, thrombosis, and inflammation. Prostanoids, including thromboxane A2 (TXA2), prostacyclin 12 (PGI2) and prostaglandin D2 (PGD2), E2 (PGE2) and F2 (PGF2) are synthesized by vascular smooth muscle, endothelium, and other tissues. TXA2 is a potent stimulator of platelet aggregation and a smooth muscle constrictor. PGI2 actions are essentially opposite to those of TXA2. PGE2 plays diverse/opposite functions as a vasodilator or vasoconstrictor based on the mediations of the subtype receptors. TXA2 and PGI2 are also mediated by their receptors. All the receptors belong to the G protein-coupled receptor family, with seven transmembrane domains, and are coupled to different signaling. The structure and function relationships of the TXA2 receptor (TP), the PGI2 receptor (IP) and four subtype PGE2 receptors (EP) are poorly defined, with little structural information on how the prostanoids recognize their receptors and signaling through G proteins specifically. The diverse receptor-mediated actions of TXA2, PGI2, and PGE2 have led us to hypothesize that the receptors have distinct 3D structures in their extracellular ligand recognition sites, and in their intracellular G protein-coupled sites. Recently, we have partially characterized the ligand-binding pockets in the extracellular domains, and parts of the intracellular G protein coupling domains of the TP and IP receptors. Further characterization of the specific ligand recognition and G protein coupling sites of the TP, IP, and a typical EP receptor should reveal their differences. The following specific aims will use recombinant receptor mutants, synthetic peptides, circular dichroism and 2D nuclear magnetic resonance spectroscopy to: 1. Identify the segments and key residues comprising the TP receptor agonist recognition pocket for comparison with the antagonist recognition pocket; 2. Determine the solution conformation of the extracellular domains of the IP receptor and define the segments and key residues making up the ligand recognition pocket; 3. Determine the solution conformation of the extracellular domains in the human EP3 receptor, and identify the residues making up its specific ligand recognition pocket for comparison with those of the TP and IP receptors; 4. Determine the solution conformation of segments comprising the intracellular domains.