STRUCTURE & FUNCTION RELATIONSHIP: PROSTANOID RECEPTORS

描述（由申请人提供）：该项目的长期目标是了解前列腺素受体如何介导人体血管和血流动力学的生理病理过程，包括止血、血栓形成和炎症。类前列腺素包括血栓素A2 （TXA2）、前列腺素12 （PGI2）和前列腺素D2 （PGD2）、E2 （PGE2）、F2 （PGF2）等由血管平滑肌、内皮等组织合成。TXA2是一种有效的血小板聚集刺激剂和平滑肌收缩剂。PGI2的作用与TXA2的作用基本相反。基于亚型受体的介导，PGE2作为血管舒张剂或血管收缩剂发挥不同/相反的功能。TXA2和PGI2也由它们的受体介导。所有受体都属于G蛋白偶联受体家族，具有7个跨膜结构域，并偶联到不同的信号。TXA2受体（TP）、PGI2受体（IP）和四种亚型PGE2受体（EP）的结构和功能关系尚不明确，关于前列腺素如何识别其受体和通过G蛋白特异性信号传导的结构信息很少。TXA2、PGI2和PGE2的不同受体介导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，我们对TP和IP受体的胞外结构域和胞内G蛋白偶联结构域的部分配体结合袋进行了部分表征。进一步表征TP、IP和一种典型EP受体的特异性配体识别和G蛋白偶联位点，将揭示它们之间的差异。以下具体目的将利用重组受体突变体、合成多肽、圆二色性和二维核磁共振波谱来：鉴定构成TP受体激动剂识别袋的片段和关键残基，以便与拮抗剂识别袋进行比较；2. 确定IP受体胞外结构域的溶液构象，确定构成配体识别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，并鉴定构成其特异性配体识别袋的残基，以便与TP和IP受体进行比较；4. 确定组成细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.