THROMBIN RECEPTOR-G PROTEIN SIGNALING MECHANISMS

说明凝血酶是在血管损伤时产生的，参与血栓形成、炎症和增殖过程。凝血酶可裂解至少三个异源三聚体G蛋白偶联多肽受体(GPCRs)，称为蛋白酶激活受体(PAR)。PAR1是典型的凝血酶受体，存在于血小板、血管平滑肌细胞、内皮细胞和许多其他类型的细胞上。PAR3和PAR4参与了小鼠和人类的血小板激活。这项研究的目的是了解G蛋白与酵母中表达的PARs之间的相互作用。这些蛋白在酵母中的表达产生了与其他哺乳动物G蛋白和受体分离的特定G蛋白-受体对。这消除了数据被哺乳动物细胞中过多的不同G蛋白污染的担忧。此外，将这些受体偶联到酵母交配途径将使人们能够有效地分析改变配体受体和G蛋白受体相互作用的突变，这些突变是传统哺乳动物细胞方法无法完成的。酵母双杂交系统将用于检测凝血酶受体细胞内环与G蛋白的相互作用，并可能发现与受体环相互作用的其他类型的蛋白质。

DESCRIPTION Thrombin is generated in response to vascular injury, and is involved in thrombotic, inflammatory, and proliferative processes. Thrombin cleaves at least three heterotrimeric G protein-coupled peptide receptors (GPCRs) known as protease-activated receptors (PAR. PAR1 is the prototypical thrombin receptor, and is found on platelets, vascular smooth muscle cells, endothelial cells, and many other cell types. PAR3 and PAR4 are involved in platelet activation in mice and humans. The goal of this research is to gain understanding of the interactions of G proteins with the PARs expressed in yeast. Expression of these proteins in yeast yields a specific G protein-receptor pair in isolation from other mammalian G proteins and receptors. This eliminates concerns of data being contaminate by the plethora of different G proteins present in mammalian cells. In addition, coupling these receptors to the yeast mating pathway will allow efficient analysis of mutations that alter ligand receptor and G protein receptor interactions with cannot be done by conventional mammalian cell methods. The yeast two-hybrid system will be used to examine interactions of the intracellular loops of the thrombin receptor with G proteins, and potentially to discover other types of proteins interacting with receptor loops.