Lerner D J, Chen M, Tram T, Coughlin S R
Cardiovascular Research Institute, Department of Medicine, and Daiichi Research Center, University of California, San Francisco, California 94143, USA.
J Biol Chem. 1996 Jun 14;271(24):13943-7.
Thrombin receptor and proteinase-activated receptor 2 (PAR2) define a family of G protein-coupled receptors that are activated by a novel proteolytic mechanism. Specific cleavage of their amino-terminal exodomains unmasks a new amino terminus which then serves as a tethered ligand, docking intramolecularly to the body of the receptor to effect signaling. Identification of the docking interactions between tethered ligand domain and receptor is critical for understanding transmembrane signaling by these receptors. Synthetic "agonist peptides" that mimic the tethered ligand domains of thrombin receptor and PAR2 act as agonists at their respective receptors. Toward defining the docking interactions which mediate receptor activation, we determined the specificity of the thrombin receptor and PAR2 for their respective agonist peptides and used receptor chimeras to identify the receptor domains responsible for such specificity. PAR2 responded to both thrombin receptor and PAR2 agonist peptides. In contrast, thrombin receptor was selective for its own agonist peptide. Substitution of the extracellular face of PAR2, its amino-terminal exodomain and three extracellular loops, for the cognate thrombin receptor structures yielded a chimeric receptor with PAR2-like agonist specificity. Substitution of individual extracellular domains revealed that the primary determinant of agonist specificity was extracellular loop 2. Strikingly, substitution of either the amino-terminal exodomain or third extracellular loop alone caused marked loss of receptor function, but the double substitution yielded a functional receptor. Thus, the extracellular domains of these G protein-coupled receptors are more than simply passive links between transmembrane domains. They participate in agonist recognition and must interact, directly or indirectly, for proper receptor function.
凝血酶受体和蛋白酶激活受体2（PAR2）定义了一类G蛋白偶联受体，它们通过一种新型蛋白水解机制被激活。其氨基末端胞外域的特异性切割会暴露出一个新的氨基末端，该末端随后作为一个拴系配体，在分子内与受体主体对接以实现信号传导。确定拴系配体结构域与受体之间的对接相互作用对于理解这些受体的跨膜信号传导至关重要。模拟凝血酶受体和PAR2拴系配体结构域的合成“激动剂肽”在各自的受体上充当激动剂。为了确定介导受体激活的对接相互作用，我们确定了凝血酶受体和PAR2对其各自激动剂肽的特异性，并使用受体嵌合体来鉴定负责这种特异性的受体结构域。PAR2对凝血酶受体和PAR2激动剂肽均有反应。相比之下，凝血酶受体对其自身的激动剂肽具有选择性。将PAR2的细胞外表面、其氨基末端胞外域和三个细胞外环替换为同源的凝血酶受体结构，产生了一种具有PAR2样激动剂特异性的嵌合受体。对单个细胞外结构域的替换表明，激动剂特异性的主要决定因素是细胞外环2。令人惊讶的是，单独替换氨基末端胞外域或第三个细胞外环会导致受体功能显著丧失，但双重替换产生了一个功能性受体。因此，这些G蛋白偶联受体的细胞外结构域不仅仅是跨膜结构域之间的被动连接。它们参与激动剂识别，并且必须直接或间接相互作用，以实现正常的受体功能。
