Chen J, Bernstein H S, Chen M, Wang L, Ishii M, Turck C W, Coughlin S R
Cardiovascular Research Institute, University of California, San Francisco 94143-0524, USA.
J Biol Chem. 1995 Oct 6;270(40):23398-401. doi: 10.1074/jbc.270.40.23398.
We exploited the mechanism underlying thrombin receptor activation to develop a novel screening method to identify peptide agonists. The thrombin receptor is activated by limited proteolysis of its amino-terminal exodomain. Thrombin cleaves this domain to unmask a new amino terminus, which then functions as a tethered peptide agonist, binding intramolecularly to the body of the receptor to trigger signaling. The thrombin receptor's amino-terminal exodomain can also donate the tethered agonist intermolecularly to activate nearby thrombin receptors. We utilized this ability by co-expressing a "tethered ligand library," which displayed the thrombin receptor's amino-terminal exodomain bearing random pentapeptides in place of the native tethered ligand together with target receptors in Xenopus oocytes. Clones that conferred thrombin-dependent signaling by intermolecular ligation of the target receptor were isolated by sib selection. Agonists for the thrombin receptor itself (GFIYF) and for the formyl peptide receptor (MMWLL) were identified. Surprisingly, the latter agonist was quite active at the formyl peptide receptor even without N-formylation, and its formylated form, fMMWLL, was more potent than the classical formyl peptide receptor agonist fMLF. In addition to identifying novel peptide agonists for targets of pharmacological interest, this method might be used to discover agonists for orphan receptors. It also suggests a possible evolutionary path from peptide to protease-activated receptors.
我们利用凝血酶受体激活的潜在机制开发了一种新型筛选方法来鉴定肽激动剂。凝血酶受体通过其氨基末端胞外域的有限蛋白水解作用而被激活。凝血酶切割该结构域以暴露一个新的氨基末端，该末端随后作为一种拴系肽激动剂，在分子内与受体主体结合以触发信号传导。凝血酶受体的氨基末端胞外域还可以在分子间提供拴系激动剂以激活附近的凝血酶受体。我们通过共表达一个“拴系配体文库”来利用这种能力，该文库展示了带有随机五肽取代天然拴系配体的凝血酶受体氨基末端胞外域，以及非洲爪蟾卵母细胞中的靶受体。通过子代选择分离出通过靶受体的分子间连接赋予凝血酶依赖性信号传导的克隆。鉴定出了凝血酶受体本身的激动剂（GFIYF）和甲酰肽受体的激动剂（MMWLL）。令人惊讶的是，即使没有N-甲酰化，后者的激动剂在甲酰肽受体上也相当活跃，并且其甲酰化形式fMMWLL比经典的甲酰肽受体激动剂fMLF更有效。除了鉴定出用于具有药理学意义的靶标的新型肽激动剂外，该方法还可用于发现孤儿受体的激动剂。它还暗示了从肽到蛋白酶激活受体的一种可能的进化途径。
