Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity
Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity
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DOI：
10.1096/fj.08-114751
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发表时间：
2009-02-01
期刊：
影响因子：
4.8
通讯作者：
Mohr, Klaus
中科院分区：
文献类型：
--
作者：
Antony, Johannes;Kellershohn, Kerstin;Mohr, Klaus
通过G蛋白偶联受体（GPCR）活化选择性调节细胞功能对于基础研究和治疗是高度期望的，但难以实现。我们提出了一种新的策略，实现这一目标，使用毒蕈碱乙酰胆碱受体作为模型。这五种亚型在受体的跨膜结构域内的高度保守的正构位点结合其生理递质。正构毒蕈碱激活剂没有结合选择性且信号传导特异性差。在结合口袋的细胞外入口处有一个不太保守的变构位点。为了获得亚型选择性受体激活，我们合成了两个杂交体融合了一个非常有效的oxotremorine样orthosteric激活剂与M-2-选择性双（氨）烷型变构片段。通过受体对接模拟补充的野生型和突变型受体中的放射性配体结合证明了M-2选择性和真正的变构/正构结合。使用正构和变构阻滞剂进行的G蛋白激活测量确定了杂交体的正构部分以产生受体激活。CHO-hM（2）细胞中杂交诱导的动态质量再分布揭示了途径特异性信号传导。在活组织制备物中验证了选择性受体活化（M-2>M-1>M-3）。随着越来越多地认识到GPCR上的变构位点，GPCR靶向的双构概念代表了一种新的途径，用于选择性受体和信号传导途径活化的有效激动剂。- 安东尼，J.，Kellershohn，K.，Mohr-Andra，M.，Kebig，A.，Prilla，S.，Muth，M.，海勒，E.，Disingrini，T.，Dallanoce，C.，Bertoni，S.，Schrobang，J.，特兰克尔角，Kostenis，E.，Christopoulos，A.，Holtje，H. D、Barocelli，E.，De Amici，M.，Holzgrabe，U.，Mohr，K. Dualsteric GPCR targeting：a novel route to binding and signaling pathway selectivity. FASEB J. 23，442-450（2009）
Selective modulation of cell function by G protein-coupled receptor (GPCR) activation is highly desirable for basic research and therapy but difficult to achieve. We present a novel strategy toward this goal using muscarinic acetylcholine receptors as a model. The five subtypes bind their physiological transmitter in the highly conserved orthosteric site within the transmembrane domains of the receptors. Orthosteric muscarinic activators have no binding selectivity and poor signaling specificity. There is a less well conserved allosteric site at the extracellular entrance of the binding pocket. To gain subtype-selective receptor activation, we synthesized two hybrids fusing a highly potent oxotremorine-like orthosteric activator with M-2-selective bis(ammonio)alkane-type allosteric fragments. Radioligand binding in wild-type and mutant receptors supplemented by receptor docking simulations proved M-2 selective and true allosteric/orthosteric binding. G protein activation measurements using orthosteric and allosteric blockers identified the orthosteric part of the hybrid to engender receptor activation. Hybrid-induced dynamic mass redistribution in CHO-hM(2) cells disclosed pathway-specific signaling. Selective receptor activation (M-2>M-1>M-3) was verified in living tissue preparations. As allosteric sites are increasingly recognized on GPCRs, the dualsteric concept of GPCR targeting represents a new avenue toward potent agonists for selective receptor and signaling pathway activation. - Antony, J., Kellershohn, K., Mohr-Andra, M., Kebig, A., Prilla, S., Muth, M., Heller, E., Disingrini, T., Dallanoce, C., Bertoni, S., Schrobang, J., Trankle, C., Kostenis, E., Christopoulos, A., Holtje, H.-D., Barocelli, E., De Amici, M., Holzgrabe, U., Mohr, K. Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity. FASEB J. 23, 442-450 (2009)