Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand
Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand
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DOI：
10.1073/pnas.0700293104
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发表时间：
2007-03-20
影响因子：
11.1
通讯作者：
Roth, Bryan L.
中科院分区：
文献类型：
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作者：
Armbruster, Blaine N.;Li, Xiang;Roth, Bryan L.
我们在酵母中进化了M受体，产生了一系列G蛋白偶联受体(GPCRs)，这些受体只被一种药理上不活泼的类药物和生物可用化合物(氯氮平-N-氧化物)激活。随后在人类细胞系中的筛选促进了适合于体外和原位研究的M胆碱乙酰胆碱GPCRs家族的建立。随后，我们建立了稳定表达所有五个家族成员的端粒酶永生化的人肺动脉平滑肌细胞系，并发现每个细胞系都忠实地概括了亲本受体的信号表型。我们还在海马神经元(HM(4)D)表达了G(I)偶联的设计者受体(HM(4)D)，并证明了它能够诱导膜超极化和神经元沉默。因此，我们设计了一种简单的方法来设计具有工程配体特异性的GPCRs家族。这种反向工程的GPCRs将被证明是在体外和体内选择性调节信号转导通路的强大工具。
We evolved muscarinic receptors in yeast to generate a family of G protein-coupled receptors (GPCRs) that are activated solely by a pharmacologically inert drug-like and bioavailable compound (clozapine-N-oxide). Subsequent screening in human cell lines facilitated the creation of a family of muscarinic acetylcholine GPCRS suitable for in vitro and in situ studies. We subsequently created lines of telomerase-immortalized human pulmonary artery smooth muscle cells stably expressing all five family members and found that each one faithfully recapitulated the signaling phenotype of the parent receptor. We also expressed a G(i)-coupled designer receptor in hippocampal neurons (hM(4)D) and demonstrated its ability to induce membrane hyperpolarization and neuronal silencing. We have thus devised a facile approach for designing families of GPCRs with engineered ligand specificities. Such reverse-engineered GPCRs will prove to be powerful tools for selectively modulating signal-transcluction pathways in vitro and in vivo.