Developing Chemical Genetic Approaches to Explore G Protein-Coupled Receptor Function: Validation of the Use of a Receptor Activated Solely by Synthetic Ligand (RASSL)
Developing Chemical Genetic Approaches to Explore G Protein-Coupled Receptor Function: Validation of the Use of a Receptor Activated Solely by Synthetic Ligand (RASSL)
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DOI：
10.1124/mol.111.074674
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发表时间：
2011-12-01
影响因子：
3.6
通讯作者：
Milligan, Graeme
中科院分区：
文献类型：
--
作者：
Alvarez-Curto, Elisa;Prihandoko, Rudi;Milligan, Graeme
分子进化和化学遗传学已被应用于产生功能配对的突变G蛋白偶联受体（GPCR）和非内源性配体。这些突变受体，被称为仅由合成配体激活的受体（RASSL）或仅由设计药物激活的设计受体（DREADD），具有巨大的潜力来定义GPCR的生理作用，并在动物模型中验证受体作为治疗人类疾病的治疗靶标。然而，不同配体在相同受体上的配体偏好性和功能选择性的评价表明RASSL可能与内源性激动剂激活的野生型受体不同地发出信号。我们通过产生野生型人M-3毒蕈碱受体和选择性地对氯氮平N-氧化物应答的RASSL变体来评估这一点。尽管RASSL受体对毒蕈碱拮抗剂（包括阿托品）的亲和力降低，但氯氮平N-氧化物刺激产生的效应与乙酰胆碱对野生型M-3受体产生的效应非常相似。这些影响包括第三胞内环和分子内荧光共振能量转移传感器的C-末端尾部的相对运动，以及野生型和进化突变体调节细胞外信号调节激酶1/2磷酸化的能力。每种形式与β-抑制蛋白2类似地相互作用，并响应于适当的配体从细胞表面内化。此外，特定丝氨酸残基的磷酸化模式内的进化受体在氯氮平N-氧化物的反应是非常相似的乙酰胆碱在野生型。这样的结果提供了信心，至少对于M-3毒蕈碱受体，在转基因表达该RASSL后获得的结果可能反映乙酰胆碱在野生型受体上的作用。
Molecular evolution and chemical genetics have been applied to generate functional pairings of mutated G protein-coupled receptors (GPCRs) and nonendogenous ligands. These mutant receptors, referred to as receptors activated solely by synthetic ligands (RASSLs) or designer receptors exclusively activated by designer drugs (DREADDs), have huge potential to define physiological roles of GPCRs and to validate receptors in animal models as therapeutic targets to treat human disease. However, appreciation of ligand bias and functional selectivity of different ligands at the same receptor suggests that RASSLs may signal differently than wild-type receptors activated by endogenous agonists. We assessed this by generating forms of wild-type human M-3 muscarinic receptor and a RASSL variant that responds selectively to clozapine N-oxide. Although the RASSL receptor had reduced affinity for muscarinic antagonists, including atropine, stimulation with clozapine N-oxide produced effects very similar to those generated by acetylcholine at the wild-type M-3-receptor. Such effects included the relative movement of the third intracellular loop and C-terminal tail of intramolecular fluorescence resonance energy transfer sensors and the ability of the wild type and evolved mutant to regulate extracellular signal-regulated kinase 1/2 phosphorylation. Each form interacted similarly with beta-arrestin 2 and was internalized from the cell surface in response to the appropriate ligand. Furthermore, the pattern of phosphorylation of specific serine residues within the evolved receptor in response to clozapine N-oxide was very similar to that produced by acetylcholine at the wild type. Such results provide confidence that, at least for the M-3 muscarinic receptor, results obtained after transgenic expression of this RASSL are likely to mirror the actions of acetylcholine at the wild type receptor.