Understanding the activation and internalization of G protein-coupled receptors (GPCRs) using conditional approaches is paramount to developing new therapeutic strategies. Here, we describe the design, synthesis, and testing of ExONatide, a benzylguanine-linked peptide agonist of the glucagon-like peptide-1 receptor (GLP-1R), a class B GPCR required for maintenance of glucose levels in humans. ExONatide covalently binds to SNAP-tagged GLP-1R-expressing cells, leading to prolonged cAMP generation, Ca²⁺ rises, and intracellular retention of the receptor. These effects were readily switched OFF following cleavage of the introduced disulfide bridge using the cell-permeable reducing agent beta-mercaptoethanol (BME). A similar approach could be extended to a class A GPCR using GhrelON, a benzylguanine-linked peptide agonist of the growth hormone secretagogue receptor 1a (GHS-R1a), which is involved in food intake and growth. Thus, ExONatide and GhrelON allow SNAP-tag-directed activation of class A and B GPCRs involved in gut hormone signaling in a reversible manner. This tactic, termed reductively cleavable agONist (RECON), may be useful for understanding GLP-1R and GHS-R1a function both in vitro and in vivo, with applicability across GPCRs.

中文翻译：

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使用束缚药理学对 A 类和 BG 蛋白偶联受体进行条件和可逆激活。

使用条件方法了解 G 蛋白偶联受体 (GPCR) 的激活和内化对于开发新的治疗策略至关重要。在这里，我们描述了 ExONatide 的设计、合成和测试，ExONatide是一种胰高血糖素样肽-1 受体 (GLP-1R) 的苄基鸟嘌呤连接肽激动剂，它是维持人体葡萄糖水平所需的 B 类 GPCR。ExONatide与 SNAP 标记的 GLP-1R 表达细胞共价结合，导致 cAMP 生成延长、Ca ²⁺升高和受体的细胞内保留。在使用细胞渗透性还原剂β裂解引入的二硫键后，这些效应很容易被关闭-巯基乙醇（BME）。类似的方法可以扩展到使用GhrelON的 A 类 GPCR ，GhrelON 是一种与食物摄入和生长有关的生长激素促分泌素受体 1a (GHS-R1a) 的苄基鸟嘌呤连接肽激动剂。因此，ExONatide和GhrelON允许 SNAP-tag 以可逆方式激活参与肠道激素信号传导的 A 类和 B 类 GPCR。这种策略称为还原性可剥离 ag ON ist ( RECON )，可能有助于了解 GLP-1R 和 GHS-R1a在体外和体内的功能，并适用于 GPCR。