G-protein coupled receptors (GPCRs) are the ligand detection machinery of a majority of extracellular signaling systems in metazoans. Novel chemical and biological tools to probe the structure-function relationships of GPCRs have impacted both basic and applied GPCR research. To better understand the structure-function of class B GPCRs, we generated receptor-ligand fusion chimeric proteins that can be activated by exogenous enzyme application. As a prototype, fusion proteins of the glucagon-like peptide-1 receptor (GLP-1R) with GLP-1(7–36) and exendin-4(1–39) peptides incorporating enterokinase-cleavable N-termini were generated. These receptors are predicted to generate fusion protein neo-epitopes upon proteolysis with enterokinase that are identical to the N-termini of GLP-1 agonists. This system was validated by measuring enterokinase-dependent GLP-1R mediated cAMP accumulation, and a structure-activity relationship for both linker length and peptide sequence was observed. Moreover, our results show this approach can be used in physiologically relevant cell systems, as GLP-1R-ligand chimeras were shown to induce glucose-dependent insulin secretion in insulinoma cells upon exposure to enterokinase. This approach suggests new strategies for understanding the structure-function of peptide-binding GPCRs.

G蛋白偶联受体（GPCR）是后生动物大多数细胞外信号系统的配体检测机制。探索GPCR结构与功能关系的新型化学和生物学工具已经影响了基础和应用GPCR研究。为了更好地了解B类GPCR的结构功能，我们生成了可以通过外源酶应用激活的受体-配体融合嵌合蛋白。作为原型，生成了胰高血糖素样肽1受体（GLP-1R）与GLP-1（7–36）和exendin-4（1–39）肽的融合蛋白，这些蛋白结合了可被肠激酶切割的N末端。预计这些受体在用肠激酶进行蛋白水解后会产生融合蛋白新表位，与GLP-1激动剂的N末端相同。通过测量肠激酶依赖的GLP-1R介导的cAMP积累来验证该系统，并观察到了接头长度和肽序列的结构-活性关系。此外，我们的结果表明该方法可用于生理相关的细胞系统，因为显示GLP-1R-配体嵌合体在暴露于肠激酶后可诱导胰岛素瘤细胞中葡萄糖依赖性胰岛素分泌。该方法提出了用于理解肽结合GPCR的结构功能的新策略。因为显示GLP-1R-配体嵌合体在暴露于肠激酶后可诱导胰岛素瘤细胞中葡萄糖依赖性胰岛素分泌。该方法提出了用于理解肽结合GPCR的结构功能的新策略。因为显示GLP-1R-配体嵌合体在暴露于肠激酶后可诱导胰岛素瘤细胞中葡萄糖依赖性胰岛素分泌。该方法提出了用于理解肽结合GPCR的结构功能的新策略。