Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta, Georgia, United States of America.
Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
PLoS One. 2021 Apr 22;16(4):e0247743. doi: 10.1371/journal.pone.0247743. eCollection 2021.
A large number of GPCRs are potentially valuable drug targets but remain understudied. Many of these lack well-validated activating ligands and are considered "orphan" receptors, and G protein coupling profiles have not been defined for many orphan GPCRs. Here we asked if constitutive receptor activity can be used to determine G protein coupling profiles of orphan GPCRs. We monitored nucleotide-sensitive interactions between 48 understudied orphan GPCRs and five G proteins (240 combinations) using bioluminescence resonance energy transfer (BRET). No receptor ligands were used, but GDP was used as a common G protein ligand to disrupt receptor-G protein complexes. Constitutive BRET between the same receptors and β-arrestins was also measured. We found sufficient GDP-sensitive BRET to generate G protein coupling profiles for 22 of the 48 receptors we studied. Altogether we identified 48 coupled receptor-G protein pairs, many of which have not been described previously. We conclude that receptor-G protein complexes that form spontaneously in the absence of guanine nucleotides can be used to profile G protein coupling of constitutively-active GPCRs. This approach may prove useful for studying G protein coupling of other GPCRs for which activating ligands are not available.
大量的 G 蛋白偶联受体（GPCRs）是具有潜在价值的药物靶点，但研究不足。其中许多缺乏经过充分验证的激活配体，被认为是“孤儿”受体，并且许多孤儿 GPCR 的 G 蛋白偶联谱尚未确定。在这里，我们想知道组成型受体活性是否可用于确定孤儿 GPCR 的 G 蛋白偶联谱。我们使用生物发光共振能量转移（BRET）监测了 48 个研究不足的孤儿 GPCR 与五种 G 蛋白（240 种组合）之间核苷酸敏感的相互作用。未使用任何受体配体，但使用 GDP 作为常见的 G 蛋白配体来破坏受体-G 蛋白复合物。还测量了同一受体与β-arrestin 之间的组成型 BRET。我们发现有足够的 GDP 敏感 BRET 可以为我们研究的 48 个受体中的 22 个生成 G 蛋白偶联谱。总的来说，我们鉴定了 48 个偶联的受体-G 蛋白对，其中许多以前没有描述过。我们得出结论，在没有鸟嘌呤核苷酸的情况下自发形成的受体-G 蛋白复合物可用于对组成型活性 GPCR 的 G 蛋白偶联进行分析。对于那些没有激活配体的其他 GPCR，这种方法可能证明是有用的。
