Receptor activity-modulating proteins (RAMPs) are accessory molecules that form complexes with specific G protein-coupled receptors (GPCRs) and modulate their functions. It is established that RAMP interacts with the glucagon receptor family of GPCRs but the underlying mechanism is poorly understood. In this study, we used a bioluminescence resonance energy transfer (BRET) approach to comprehensively investigate such interactions. In conjunction with cAMP accumulation, Gα_q activation and β-arrestin1/2 recruitment assays, we not only verified the GPCR–RAMP pairs previously reported, but also identified new patterns of GPCR–RAMP interaction. While RAMP1 was able to modify the three signaling events elicited by both glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), and RAMP2 mainly affected β-arrestin1/2 recruitment by GCGR, GLP-1R and glucagon-like peptide-2 receptor, RAMP3 showed a widespread negative impact on all the family members except for growth hormone-releasing hormone receptor covering the three pathways. Our results suggest that RAMP modulates both G protein dependent and independent signal transduction among the glucagon receptor family members in a receptor-specific manner. Mapping such interactions provides new insights into the role of RAMP in ligand recognition and receptor activation.

受体活性调节蛋白 (RAMP) 是与特定 G 蛋白偶联受体 (GPCR) 形成复合物并调节其功能的辅助分子。已确定 RAMP 与 GPCR 的胰高血糖素受体家族相互作用，但其潜在机制知之甚少。在这项研究中，我们使用生物发光共振能量转移 (BRET) 方法来全面研究这种相互作用。结合 cAMP 积累、G α _q激活和β-arrestin1/2 招募分析，我们不仅验证了先前报道的 GPCR-RAMP 对，而且还确定了 GPCR-RAMP 相互作用的新模式。虽然 RAMP1 能够改变由胰高血糖素受体 (GCGR) 和胰高血糖素样肽-1 受体 (GLP-1R) 引发的三种信号传导事件，但 RAMP2 主要影响β-arrestin1/2 通过 GCGR、GLP-1R 和胰高血糖素样肽 2 受体募集，RAMP3 对所有家族成员显示出广泛的负面影响，除了覆盖这三种途径的生长激素释放激素受体。我们的结果表明，RAMP 以受体特异性方式调节胰高血糖素受体家族成员之间的 G 蛋白依赖性和独立信号转导。映射这种相互作用为 RAMP 在配体识别和受体激活中的作用提供了新的见解。