Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6^th transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface. We determined the cryo-EM structure of the GCGR-G_s complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner; however, the receptor ECD is indeed largely disordered along with rearrangements of several intracellular hallmarks of activation. Our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.

受体活性修饰蛋白 (RAMP) 调节许多 B 族 GPCR 的活性。我们发现 RAMP2 直接与胰高血糖素受体 (GCGR) 相互作用，胰高血糖素受体是一种负责血糖稳态的 B 族 GPCR，并广泛抑制受体诱导的下游信号传导。 HDX-MS 实验表明，RAMP2 增强了受体胞外域 (ECD) 内和附近的选定位置以及^第六跨膜螺旋的局部灵活性，而 smFRET 实验表明，这种 ECD 紊乱会导致受体胞外域 (ECD) 的活性和中间状态受到抑制。细胞内表面。我们在 RAMP2 存在的情况下以 2.9 Å 分辨率确定了 GCGR-G _s复合物的冷冻电镜结构。 RAMP2 显然不以有序的方式与 GCGR 相互作用；然而，受体 ECD 确实在很大程度上受到紊乱，同时还伴随着一些细胞内激活标志的重排。我们的研究表明 RAMP2 通过增强 ECD 的构象采样而充当 GCGR 的负变构调节剂。