肾上腺髓质素2/中介素(AM2/IMD),肾上腺髓质素(AM),和降钙素基因相关肽(CGRP)在心血管信号功能,淋巴管,通过激活由B类GPCRCLR和RAMP1、-2或-3调节亚基组成的三种异二聚体受体来实现和神经系统。CGRP和AM更喜欢RAMP1和RAMP2/3复合物,分别,而AM2/IMD被认为是相对非选择性的。因此,AM2/IMD表现出与CGRP和AM的重叠动作,因此CLR-RAMP复合物的第三种激动剂的基本原理尚不清楚.这里,我们报道AM2/IMD对CLR-RAMP3具有动力学选择性,称为AM2R,我们定义了其独特动力学的结构基础。在活细胞生物传感器检测中,AM2/IMD-AM2R引发的cAMP信号传导持续时间明显长于8种其他肽-受体组合。AM2/IMD和AM以相似的平衡亲和力约束AM2R,但AM2/IMD具有更慢的解离速率和更长的受体停留时间,从而解释了它延长的信令容量。使用肽和受体嵌合体和诱变将负责不同结合和信号传导动力学的区域定位到AM2/IMD中间区域和RAMP3胞外域(ECD)。分子动力学模拟揭示了前者如何在CLRECD-跨膜结构域界面形成稳定的相互作用,以及后者如何增强CLRECD结合口袋以锚定AM2/IMDC末端。这两种强结合组分仅在AM2R中结合。我们的发现揭示了AM2/IMD-AM2R作为具有独特时间特征的同源对,揭示AM2/IMD和RAMP3如何合作塑造CLR信号,对AM2/IMD生物学有重要意义。
Adrenomedullin 2/intermedin (AM2/IMD), adrenomedullin (AM), and calcitonin gene-related peptide (CGRP) have functions in the cardiovascular, lymphatic, and nervous systems by activating three heterodimeric receptors comprising the class B GPCR CLR and a RAMP1, -2, or -3 modulatory subunit. CGRP and AM prefer the RAMP1 and RAMP2/3 complexes, respectively, whereas AM2/IMD is thought to be relatively nonselective. Accordingly, AM2/IMD exhibits overlapping actions with CGRP and AM, so the rationale for this third agonist for the CLR-RAMP complexes is unclear. Here, we report that AM2/IMD is kinetically selective for CLR-RAMP3, known as the AM2R, and we define the structural basis for its distinct kinetics. In live cell biosensor assays, AM2/IMD-AM2R elicited longer-duration cAMP signaling than the other peptide-receptor combinations. AM2/IMD and AM bound the AM2R with similar equilibrium affinities, but AM2/IMD had a slower off-rate and longer receptor residence time, thus explaining its prolonged signaling capacity. Peptide and receptor chimeras and mutagenesis were used to map the regions responsible for the distinct binding and signaling kinetics to the AM2/IMD mid-region and the RAMP3 extracellular domain (ECD). Molecular dynamics simulations revealed how the former forms stable interactions at the CLR ECD-transmembrane domain interface and how the latter augments the CLR ECD binding pocket to anchor the AM2/IMD C terminus. These strong binding components only combine in the AM2R. Our findings uncover AM2/IMD-AM2R as a cognate pair with unique temporal features, reveal how AM2/IMD and RAMP3 collaborate to shape CLR signaling, and have significant implications for AM2/IMD biology.