Adrenomedullin 2/intermedin is a slow off-rate, long-acting endogenous agonist of the adrenomedullin(2) G protein–coupled receptor

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 AM(2)R, and we define the structural basis for its distinct kinetics. In live cell biosensor assays, AM2/IMD–AM(2)R elicited longer-duration cAMP signaling than the other peptide–receptor combinations. AM2/IMD and AM bound the AM(2)R 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 AM(2)R. Our findings uncover AM2/IMD–AM(2)R 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.