Amylin receptor subunit interactions are modulated by agonists and determine signaling

Three amylin receptors (AMYRs) mediate the metabolic actions of the peptide hormone amylin and are drug targets for diabetes and obesity. AMY₁R, AMY₂R, and AMY₃R are heterodimers consisting of the calcitonin receptor (CTR), a G protein–coupled receptor, paired with a RAMP1, RAMP2, or RAMP3 accessory subunit, respectively, which increases amylin potency. Here, we found that the AMYRs had distinct basal subunit equilibria that were modulated by peptide agonists and determined the extent of cAMP signaling downstream of receptor activation. By developing a biochemical assay that resolves the AMYR heterodimers and free subunits, we found that the AMY₁R and AMY₂R subunit distributions favored free CTR and RAMPs and that rat amylin promoted association of the constituent subunits of AMY₁R and AMY₂R. The agonist αCGRP also induced AMY₁R subunit association. A stronger interaction between the CTR and the RAMP3 transmembrane domains yielded a more stable AMY₃R, and human and salmon calcitonin agonists promoted AMY₃R dissociation. Similar changes in subunit association and dissociation were observed in live-cell membranes, and G protein coupling and cAMP signaling assays showed how these changes altered signaling. Our findings have implications for AMYR biology and drug development and reveal regulation of heteromeric GPCR signaling through subunit interaction dynamics.