LAUSR

Amylin receptors (AMYRs) are heterodimers of the calcitonin (CT) receptor (CTR) and one of three receptor activity–modifying proteins (RAMPs), AMY1R, AMY2R, and AMY3R. Selective AMYR agonists and dual AMYR/CTR agonists are being developed as obesity treatments; however, the molecular basis for peptide binding and selectivity is unknown. We determined the structure and dynamics of active AMYRs with amylin, AMY1R with salmon CT (sCT), AMY2R with sCT or human CT (hCT), and CTR with amylin, sCT, or hCT. The conformation of amylin-bound complexes was similar for all AMYRs, constrained by the RAMP, and an ordered midpeptide motif that we call the bypass motif. The CT-bound AMYR complexes were distinct, overlapping the CT-bound CTR complexes. Our findings indicate that activation of AMYRs by CT-based peptides is distinct from their activation by amylin-based peptides. This has important implications for the development of AMYR therapeutics. Description Complex responses of an obesity target Amylin receptors (AMYRs), which respond to the peptide hormones amylin and calcitonin, are targets for treating obesity and metabolic disorders. They are heterodimers comprising the calcitonin receptor, which is a G protein–coupled receptor, and one of three receptor-modifying proteins. An impediment to functional studies is that it is difficult to separate AMYR phenotypes from calcitonin receptor phenotypes. Cao et al. present six cryo–electron microscopy structures of active AMYRs bound to amylin or calcitonin. The structures reveal that the two peptide hormones activate AMYRs by distinct mechanisms. The structural and mechanistic insights will be valuable in designing both specific agonists and agonists with dual action. —VV There are distinct modes of activation of an obesity target, the amylin receptor, by calcitonin and amylin peptide agonists. INTRODUCTION Amylin receptors (AMYRs) are heterodimers of the calcitonin (CT) receptor (CTR) and one of three receptor activity-modifying proteins (RAMPs), AMY1R, AMY2R, and AMY3R. AMYRs have emerged as important potential disease targets, and selective AMYR agonists and dual AMYR/CTR agonists (DACRAs) are being developed as obesity treatments. However, it is unclear whether DACRAs based on amylin-template peptides versus calcitonin-template peptides activate target receptors by similar or distinct molecular mechanisms. Understanding the structural basis for the binding and selectivity of peptides to CTRs and AMYRs is important for future drug discovery and development. RATIONALE Currently, no structural information exists on any of the AMYRs. To address this gap, we applied cryo–electron microscopy (cryo-EM) to determine structures of AMY1R, AMY2R, AMY3R bound to rat amylin (rAmy), AMY1R and AMY2R bound to salmon CT (sCT), AMY2R with human CT (hCT), and the CTR bound to rAmy, hCT, or sCT, all in complex with Gs. RESULTS Purified samples for each complex were vitrified and imaged by cryo-EM to yield consensus maps at gold standard Fourier shell correlation 0.143 of 2.2 Å (rAmy-AMY1R), 2.6 Å (rAmy-AMY2R), 2.4 Å (rAmy-AMY3R), 3.0 Å (sCT-AMY1R), 3.0 Å (sCT-AMY2R), 3.3 Å (hCT-AMY2R), 3.3 Å (rAmy-CTR), 2.6 Å (sCT-CTR), or 2.7 Å (hCT-CTR). The CT-CTR co-complexes had similar backbone conformations, and, similarly, the rAmy-bound AMY1R, AMY2R, and AMY3R complex structures exhibited almost identical backbone conformations. However, the ECD of the CTR subunit in rAmy-bound AMYRs undergoes an ~12-Å rigid-body translation relative to CT-CTR complexes. Although there was a high degree of overlap in the location of the N-terminal loop and midregion helical extension of both rAmy and CT peptides within the transmembrane domain (TMD), the peptides diverge as they exit the receptor core. The CT peptides have an unstructured C-terminal region that extends in the same vertical plane as the helix. By contrast, the α-helix of the rAmy peptide terminates half a turn earlier, extending in the plane of the membrane toward the top of the transmembrane helix 1 (TM1) (bypass motif) before forming a tight turn that extends to interact with the ECD. In the sCT-AMY1R and sCT- or hCT-bound AMY2R complexes, the backbone structure and orientation of the CTR protomer, including the ECD, and CT peptides were effectively equivalent to those in the CT-CTR complexes, with a consequent reorientation of the RAMP ECD compared with rAmy occupied receptors. RAMP2 has a weaker TMD interface than the other two RAMPs, and this becomes destabilized, allowing repositioning of the RAMP ECD in CT-bound complexes. By contrast, the tighter TMD interface for RAMP1 and CTR leads to only partial disengagement of the top of the RAMP TMD helix with greater mobility of the RAMP ECD to accommodate the position of the CTR ECD. The tighter TMD constraint for this RAMP likely contributes to lower potency of hCT at AMY1R and AMY3R. The rAmy-bound CTR complex could be resolved into two major classes, CT-like and rAmy bypass-like, differing in the ECD orientations and peptide conformations. CONCLUSION Our data provide details of the molecular basis for activation of AMYRs and reveal that CT peptides activate AMYRs by distinct mechanisms compared with activation by Amy peptides. Our work provides a template for the future development of selective and nonselective agonists of CT and AMY receptors. CT and Amy peptides have distinct modes of amylin receptor activation. Left: overlay of the rAmy-AMY1R, rAmy -AMY2R, and rAmy-AMY3R complexes. rAmy, gold; CTR, blue; RAMP1 purple; RAMP2, green; RAMP3, orange. Right: overlay of the sCT (cyan)-CTR (yellow) and sCT (green)-AMY2R (CTR, grey; RAMP2, dark pink) complexes. Amylin peptides display a midregion “bypass” motif (black dashed oval) that reorients the extracellular domains of CTR and RAMP relative to CT-bound receptor (red dashed rectangle).