Cryo-EM Structure of the Human Amylin 1 Receptor in Complex with CGRP and Gs Protein.

Inhibition of calcitonin gene-related peptide (CGRP) or its cognate CGRP receptor (CGRPR) has arisen as a major breakthrough in the treatment of migraine. However, a second CGRP-responsive receptor exists, the amylin (Amy) 1 receptor (AMY1R), yet its involvement in the pathology of migraine is poorly understood. AMY1R and CGRPR are heterodimers consisting of receptor activity-modifying protein 1 (RAMP1) with the calcitonin receptor (CTR) and the calcitonin receptor-like receptor (CLR), respectively. Here, we present the structure of AMY1R in complex with CGRP and Gs protein and compare it with the reported structures of the AMY1R complex with rat amylin (rAmy) and the CGRPR in complex with CGRP. Despite similar protein backbones observed within the receptors and the N- and C-termini of the two peptides bound to the AMY1R complexes, they have distinct organization in the peptide midregions (the bypass motif) that is correlated with differences in the dynamics of the respective receptor extracellular domains. Moreover, divergent conformations of extracellular loop (ECL) 3, intracellular loop (ICL) 2, and ICL3 within the CTR and CLR protomers are evident when comparing the CGRP bound to the CGRPR and AMY1R, which influences the binding mode of CGRP. However, the conserved interactions made by the C-terminus of CGRP to the CGRPR and AMY1R are likely to account for cross-reactivity of nonpeptide CGRPR antagonists observed at AMY1R, which also extends to other clinically used CGRPR blockers, including antibodies.

A structural basis for amylin receptor phenotype.

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.

The dual amylin and calcitonin receptor agonist KBP-089 and the GLP-1 receptor agonist liraglutide act complimentarily on body weight reduction and metabolic profile.

BACKGROUND: Weight loss therapy is becoming more and more important, and two classes of molecules, namely amylin receptor and GLP-1 receptor agonists, have shown promise in this regard. Interestingly, these molecules have several overlapping pharmacological effects, such as suppression of gastric emptying, reduction of glucagon secretion and weight loss in common; however, they also have distinct effects on prandial insulin secretion. Hence, a combination of these two mechanisms is of significant interest. METHODS: In this study, we investigated the add-on potential of the dual amylin and calcitonin receptor agonist (DACRA) KBP-089 in combination with the GLP-1 receptor agonist liraglutide as obesity treatment in high-fat diet (HFD) fed rats. RESULTS: Increasing doses of KBP-089 and liraglutide alone and in combination were studied with respect to their effects on body weight, food intake and glucose metabolism during a 9-week intervention study conducted in HFD rats. Further, the gastric emptying rate during an oral glucose tolerance was assessed. Treatment with KBP-089 and liraglutide dose-dependently lowered body weight 15% (at 2.5 µg/kg/day) and 7% (at 400 µg/kg/day) in HFD rats, respectively, while the combination resulted in a 21% body weight reduction, which was mirrored by reduction in fat depot sizes. Gastric emptying and glucose metabolism were improved, primarily by KBP-089, although liraglutide led to a reduction in fasting plasma glucagon. CONCLUSION: DACRAs complement GLP-1 on food intake, body weight, and glucose tolerance indicating the potential for an add-on therapy.

Calcitonin and Amylin Receptor Peptide Interaction Mechanisms: INSIGHTS INTO PEPTIDE-BINDING MODES AND ALLOSTERIC MODULATION OF THE CALCITONIN RECEPTOR BY RECEPTOR ACTIVITY-MODIFYING PROTEINS.

Receptor activity-modifying proteins (RAMP1-3) determine the selectivity of the class B G protein-coupled calcitonin receptor (CTR) and the CTR-like receptor (CLR) for calcitonin (CT), amylin (Amy), calcitonin gene-related peptide (CGRP), and adrenomedullin (AM) peptides. RAMP1/2 alter CLR selectivity for CGRP/AM in part by RAMP1 Trp-84 or RAMP2 Glu-101 contacting the distinct CGRP/AM C-terminal residues. It is unclear whether RAMPs use a similar mechanism to modulate CTR affinity for CT and Amy, analogs of which are therapeutics for bone disorders and diabetes, respectively. Here, we reproduced the peptide selectivity of intact CTR, AMY1 (CTR·RAMP1), and AMY2 (CTR·RAMP2) receptors using purified CTR extracellular domain (ECD) and tethered RAMP1- and RAMP2-CTR ECD fusion proteins and antagonist peptides. All three proteins bound salmon calcitonin (sCT). Tethering RAMPs to CTR enhanced binding of rAmy, CGRP, and the AMY antagonist AC413. Peptide alanine-scanning mutagenesis and modeling of receptor-bound sCT and AC413 supported a shared non-helical CGRP-like conformation for their TN(T/V)G motif prior to the C terminus. After this motif, the peptides diverged; the sCT C-terminal Pro was crucial for receptor binding, whereas the AC413/rAmy C-terminal Tyr had little or no influence on binding. Accordingly, mutant RAMP1 W84A- and RAMP2 E101A-CTR ECD retained AC413/rAmy binding. ECD binding and cell-based signaling assays with antagonist sCT/AC413/rAmy variants with C-terminal residue swaps indicated that the C-terminal sCT/rAmy residue identity affects affinity more than selectivity. rAmy(8-37) Y37P exhibited enhanced antagonism of AMY1 while retaining selectivity. These results reveal unexpected differences in how RAMPs determine CTR and CLR peptide selectivity and support the hypothesis that RAMPs allosterically modulate CTR peptide affinity.