Deconvoluting the Molecular Control of Binding and Signaling at the Amylin 3 Receptor: RAMP3 Alters Signal Propagation through Extracellular Loops of the Calcitonin Receptor

Abstract

Amylin is coexpressed with insulin in pancreatic islet βcells and has potent effects on gastric emptying and food intake. The effect of amylin on satiation has been postulated to involve AMY 3 receptors (AMY 3 R) that are heteromers of the calcitonin receptor (CTR) and receptor activity-modifying protein 3 (RAMP3). Understanding the molecular control of signaling through the AMY 3 R is thus important for peptide drug targeting of this receptor. We have previously used alanine scanning mutagenesis to study the contribution of the extracellular surface of the CTR to binding and signaling initiated by calcitonin (CT) and related peptides (Dal Maso, E., et al. (2019) The molecular control of calcitonin receptor signaling. ACS Pharmacol. Transl. Sci. 2, 31−51). That work revealed ligand-and pathway-specific effects of mutation, with extracellular loops (ECLs) 2 and 3 particularly important in the distinct propagation of signaling mediated by individual peptides. In the current study, we have used equivalent alanine scanning of ECL2 and ECL3 of the CTR in the context of coexpression with RAMP3 to form AMY 3 Rs, to examine functional affinity and efficacy of peptides in cAMP accumulation and extracellular signal-regulated kinase (ERK) phosphorylation (pERK). The effect of mutation was determined on representatives of the three major distinct classes of CT peptide, salmon CT (sCT), human CT (hCT), and porcine CT (pCT), as well as rat amylin (rAmy) or human α-CGRP (calcitonin gene-related peptide, hCGRP) whose potency is enhanced by RAMP interaction. We demonstrate that the dynamic nature of CTR ECL2 and ECL3 in propagation of signaling is fundamentally altered when complexed with RAMP3 to form the AMY 3 R, despite only having predicted direct interactions with ECL2. Moreover, the work shows that the role of these loops in receptor signaling is highly peptide dependent, illustrating that even subtle changes to peptide sequence may change signaling output downstream of the receptor.

Structure and Dynamics of Adrenomedullin Receptors AM1 and AM2 Reveal Key Mechanisms in the Control of Receptor Phenotype by Receptor Activity-Modifying Proteins

“…Interestingly, the interactions of AM, AM2, and CGRP with ECL2 occur principally at the mid-distal (TM5) end of the helix, similar to what is also observed for the related CTR but distinct from other class B GPCRs that bind peptides with extended helices. In those receptors, the interactions are greater with more proximal ECL2 residues, and this distinction may, in part, explain the large differences in the effect of Ala mutations of ECL2 on peptide efficacy seen between GLP-1R and CTR, − which can be further modified by RAMP interaction …”

Toward a Structural Understanding of Class B GPCR Peptide Binding and Activation

“…Nonetheless, despite the conserved secondary structure of ECL2, previous mutational analysis of CTR versus GLP-1R demonstrates that the peptide-mediated propagation of signaling through this domain was distinct (Dal Maso et al, 2018;Wootten et al, 2016), and this can be further affected by interaction of receptors with RAMPs (Pham et al, 2019).…”

Exploring Ligand Binding to Calcitonin Gene-Related Peptide Receptors

“…GPCRs are characterized by a 7-transmembrane helix domain (TMD) and, in class B1, by a N-terminal extracellular domain that contributes to agonist binding, receptor activation, and signaling ( de Graaf et al, 2017 ) ( Figure 1A ). Heterodimerization with accessory receptor activity-modifying proteins (RAMPs, Figure 1A ) can modulate hormone binding and signaling ( Hay and Pioszak, 2016 ) through an allosteric mechanism ( Liang et al, 2020a ; Pham et al, 2019 ). The transmembrane spanning RAMPs interact with the calcitonin receptor (CTR) and CTR-like (CLR) receptors at the transmembrane level, making contacts with TM3, TM4 and TM5, and at the extracellular level, where they interface with the ECD ( Figures 1A,D ).…”