title: Calcitonin and Amylin Receptor Peptide Interaction Mechanisms: INSIGHTS INTO PEPTIDE-BINDING MODES AND ALLOSTERIC MODULATION OF THE CALCITONIN RECEPTOR BY RECEPTOR ACTIVITY-MODIFYING PROTEINS.
abstract: 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.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104 and.
authors.forename: Sang-Min
authors.initials: SM
authors.lastname: Lee
authors.affiliation: the School of Biological Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland 1142, New Zealand.
authors.forename: Debbie L
authors.initials: DL
authors.lastname: Hay
authors.affiliation: From the Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104 and augen-pioszak@ouhsc.edu.
authors.forename: Augen A
authors.initials: AA
authors.lastname: Pioszak
mesh_terms: D000494:Allosteric Regulation / Q000502:physiology; D020816:Amino Acid Motifs; D057809:HEK293 Cells; D006801:Humans; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D058262:Receptor Activity-Modifying Protein 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D058263:Receptor Activity-Modifying Protein 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018003:Receptors, Calcitonin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D058287:Receptors, Islet Amyloid Polypeptide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D010455:Peptides; C545502:RAMP1 protein, human; C545503:RAMP2 protein, human; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D018003:Receptors, Calcitonin; D058287:Receptors, Islet Amyloid Polypeptide
keywords: G protein-coupled receptor (GPCR); RAMP; allosteric regulation; cell surface receptor; glycoprotein; mutagenesis; peptide hormone; structure-function