Variable CGRP family peptide signaling durations and the structural determinants thereof.

Calcitonin gene-related peptides alpha and beta (αCGRP, ßCGRP), adrenomedullin (AM), and adrenomedullin 2/intermedin (AM2/IMD) function in pain signaling, neuroimmune communication, and regulation of the cardiovascular and lymphatic systems by activating either of two class B GPCRs, CLR and CTR, in complex with a RAMP1, -2, or -3 modulatory subunit. Inspired by our recent discovery that AM2/IMD(1-47) activation of CLR-RAMP3 elicits long duration cAMP signaling, here we used a live-cell cAMP biosensor assay to characterize the signaling kinetics of the two CGRP peptides and several bioactive AM and AM2/IMD fragments with variable N-terminal extensions. Remarkably, AM2/IMD(8-47) and AM2/IMD-53 exhibited even longer duration signaling than the 1-47 fragment. AM2/IMD(8-47) was a striking 8-fold longer acting than AM(13-52) at CLR-RAMP3. In contrast, the N-terminal extension of AM had no effect on signaling duration. AM(1-52) and (13-52) were equally short-acting. Analysis of AM2/IMD-AM mid-region chimeras and AM2/IMD R23 and R33 point mutants showed the importance of these residues for long-duration signaling and identified AM2/IMD peptides that exhibited up to 17-fold diminished signaling duration at CLR-RAMP3, while retaining near wildtype signaling potencies. ßCGRP was âˆ¼ 3-fold longer acting than αCGRP at the CGRP (CLR-RAMP1) and the amylin1 (CTR-RAMP1) receptors. Chimeric CGRP peptides showed that the single residue difference near the N-terminus, and the two differences in the mid-region, equally contributed to the longer duration of ßCGRP signaling. This work uncovers key temporal differences in cAMP signaling among the CGRP family peptides, elucidates the structural bases thereof, and provides pharmacological tools for studying long-duration AM2/IMD signaling.

Adrenomedullin 2/intermedin is a slow off-rate, long-acting endogenous agonist of the adrenomedullin2 G protein-coupled receptor.

Adrenomedullin 2/intermedin (AM2/IMD), adrenomedullin (AM), and calcitonin gene-related peptide (CGRP) have functions in the cardiovascular, lymphatic, and nervous systems by activating three heterodimeric receptors comprising the class B GPCR CLR and a RAMP1, -2, or -3 modulatory subunit. CGRP and AM prefer the RAMP1 and RAMP2/3 complexes, respectively, whereas AM2/IMD is thought to be relatively nonselective. Accordingly, AM2/IMD exhibits overlapping actions with CGRP and AM, so the rationale for this third agonist for the CLR-RAMP complexes is unclear. Here, we report that AM2/IMD is kinetically selective for CLR-RAMP3, known as the AM2R, and we define the structural basis for its distinct kinetics. In live cell biosensor assays, AM2/IMD-AM2R elicited longer-duration cAMP signaling than the other peptide-receptor combinations. AM2/IMD and AM bound the AM2R with similar equilibrium affinities, but AM2/IMD had a slower off-rate and longer receptor residence time, thus explaining its prolonged signaling capacity. Peptide and receptor chimeras and mutagenesis were used to map the regions responsible for the distinct binding and signaling kinetics to the AM2/IMD mid-region and the RAMP3 extracellular domain (ECD). Molecular dynamics simulations revealed how the former forms stable interactions at the CLR ECD-transmembrane domain interface and how the latter augments the CLR ECD binding pocket to anchor the AM2/IMD C terminus. These strong binding components only combine in the AM2R. Our findings uncover AM2/IMD-AM2R as a cognate pair with unique temporal features, reveal how AM2/IMD and RAMP3 collaborate to shape CLR signaling, and have significant implications for AM2/IMD biology.

Adrenomedullin 2/intermedin is a slow off-rate, long-acting endogenous agonist of the adrenomedullin 2 G protein-coupled receptor.

The signaling peptides adrenomedullin 2/intermedin (AM2/IMD), adrenomedullin (AM), and CGRP have overlapping and distinct functions in the cardiovascular, lymphatic, and nervous systems by activating three shared receptors comprised of the class B GPCR CLR in complex with a RAMP1, -2, or -3 modulatory subunit. Here, we report that AM2/IMD, which is thought to be a non-selective agonist, is kinetically selective for CLR-RAMP3, known as the AM 2 R. AM2/IMD-AM 2 R elicited substantially longer duration cAMP signaling than the eight other peptide-receptor combinations due to AM2/IMD slow off-rate binding kinetics. The regions responsible for the slow off-rate were mapped to the AM2/IMD mid-region and the RAMP3 extracellular domain. MD simulations revealed how these bestow enhanced stability to the complex. Our results uncover AM2/IMD-AM 2 R as a cognate pair with unique temporal features, define the mechanism of kinetic selectivity, and explain how AM2/IMD and RAMP3 collaborate to shape the signaling output of a clinically important GPCR.

Time- and cost-efficient bacterial expression and purification of potato apyrase.

Apyrase from potato (Solanum tuberosum) is a divalent metal ion-dependent enzyme that catalyzes the hydrolysis of nucleoside di- and tri-phosphates with broad substrate specificity. The enzyme is widely used to manipulate nucleotide levels such as in the G protein-coupled receptor (GPCR) field where it is used to deplete guanine nucleotides to stabilize nucleotide-free ternary agonist-GPCR-G protein complexes. Potato apyrase is available commercially as the native enzyme purified from potatoes or as a recombinant protein, but these are prohibitively expensive for some research applications. Here, we report a relatively simple method for the bacterial production of soluble, active potato apyrase. Apyrase has several disulfide bonds, so we co-expressed the enzyme bearing a C-terminal (His)6 tag with the E. coli disulfide isomerase DsbC at low temperature (18 °C) in the oxidizing cytoplasm of E. coli Origami B (DE3). This allowed low level production of soluble apyrase. A two-step purification procedure involving Ni-affinity followed by Cibacron Blue-affinity chromatography yielded highly purified apyrase at a level of ∼0.5 mg per L of bacterial culture. The purified enzyme was functional for ATP hydrolysis in an ATPase assay and for GTP/GDP hydrolysis in a GPCR-G protein coupling assay. This methodology enables the time- and cost-efficient production of recombinant apyrase for various research applications.

A Native PAGE Assay for the Biochemical Characterization of G Protein Coupling to GPCRs.

G protein-coupled receptors (GPCRs) are a large family of membrane-embedded receptors that have diverse roles in physiology and are major drug targets. GPCRs transduce an agonist binding signal across the membrane to activate intracellular heterotrimeric G proteins. The dynamic nature of the receptors and the complexity of their interactions with agonists and G proteins present significant challenges for biochemical studies. Most biochemical/biophysical methods that have been employed to study GPCR-G protein coupling require purified receptors and are technically difficult. Here, we provide a protocol for a relatively simple and time- and cost-effective membrane protein native PAGE assay, to visualize and biochemically characterize agonist-dependent coupling of detergent-solubilized GPCRs to purified G protein surrogate "mini-G" proteins, which stabilize the receptor in an active state. The assay was developed for our studies of the calcitonin receptor-like receptor, a class B GPCR that mediates the actions of calcitonin gene-related peptide and adrenomedullin peptide agonists. It does not require a purified receptor and it can be used in a screening format with transiently-transfected adherent mammalian cell cultures, to quickly identify detergent-stable complexes amenable to study, or in a quantitative format with membrane preparations, to determine apparent affinities of agonists for the mini-G-coupled receptor and apparent affinities of mini-G proteins for the agonist-occupied receptor. The latter provides a partial measure of agonist efficacy. The method should be applicable to other GPCRs, and has the potential to be adapted to the study of other challenging membrane proteins and their complexes with binding partners. Graphic abstract: Visualizing agonist-dependent mini-G protein coupling and determining apparent binding affinities using the native PAGE assay quantitative formats.

Molecular interaction of an antagonistic amylin analog with the extracellular domain of receptor activity-modifying protein 2 assessed by fluorescence polarization.

The peptide hormone amylin receptor is a complex of the calcitonin receptor (CTR) and an accessory protein called receptor activity-modifying proteins (RAMPs). The soluble extracellular domain (ECD) of CTR is an important binding site of peptide hormone calcitonin. RAMPs also have an ECD and the association of CTR ECD with RAMP ECD enhances the affinity of peptide hormone amylin. However, the mechanism of how RAMP ECD association enhances amylin affinity remains elusive. Here, we report evidence supporting direct molecular interaction between an antagonistic amylin analog AC413 and RAMP2 ECD. We measured FITC-labeled peptide affinity for purified receptor ECD using fluorescence polarization (FP). We first found that RAMP2 ECD addition to maltose-binding protein (MBP)-tagged CTR ECD and an engineered MBP-tagged RAMP2 ECD-CTR ECD fusion protein (MBP-RAMP2-CTR ECD fusion) enhanced AC413 affinity. This suggests that these recombinant ECD systems represent functional amylin receptors. Interestingly, AC413 C-terminal residue Tyr25 (Y25) to Pro mutation eliminated its selective affinity for the MBP-RAMP2-CTR ECD fusion suggesting the critical role of the AC413 C-terminal residue in amylin receptor selectivity. Our structural model of the RAMP2 ECD:CTR ECD complex predicted molecular interaction of AC413 C-terminal residue Y25 with RAMP2 Glu101 (E101). Our FP peptide-binding assay showed that the RAMP2 E101A mutation of MBP-RAMP2-CTR ECD fusion decreased AC413 affinity by 7-fold, while the affinity of AC413 with the Y25P mutation was minimally changed. Consistently, AC413 binding affinity for the MBP-free RAMP2-CTR ECD fusion protein was also markedly decreased by the RAMP2 E101A mutation, while the affinity of AC413 with the Y25P mutation was moderately decreased. Together, our results support the molecular interaction between the AC413 C-terminal residue Y25 and RAMP2 E101 expanding our understanding of how the accessory protein RAMP2 enhances affinity of peptide hormone amylin for its receptor.

Picomolar Affinity Antagonist and Sustained Signaling Agonist Peptide Ligands for the Adrenomedullin and Calcitonin Gene-Related Peptide Receptors.

The calcitonin receptor-like class B G protein-coupled receptor (CLR) mediates adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) functions including vasodilation, cardioprotection, and nociception. Receptor activity-modifying proteins (RAMP1-3) form heterodimers with CLR and determine its peptide ligand selectivity through an unresolved mechanism. The CGRP (RAMP1:CLR) and AM (RAMP2/3:CLR) receptors are proven or promising drug targets, but short AM and CGRP plasma half-lives limit their therapeutic utility. Here, we used synthetic peptide combinatorial library and rational design approaches to probe the ligand selectivity determinants and develop truncated AM and CGRP antagonist variants with receptor extracellular domain binding affinities that were enhanced ∼1000-fold into the low nanomolar range. Receptor binding studies and a high-resolution crystal structure of a novel library-identified AM variant bound to the RAMP2-CLR extracellular domain complex explained the increased affinities and defined roles for AM Lys46 and RAMP modulation of CLR conformation in the ligand selectivity mechanism. In longer AM and CGRP scaffolds that also bind the CLR transmembrane domain, the variants generated picomolar affinity antagonists, one with an estimated 12.5 h CGRP receptor residence time, and sustained signaling agonists "ss-AM" and "ss-CGRP" that exhibited persistent cAMP signaling after ligand washout. Sustained signaling was demonstrated in primary human umbilical vein endothelial cells and the SK-N-MC cell line, which endogenously express AM and CGRP receptors, respectively. This work clarifies the RAMP-modulated CLR ligand selectivity mechanism and provides AM and CGRP variants that are valuable pharmacological tools and may have potential as long-acting therapeutics.

Biochemical characterization of G protein coupling to calcitonin gene-related peptide and adrenomedullin receptors using a native PAGE assay.

Calcitonin gene-related peptide (CGRP), adrenomedullin (AM), and adrenomedullin 2/intermedin (AM2/IMD) have overlapping and unique functions in the nervous and circulatory systems including vasodilation, cardioprotection, and pain transmission. Their actions are mediated by the class B calcitonin-like G protein-coupled receptor (CLR), which heterodimerizes with three receptor activity-modifying proteins (RAMP1-3) that determine its peptide ligand selectivity. How the three agonists and RAMPs modulate CLR binding to transducer proteins remains poorly understood. Here, we biochemically characterized agonist-promoted G protein coupling to each CLR·RAMP complex. We adapted a native PAGE method to assess the formation and thermostabilities of detergent-solubilized fluorescent protein-tagged CLR·RAMP complexes expressed in mammalian cells. Addition of agonist and the purified Gs protein surrogate mini-Gs (mGs) yielded a mobility-shifted agonist·CLR·RAMP·mGs quaternary complex gel band that was sensitive to antagonists. Measuring the apparent affinities of the agonists for the mGs-coupled receptors and of mGs for the agonist-occupied receptors revealed that both ligand and RAMP control mGs coupling and defined how agonist engagement of the CLR extracellular and transmembrane domains affects transducer recruitment. Using mini-Gsq and -Gsi chimeras, we observed a coupling rank order of mGs > mGsq > mGsi for each receptor. Last, we demonstrated the physiological relevance of the native gel assays by showing that they can predict the cAMP-signaling potencies of AM and AM2/IMD chimeras. These results highlight the power of the native PAGE assay for membrane protein biochemistry and provide a biochemical foundation for understanding the molecular basis of shared and distinct signaling properties of CGRP, AM, and AM2/IMD.

RAMPs as allosteric modulators of the calcitonin and calcitonin-like class B G protein-coupled receptors.

Receptor activity-modifying proteins (RAMPs) are a family of three single span transmembrane proteins in humans that interact with many GPCRs and can modulate their function. RAMPs were discovered as key components of the calcitonin gene-related peptide and adrenomedullin receptors. They are required for transport of this class B GPCR, calcitonin receptor-like receptor (CLR), to the cell surface and determine its peptide ligand binding preferences. Soon thereafter RAMPs were shown to modulate the binding of calcitonin and amylin peptides to the related calcitonin receptor (CTR) and in the years since an ever-growing number of RAMP-interacting receptors have been identified including most if not all of the 15 class B GPCRs and several GPCRs from other families. Studies of CLR, CTR, and a handful of other GPCRs revealed that RAMPs are able to modulate various aspects of receptor function including trafficking, ligand binding, and signaling. Here, we review RAMP interactions and functions with an emphasis on class B receptors for which our understanding is most advanced. A key focus is to discuss recent evidence that RAMPs serve as endogenous allosteric modulators of CLR and CTR. We discuss structural studies of RAMP-CLR complexes and CTR and biochemical and pharmacological studies that collectively have significantly expanded our understanding of the mechanistic basis for RAMP modulation of these class B GPCRs. Last, we consider the implications of these findings for drug development targeting RAMP-CLR/CTR complexes.

Identification of Small-Molecule Positive Modulators of Calcitonin-like Receptor-Based Receptors.

Class B G protein-coupled receptors are highly therapeutically relevant but challenges remain in identifying suitable small-molecule drugs. The calcitonin-like receptor (CLR) in particular is linked to conditions such as migraine, cardiovascular disease, and inflammatory bowel disease. The CLR cannot act as a cell-surface receptor alone but rather must couple to one of three receptor activity-modifying proteins (RAMPs), forming heterodimeric receptors for the peptides adrenomedullin and calcitonin gene-related peptide. These peptides have extended binding sites across their receptors. This is one reason why there are few small-molecule ligands that can modulate these receptors. Here we describe small molecules that are able to positively modulate the signaling of the CLR with all three RAMPs but are not active at the related calcitonin receptor. These compounds were selected from a ß-arrestin recruitment screen, coupled with rounds of medicinal chemistry to improve their activity. Translational potential is shown as the compounds can positively modulate cAMP signaling in a vascular cell line model. Binding experiments do not support an extracellular domain binding site; however, molecular modeling reveals potential allosteric binding sites in multiple receptor regions. These are the first small-molecule positive modulators described for the CLR:RAMP complexes.

Calcitonin Receptor N-Glycosylation Enhances Peptide Hormone Affinity by Controlling Receptor Dynamics.

The class B G protein-coupled receptor (GPCR) calcitonin receptor (CTR) is a drug target for osteoporosis and diabetes. N-glycosylation of asparagine 130 in its extracellular domain (ECD) enhances calcitonin hormone affinity with the proximal GlcNAc residue mediating this effect through an unknown mechanism. Here, we present two crystal structures of salmon calcitonin-bound, GlcNAc-bearing CTR ECD at 1.78 and 2.85 Å resolutions and analyze the mechanism of the glycan effect. The N130 GlcNAc does not contact the hormone. Surprisingly, the structures are nearly identical to a structure of hormone-bound, N-glycan-free ECD, which suggested that the GlcNAc might affect CTR dynamics not observed in the static crystallographic snapshots. Hydrogen-deuterium exchange mass spectrometry and molecular dynamics simulations revealed that glycosylation stabilized a ß-sheet adjacent to the N130 GlcNAc and the N-terminal α-helix near the peptide-binding site while increasing flexibility of the peptide-binding site turret loop. These changes due to N-glycosylation increased the ligand on-rate and decreased its off-rate. The glycan effect extended to RAMP-CTR amylin receptor complexes and was also conserved in the related CGRP receptor. These results reveal that N-glycosylation can modulate GPCR function by altering receptor dynamics.

Structure-function analyses reveal a triple ß-turn receptor-bound conformation of adrenomedullin 2/intermedin and enable peptide antagonist design.

The cardioprotective vasodilator peptide adrenomedullin 2/intermedin (AM2/IMD) and the related adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) signal through three heterodimeric receptors comprising the calcitonin receptor-like class B G protein-coupled receptor (CLR) and a variable receptor activity-modifying protein (RAMP1, -2, or -3) that determines ligand selectivity. The CGRP receptor (RAMP1:CLR) favors CGRP binding, whereas the AM1 (RAMP2:CLR) and AM2 (RAMP3:CLR) receptors favor AM binding. How AM2/IMD binds the receptors and how RAMPs modulate its binding is unknown. Here, we show that AM2/IMD binds the three purified RAMP-CLR extracellular domain (ECD) complexes with a selectivity profile that is distinct from those of CGRP and AM. AM2/IMD bound all three ECD complexes but preferred the CGRP and AM2 receptor complexes. A 2.05 Å resolution crystal structure of an AM2/IMD antagonist fragment-bound RAMP1-CLR ECD complex revealed that AM2/IMD binds the complex through a unique triple ß-turn conformation that was confirmed by peptide and receptor mutagenesis. Comparisons of the receptor-bound conformations of AM2/IMD, AM, and a high-affinity CGRP analog revealed differences that may have implications for biased signaling. Guided by the structure, enhanced-affinity AM2/IMD antagonist variants were developed, including one that discriminates the AM1 and AM2 receptors with ∼40-fold difference in affinities and one stabilized by an intramolecular disulfide bond. These results reveal differences in how the three peptides engage the receptors, inform development of AM2/IMD-based pharmacological tools and therapeutics, and provide insights into RAMP modulation of receptor pharmacology.

Probing the Mechanism of Receptor Activity-Modifying Protein Modulation of GPCR Ligand Selectivity through Rational Design of Potent Adrenomedullin and Calcitonin Gene-Related Peptide Antagonists.

Binding of the vasodilator peptides adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) to the class B G protein-coupled receptor calcitonin receptor-like receptor (CLR) is modulated by receptor activity-modifying proteins (RAMPs). RAMP1 favors CGRP, whereas RAMP2 and RAMP3 favor AM. Crystal structures of peptide-bound RAMP1/2-CLR extracellular domain (ECD) heterodimers suggested RAMPs alter ligand preference through direct peptide contacts and allosteric modulation of CLR. Here, we probed this dual mechanism through rational structure-guided design of AM and CGRP antagonist variants. Variants were characterized for binding to purified RAMP1/2-CLR ECD and for antagonism of the full-length CGRP (RAMP1:CLR), AM1 (RAMP2:CLR), and AM2 (RAMP3:CLR) receptors. Short nanomolar affinity AM(37-52) and CGRP(27-37) variants were obtained through substitutions including AM S45W/Q50W and CGRP K35W/A36S designed to stabilize their ß-turn. K46L and Y52F substitutions designed to exploit RAMP allosteric effects and direct peptide contacts, respectively, yielded AM variants with selectivity for the CGRP receptor over the AM1 receptor. AM(37-52) S45W/K46L/Q50W/Y52F exhibited nanomolar potency at the CGRP receptor and micromolar potency at AM1 A 2.8-Å resolution crystal structure of this variant bound to the RAMP1-CLR ECD confirmed that it bound as designed. CGRP(27-37) N31D/S34P/K35W/A36S exhibited potency and selectivity comparable to the traditional antagonist CGRP(8-37). Giving this variant the ability to contact RAMP2 through the F37Y substitution increased affinity for AM1, but it still preferred the CGRP receptor. These potent peptide antagonists with altered selectivity inform the development of AM/CGRP-based pharmacological tools and support the hypothesis that RAMPs alter CLR ligand selectivity through allosteric effects and direct peptide contacts.

Molecular Signature for Receptor Engagement in the Metabolic Peptide Hormone Amylin.

The pancreatic peptide hormone, amylin, plays a critical role in the control of appetite, and synergizes with other key metabolic hormones such as glucagon-like peptide 1 (GLP-1). There is opportunity to develop potent and long-acting analogues of amylin or hybrids between these and GLP-1 mimetics for treating obesity. To achieve this, interrogation of how the 37 amino acid amylin peptide engages with its complex receptor system is required. We synthesized an extensive library of peptides to profile the human amylin sequence, determining the role of its disulfide loop, amidated C-terminus and receptor "capture" and "activation" regions in receptor signaling. We profiled four signaling pathways with different ligands at multiple receptor subtypes, in addition to exploring selectivity determinants between related receptors. Distinct roles for peptide subregions in receptor binding and activation were identified, resulting in peptides with greater activity than the native sequence. Enhanced peptide activity was preserved in the brainstem, the major biological target for amylin. Interpretation of our data using full-length active receptor models supported by molecular dynamics, metadynamics, and supervised molecular dynamics simulations guided the synthesis of a potent dual agonist of GLP-1 and amylin receptors. The data offer new insights into the function of peptide amidation, how allostery drives peptide-receptor interactions, and provide a valuable resource for the development of novel amylin agonists for treating diabetes and obesity.

N-Glycosylation of Asparagine 130 in the Extracellular Domain of the Human Calcitonin Receptor Significantly Increases Peptide Hormone Affinity.

The calcitonin receptor (CTR) is a class B G protein-coupled receptor that is activated by the peptide hormones calcitonin and amylin. Calcitonin regulates bone remodeling through CTR, whereas amylin regulates blood glucose and food intake by activating CTR in complex with receptor activity-modifying proteins (RAMPs). These receptors are targeted clinically for the treatment of osteoporosis and diabetes. Here, we define the role of CTR N-glycosylation in hormone binding using purified calcitonin and amylin receptor extracellular domain (ECD) glycoforms and fluorescence polarization/anisotropy and isothermal titration calorimetry peptide-binding assays. N-Glycan-free CTR ECD produced in Escherichia coli exhibited ∼10-fold lower peptide affinity than CTR ECD produced in HEK293T cells, which yield complex N-glycans, or in HEK293S GnTI- cells, which yield core N-glycans (Man5GlcNAc2). PNGase F-catalyzed removal of N-glycans at N73, N125, and N130 in the CTR ECD decreased peptide affinity ∼10-fold, whereas Endo H-catalyzed trimming of the N-glycans to single GlcNAc residues had no effect on peptide binding. Similar results were observed for an amylin receptor RAMP2-CTR ECD complex. Characterization of peptide-binding affinities of purified N → Q CTR ECD glycan site mutants combined with PNGase F and Endo H treatment strategies and mass spectrometry to define the glycan species indicated that a single GlcNAc residue at CTR N130 was responsible for the peptide affinity enhancement. Molecular modeling suggested that this GlcNAc functions through an allosteric mechanism rather than by directly contacting the peptide. These results reveal an important role for N-linked glycosylation in the peptide hormone binding of a clinically relevant class B GPCR.

An allosteric role for receptor activity-modifying proteins in defining GPCR pharmacology.

G protein-coupled receptors are allosteric proteins that control transmission of external signals to regulate cellular response. Although agonist binding promotes canonical G protein signalling transmitted through conformational changes, G protein-coupled receptors also interact with other proteins. These include other G protein-coupled receptors, other receptors and channels, regulatory proteins and receptor-modifying proteins, notably receptor activity-modifying proteins (RAMPs). RAMPs have at least 11 G protein-coupled receptor partners, including many class B G protein-coupled receptors. Prototypic is the calcitonin receptor, with altered ligand specificity when co-expressed with RAMPs. To gain molecular insight into the consequences of this protein-protein interaction, we combined molecular modelling with mutagenesis of the calcitonin receptor extracellular domain, assessed in ligand binding and functional assays. Although some calcitonin receptor residues are universally important for peptide interactions (calcitonin, amylin and calcitonin gene-related peptide) in calcitonin receptor alone or with receptor activity-modifying protein, others have RAMP-dependent effects, whereby mutations decreased amylin/calcitonin gene-related peptide potency substantially only when RAMP was present. Remarkably, the key residues were completely conserved between calcitonin receptor and AMY receptors, and between subtypes of AMY receptor that have different ligand preferences. Mutations at the interface between calcitonin receptor and RAMP affected ligand pharmacology in a RAMP-dependent manner, suggesting that RAMP may allosterically influence the calcitonin receptor conformation. Supporting this, molecular dynamics simulations suggested that the calcitonin receptor extracellular N-terminal domain is more flexible in the presence of receptor activity-modifying protein 1. Thus, RAMPs may act in an allosteric manner to generate a spectrum of unique calcitonin receptor conformational states, explaining the pharmacological preferences of calcitonin receptor-RAMP complexes. This provides novel insight into our understanding of G protein-coupled receptor-protein interaction that is likely broadly applicable for this receptor class.

Erratum: An allosteric role for receptor activity-modifying proteins in defining GPCR pharmacology.

[This corrects the article DOI: 10.1038/celldisc.2016.12.].

Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties.

Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins 2 and 3 (RAMP2 and RAMP3), respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMP2 and -3 on the activation and conformation of the CLR subunit of AM receptors, we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors, and determined the effects on cAMP signaling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modeling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function.

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.