Calcitonin Family Of Peptides Research Articles

A narrative review of the calcitonin peptide family and associated receptors as migraine targets: Calcitonin gene-related peptide and beyond.

To summarize the pharmacology of the calcitonin peptide family of receptors and explore their relationship to migraine and current migraine therapies. Therapeutics that dampen calcitonin gene-related peptide (CGRP) signaling are now in clinical use to prevent or treat migraine. However, CGRP belongs to a broader peptide family, including the peptides amylin and adrenomedullin. Receptors for this family are complex, displaying overlapping pharmacologic profiles. Despite the focus on CGRP and the CGRP receptor in migraine research, recent evidence implicates related peptides and receptors in migraine. This narrative review summarizes literature encompassing the current pharmacologic understanding of the calcitonin peptide family, and the evidence that links specific members of this family to migraine and migraine-like behaviors. Recent work links amylin and adrenomedullin to migraine-like behavior in rodent models and migraine-like attacks in individuals with migraine. We collate novel information that suggests females may be more sensitive to amylin and CGRP in the context of migraine-like behaviors. We report that drugs designed to antagonize the canonical CGRP receptor also antagonize a second CGRP-responsive receptor and speculate as to whether this influences therapeutic efficacy. We also discuss the specificity of current drugs with regards to CGRP isoforms and how this may influence therapeutic profiles. Lastly, we emphasize that receptors related to, but distinct from, the canonical CGRP receptor may represent underappreciated and novel drug targets. Multiple peptides within the calcitonin family have been linked to migraine. The current focus on CGRP and its canonical receptor may be obscuring pathways to further therapeutics. Drug discovery schemes that take a wider view of the receptor family may lead to the development of new anti-migraine drugs with favorable clinical profiles. We also propose that understanding these related peptides and receptors may improve our interpretation regarding the mechanism of action of current drugs.

Beyond CGRP: The calcitonin peptide family as targets for migraine and pain.

The CGRP system has emerged as a key pharmacological target for the treatment of migraine. However, some individuals who suffer from migraine have low or no response to anti-CGRP or other treatments, suggesting the need for additional clinical targets. CGRP belongs to the calcitonin family of peptides, which includes calcitonin, amylin, adrenomedullin and adrenomedullin 2. These peptides display a range of pro-nociceptive and anti-nociceptive actions, in primary headache conditions such as migraine. Calcitonin family peptides also show expression at sites relevant to migraine and pain. This suggests that calcitonin family peptides and their receptors, beyond CGRP, may be therapeutically useful in the treatment of migraine and other pain disorders. This review considers the localisation of the calcitonin family in peripheral pain pathways and discusses how they may contribute to migraine and pain. LINKED ARTICLES: This article is part of a themed issue on Advances in Migraine and Headache Therapy (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.3/issuetoc.

2037-P: Characterization of Novel Unimolecular Amylin-Adrenomedullin Dual Agonists

Amylin and Adrenomedullin both belong to the calcitonin peptide family. While amylin plays an important role in maintaining glucose and energy homeostasis, adrenomedullin is primarily known for its beneficial vasoactive effects. We hypothesize that combining the glucoregulatory and anti-obesity properties of amylin with the cardioprotective effects of adrenomedullin could be an effective treatment strategy to address type 2 diabetes, obesity and cardiovascular diseases in one molecule. Here we report the design and receptor potencies of unimolecular peptide agonists with dual activity at the amylin and adrenomedullin receptors. Using solid-phase peptide synthesis, an array of hybrid peptides was rationally designed by substituting amino acids essential for amylin activity into the native adrenomedullin sequence. The pharmacokinetic profile was optimized by lipidation with a C20 diacid. Functional activities of the analogues were measured using a cAMP accumulation assay in cells overexpressing the human amylin receptor subtype 3 (hAMY3-R) or human adrenomedullin receptor subtype 1 (hAM1-R). All analogues were full agonists on both receptors. hAMY3R potencies were comparable to human native amylin, whereas hAM1-R potencies ranged from equipotent to 100-fold less potent compared to human native adrenomedullin. In summary, we have synthesized novel hybrid amylin-adrenomedullin peptide agonists with dual activity at the hAMY3R and hAM1-R. Ongoing studies address in vivo efficacy and potency of amylin-adrenomedullin dual agonists in rodent models of obesity and diabetes. Disclosure L.S. Dalboege: None. P. Magotti: None. N. Buch-Månson: None. E.M. Bech: None. L.N. Fink: Employee; Self; Gubra. Stock/Shareholder; Self; Novo Nordisk A/S. S.L. Pedersen: None. K. Fosgerau: Stock/Shareholder; Self; Gubra.

Intrinsically Disordered Protein Dynamics Uncovered through Dynamic Flexibility Index (DFI) Analysis

Calcitonin gene-related peptide (CGRP) and islet amyloid polypeptide (IAPP) are two intrinsically disordered proteins (IDP) of the calcitonin peptide family. CGRP is an important new target for antimigraine drugs, it is involved in vasodilation and transmission of pain signals. IAPP regulates blood glucose levels and forms amyloid fibers in type II diabetes. They share up to 40% sequence similarity and bind to very similar receptors, but are found in different types of tissue and carry out different functions. Recent experimental studies measuring end-to-end contact formation by tryptophan triplet quenching, show a significant difference in the conformational dynamics of CGRP and IAPP. We investigate this through Dynamic Flexibility Index (DFI). DFI quantifies the resilience of a given position to the perturbations that occur at different parts of the chain, using linear response theory. This captures multi-dimensional effects when the chain is displaced out of equilibrium. Thus, it identifies flexible and rigid positions that govern the function. Our DFI profiles obtained from equilibrated atomistic MD simulations reveal distinct patterns for CGRP and IAPP. We investigate the effect of amino acid substitutions on the conformational dynamics of the unbound disordered IDP, and pinpoint the positions that give a significant difference in dynamics.

Calcitonin Peptide Family Members Are Differentially Regulated by LPS and Inhibit Functions of Rat Alveolar NR8383 Macrophages.

Members of the calcitonin peptide family—calcitonin gene-related peptide (CGRP), adrenomedullin (AM), and adrenomedullin2/intermedin (IMD)–exert modulatory effects upon monocytes and macrophages of various extrapulmonary origins. Utilizing the rat alveolar macrophage (AMφ) cell line NR8383, we here set out to determine to which extent these three peptides and their receptors are differentially regulated in AMφ and what specific effects they have on AMφ key functions. LPS treatment differentially up-regulated expression of the peptides and receptors. Among the three peptides, IMD mRNA content was lowest both in primary rat AMφ and NR8383 cells, whereas IMD peptide dominated in basal and LPS-stimulated secretion from NR8383 cells. Fcγ receptor-mediated phagocytosis and TNF-α production were inhibited by AM, IMD, and CGRP, whereas pro-IL-1β mRNA was slightly down-regulated exclusively by CGRP. Neither of these peptides affected IL-6 or IL-10 production. None increased intracellular calcium concentration, but AM significantly inhibited store-operated calcium entry. In conclusion, the rat AMφ cell line NR8383 is both a source and a target of the calcitonin peptide family members AM, IMD, and CGRP. Despite sharing proteins of the receptor complexes, AM, IMD, and CGRP each showed a characteristic pattern of effects and regulation, suggesting that these closely related peptides are not just redundant members of one common signaling pathway but act in concert by addressing parallel signaling cascades. Since peptide and receptor expression are up-regulated by LPS, these signaling pathways might act as inhibitory feedback mechanisms in pulmonary bacterial infection.

Slow Internal Dynamics and Structural Properties of IDPs of the Ct Family: Comparing Amyloid and Non-Amyloid Variants

The calcitonin peptide (Ct) family includes intrinsically disordered peptides which function as hormones (regulating glucose levels in the blood, calcium resorption in the bone, vasodilation and the transmission of pain signals, and triggering migraine attacks). They have highly conserved sequence elements, share key structural features, and bind to similar receptors. Their characterization is extremely challenging due to their low molecular weight and size, the absence of persistent secondary structure, and the high propensity of some Ct sequences to aggregate into pathological amyloid fibers. Using nanosecond laser-pump spectroscopy, in combination with other techniques, allows quantifying structural and dynamical features of these IDPs which are important for their function and pathological aggregation.

Slow Internal Dynamics and Charge Expansion in the Disordered Protein CGRP: A Comparison with Amylin

We provide the first direct experimental comparison, to our knowledge, between the internal dynamics of calcitonin-gene-related peptide (CGRP) and amylin (islet amyloid polypeptide, IAPP), two intrinsically disordered proteins of the calcitonin peptide family. Our end-to-end contact formation measurements reveal that in aqueous solution (i.e., in the absence of structure-inducing organic solvents) CGRP preferentially populates conformations with short end-to-end distances. However, the end-to-end distance of CGRP is larger than that of IAPP. We find that electrostatic interactions can account for such a difference. At variance with previous reports on the secondary structure of CGRP, we find that the end-to-end distance of the peptide increases with decreasing pH and salt concentration, due to Coulomb repulsion by charged residues. Interestingly, our data show that the reconfiguration dynamics of CGRP is significantly slower than that of human IAPP in water but not in denaturant, providing experimental evidence for roughness in the energy landscape, or internal friction, in these peptides. The data reported here provide both structural and dynamical information that can be used to validate results from molecular simulations of calcitonin family peptides in aqueous solution.

Evidence for Internal Friction in IDPs of the Calcitonin Peptide Family

The rate at which Intrinsically Disordered Proteins (IDPs) undergo large conformational changes in solution is expected to substantially affect their function. The roughness in IDPs energy landscape can significantly slow down this diffusional dynamics, giving rise to internal friction. Both experiments and simulations of unfolded states of proteins show that compact states exhibit greater internal friction than denaturant-expanded states1,2. Yet it is unclear to what extent, and in which way, changes in a protein sequence may affect the internal friction. Using a nanosecond laser-pump spectroscopy technique we quantify the rate at which two ends of a polypeptide chain come into contact, while also quantifying the relative end-to-end distance (or compactness) of the chain. We compare these values for different IDPs of the Calcitonin peptide (Ct) family. Our previous studies showed that these IDPs populate highly compact states in near native conditions3,4 and become more expanded at increasing denaturant concentrations5,6. We find that, under conditions in which the end-to-end distances are the same, certain sequences exhibit significantly slower contact formation rates than others, indicating greater internal friction. As expected, such differences are apparent in water but not in denaturant. We experimentally investigate the possible causes for the observed sequence-dependence.1. Soranno, Andrea, et al. PNAS 2012; 109:17800-17806.2. Echeverria, Ignacia, et al. JACS 2014; 136:8708-8713.3. Vaiana, Sara M., et al. BIOPHYSICAL J 2009; 97:2948-2957.4. Sizemore, Sara M., et al. BIOPHYSICAL J 2013; 104:54A-55A.5. Sizemore, Sara M., et al. BIOPHYSICAL J 2014; 106:270A.6. Cope, Stephanie M., et al. BIOPHYSICAL J 2014; 106:687A.

Comparing Solution Structures of Amylin and CGRP by Nanosecond Laser-Pump Spectroscopy and Atomistic Simulations

Amylin and calcitonin gene-related peptide (CGRP) are intrinsically disordered proteins, members of the calcitonin (Ct) peptide family. They are found with their respective receptors in different organs and carry out different functions. Amylin is involved in regulating glucose metabolism and is implicated in type II diabetes, while CGRP is a vasodilator involved in transmitting pain signals in the nervous system, and triggers migraine attacks. Amylin and CGRP share 47% sequence homology and are able to bind to each other's receptors and activate cell response. Such cross-reactivity is attributed to their possible structural similarity. Solution state NMR experiments show that both peptides are disordered and locally sample transient helical states close to the N-terminus. While such short-range structural properties have been compared, it is not clear whether the long-range properties are affected or not. Here we combine results from experiments, probing both long- and short-range properties of the two peptides, with results from replica exchange molecular dynamics (REMD) simulations. To measure a long-range property directly comparable to simulations, we use a nanosecond laser-pump spectroscopy technique based on tryptophan triplet quenching. This allows probing both the end-to-end distance and the rate of end-to-end contact formation in IDPs, without using prosthetic dyes. Because of the short length of our peptides and the high aggregation propensity of amylin, this information cannot be obtained using other techniques such as FRET. Our data show that both the secondary structure content and the end-to-end distance of the two peptides differ significantly, and that such differences are affected by electrostatic interactions. Both our experiments and REMD simulations indicate that long-range interactions (i.e. interactions between residues that are far away in the sequence), play a significant role in determining the peptide structural ensemble in solution.

Charge Patterning, Salt Screening and Denaturant Expansion in the CGRP Neuropeptide

Calcitonin gene related peptide (CGRP) is a neuropeptide of the calcitonin peptide family, which acts as a vasodilator and is involved in the transmission of pain signals in the nervous system upon binding to the correspondent receptor. It also triggers migraine attacks, and is a major therapeutic target for the prevention of migraines. In physiological conditions, CGRP is intrinsically disordered and, therefore, the binding to its receptor (or to drugs) will depend strongly on the structural and dynamical properties of the disordered unbound state. Such properties can be affected in vivo by changes in salt concentration and pH. However, while some information is available on CGRP's sampling of local secondary structural elements, very little is known about its long-range (“tertiary”) structural and dynamical properties. Detecting such properties is challenging because CGRP has a low molecular weight and samples many different conformations on very fast time scales. We use a nanosecond laser-pump spectroscopy technique, based on tryptophan triplet quenching, which allows probing the end-to-end distance and the rate of end-to-end contact formation in IDPs. This provides similar information to FRET, but without the use of prosthetic dyes. Our data show that CGRP populates compact states in buffer, which are extremely sensitive to pH and salt concentration. We find that a change from pH 8 to pH 3 can induce a significant expansion of conformations due to the modulation of charge interactions, with a dramatic change of the corresponding salt screening effects. This suggests a key role of specific charged residues in CGRP. In addition, we find a “denaturant expansion” effect that depends on the nature of the denaturant. The observations can be rationalized in terms of polymer models where the polyelectrolyte/polyampholyte nature of the peptide is taken into account.

Structure and Internal Dynamics of Calcitonin Family Peptides: Implications for Amyloid Formation

The calcitonin peptide (Ct) family comprises the intrinsically disordered proteins amylin (IAPP), calcitonin gene-related peptide (CGRP), calcitonin, and adrenomedullins. These are genetically and structurally related hormone peptides that are able to bind to each other's receptors, though with varying degrees of affinity. Some of these peptides form amyloid fibers, while others do not. They contain highly conserved sequence elements that have been experimentally shown to affect the secondary structural preferences of these peptides. The effect of such conserved elements on tertiary structure has not been experimentally explored to the same extent. Detecting tertiary structural properties of IDPs is considerably more challenging due to fast reconfigurations of the backbone over a wide range of possible conformations. High resolution time-resolved techniques are needed. We use a nanosecond laser spectroscopy technique to measure transient tertiary contact formation. This technique reveals information on the structure and internal dynamics of IDPs. We compare members of the Ct family which differ in hydrophobicity and net charge, and study the effect of proline mutations on contact formation rates. We find that functionally required, conserved sequence elements play an important role in determining the structure, internal dynamics and aggregation properties of these peptides.

Solvent Effects on the Structure and Internal Dynamics of Calcitonin Gene-Related Peptide

Calcitonin gene-related peptide (CGRP) is an intrinsically disordered, 37 residue neuropeptide that acts as a potent vasodilator. It is a member of the calcitonin peptide (Ct) family, together with amylin, calcitonin and adrenomedullin. Understanding how sequence and solvent variations affect the conformation and dynamics of these genetically and functionally related IDPs is of considerable interest. We use a nanosecond-resolved spectroscopic technique based on tryptophan triplet quenching by cystine to detect transient tertiary contact formation in CGRP under varying solvent and temperature conditions. Using this technique, we had previously found that electrostatic interactions modulate the degree of compaction in CGRP1. Here we explore the effect of solvent on CGRP structure and dynamics. We find that, though disordered, CGRP is very sensitive to small variations in the solution environment. Our findings can be rationalized in terms of polymer models and residual secondary structure content.1 Sara M. Sizemore et al. 2013 Biophys. J, Supplement, Abstract.

Cyclic N-Terminal Loop of Amylin Forms Non Amyloid Fibers

We report for the first time, to our knowledge, that the N-terminal loop (N_loop) of amylin (islet amyloid polypeptide (IAPP) residues 1–8) forms extremely long and stable non-β-sheet fibers in solution under the same conditions in which human amylin (hIAPP) forms amyloid fibers. This observation applies to the cyclic, oxidized form of the N_loop but not to the linear, reduced form, which does not form fibers. Our findings indicate a potential role of direct N_loop-N_loop interactions in hIAPP aggregation, which has not been previously explored, with important implications for the mechanism of hIAPP amyloid fiber formation, the inhibitory action of IAPP variants, and the competition between ordered and disordered aggregation in peptides of the calcitonin peptide family.

Electrostatic Interactions Modulate the Structure and Dynamics of Calcitonin Gene-Related Peptide

Calcitonin gene-related peptide (CGRP) is an intrinsically disordered, 37 residue neuropeptide that acts as a potent vasodilator. It is a member of the calcitonin peptide (Ct) family, together with amylin, calcitonin and adrenomedullin. Understanding how sequence variations affect the conformation and dynamics of these genetically and functionally related IDPs is of considerable interest. While the secondary structural preferences of these peptides have been experimentally determined, the tertiary structural preferences are more challenging to detect due to fast reconfigurations of the backbone over a wide range of conformations, and to the very low solubility of certain Ct family peptides. High resolution time-resolved techniques are needed.We use a nanosecond-resolved spectroscopic technique based on tryptophan triplet quenching by cystine to detect tertiary contact formation in CGRP under varying solvent and temperature conditions. Using this technique we had previously found that conserved structural elements of amylin and CGRP induce compact states, characterized by short end-to-end distances(1,2). Here we compare contact formation rates of wild type and mutant CGRPs with different net charge. We find that electrostatic interactions modulate the degree of compaction of CGRP. The observed difference between CGRP and amylin can be rationalized in terms of electrostatic interactions. Interestingly, even with a net charge of +6, CGRP remains more compact in aqueous solvent than in denaturant with high ionic strength, indicating strong attractive intra-molecular interactions. Overall our data are consistent with a common mechanism driving compaction in CGRP and amylin, with electrostatic interactions modulating the degree of compaction in CGRP. We discuss our findings in relation to secondary structural preferences of these peptides and discuss their possible functional role.1 Vaiana S.M. et al. Biophys. J. 97 2009.2 Sara M. Sizemore et al.2012 Biophys. J, Supplement, Abstract.

The pharmacology of Adrenomedullin 2/Intermedin

Adrenomedullin 2 (AM2) or intermedin is a member of the calcitonin gene-related peptide (CGRP)/calcitonin family of peptides and was discovered in 2004. Unlike other members of this family, no unique receptor has yet been identified for it. It is extensively distributed throughout the body. It causes hypotension when given peripherally, but when given into the CNS, it increases blood pressure and causes sympathetic activation. It also increases prolactin release, is anti-diuretic and natriuretic and reduces food intake. Whilst its effects resemble those of AM, it is frequently more potent. Some characterization of AM2 has been done on molecularly defined receptors; the existing data suggest that it preferentially activates the AM(2) receptor formed from calcitonin receptor-like receptor and receptor activity modifying protein 3. On this complex, its potency is generally equivalent to that of AM. There is no known receptor-activity where it is more potent than AM. In tissues and in animals it is frequently antagonised by CGRP and AM antagonists; however, situations exist in which an AM2 response is maintained even in the presence of supramaximal concentrations of these antagonists. Thus, there is a partial mismatch between the pharmacology seen in tissues and that on cloned receptors. The only AM2 antagonists are peptide fragments, and these have limited selectivity. It remains unclear as to whether novel AM2 receptors exist or whether the mismatch in pharmacology can be explained by factors such as metabolism.

Localization of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) in human gastrointestinal tract

Calcitonin gene-related peptide (CGRP) exerts its diverse effects on vasodilation, nociception, secretion, and motor function through a heterodimeric receptor comprising of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). Despite the importance of CLR·RAMP1 in human disease, little is known about its distribution in the human gastrointestinal (GI) tract, where it participates in inflammation and pain. In this study, we determined that CLR and RAMP1 mRNAs are expressed in normal human stomach, ileum and colon by RT-PCR. We next characterized antibodies that we generated to rat CLR and RAMP1 in transfected HEK cells. Having characterized these antibodies in vitro, we then localized CLR-, RAMP1-, CGRP- and intermedin-immunoreactivity (IMD-IR) in various human GI segments. In the stomach, nerve bundles in the myenteric plexus and nerve fibers throughout the circular and longitudinal muscle had prominent CLR-IR. In the proximal colon and ileum, CLR was found in nerve varicosities of the myenteric plexus and surrounding submucosal neurons. Interestingly, CGRP expressing fibers did not co-localize, but were in close proximity to CLR. However, CLR and RAMP1, the two subunits of a functional CGRP receptor were clearly localized in myenteric plexus, where they may form functional cell-surface receptors. IMD, another member of calcitonin peptide family was also found in close proximity to CLR, and like CGRP, did not co-localize with either CLR or RAMP1 receptors. Thus, CGRP and IMD appear to be released locally, where they can mediate their effect on their receptors regulating diverse functions such as inflammation, pain and motility.

Transient Tertiary Contact Formation in the CGRP Neuropeptide Revealed by Nanosecond Laser Spectroscopy

Calcitonin gene-related peptide (CGRP) is an intrinsically disordered, 37 residue neuropeptide that acts as a potent vasodilator, which is of considerable interest in migrane research. It is a member of the calcitonin peptide (Ct) family, together with amylin, calcitonin and adrenomedullin. These are genetically and structurally related intrinsically disordered hormone peptides that are able to bind to each other’s receptors, though with varying degrees of affinity. They contain highly conserved sequence elements that have been experimentally shown to affect the secondary structural preferences of these peptides. The effect of such conserved elements on tertiary structure has not been experimentally explored to the same extent. Detecting tertiary structural properties of IDPs is considerably more challenging due to fast reconfigurations of the backbone over a wide range of possible conformations. High resolution time-resolved techniques are needed.We use a nanosecond-resolved spectroscopic technique based on tryptophan triplet quenching by cystine to detect tertiary contact formation in CGRP under varying solvent and temperature conditions. Using this technique, Vaiana et al.1 have previously shown that conserved structural elements of amylin induce compact states characterized by short end-to-end distances, and that compaction is not driven by hydrophobic side-chains. Using triplet quenching we explore the effect of these conserved elements on the conformation and dynamics of CGRP, a peptide with higher mean hydrophobicity and net charge per residue than amylin. We discuss our findings in relation to secondary structural preferences of these peptides and discuss their possible functional role.Footnote1 Vaiana S.M. et al. Biophys. J. 97 2009.

Comparisons between the effects of calcitonin receptor‐stimulating peptide and intermedin and other peptides in the calcitonin family on bone resorption and osteoclastogenesis

Calcitonin receptor-stimulating peptide (CRSP) and intermedin (IMD) are two recently discovered peptides in the calcitonin (CT) family of peptides. CRSP and IMD, similar to CT, calcitonin gene-related peptide (CGRP), and amylin (AMY), but in contrast to adrenomedullin (ADM), inhibited bone resorption in mouse calvarial bones. CRSP and IMD, similar to CT, CGRP, AMY, but in contrast to ADM, decreased formation of osteoclasts and number of pits in bone marrow macrophage cultures stimulated by M-CSF and RANKL, with no effect on the expression of a number of genes associated with osteoclast progenitor cell differentiation. CRSP and IMD inhibited osteoclastogenesis at a late stage but had no effect on DC-STAMP mRNA. IMD, similar to CGRP, AMY, and ADM stimulated cyclic AMP formation in M-CSF expanded osteoclast progenitor cells lacking CT receptors (CTRs). RANKL induced CTRs and a cyclic AMP response also to CT and CRSP, and increased the cyclic AMP response to CGRP, AMY, and IMD but decreased the response to ADM. Our data demonstrates that CRSP and IMD share several functional properties of peptides in the CT family of peptides, including inhibition of bone resorption and osteoclast formation. The data also show that the reason why ADM does not inhibit osteoclast activity or formation is related to the fact that RANKL decreases ADM receptor signaling through the adenylate cyclase-cyclic AMP pathway. Finally, the findings indicate that activation by CGRP, AMY, and IMD may include activation of both CT and CT receptor-like receptors.

Structure–function relationships of the N‐terminus of receptor activity‐modifying proteins

The receptor activity-modifying proteins (RAMPs) are a family of three single transmembrane proteins that have been identified as accessory proteins to some G-protein-coupled receptors (GPCRs). They can regulate their pharmacology, forward trafficking and recycling, depending on the GPCR. The best characterized receptor complexes formed by RAMPs and GPCRs are the calcitonin peptide family receptors. The association of RAMP1 with the calcitonin receptor-like receptor (CL) constitutes the calcitonin gene-related peptide receptor, whereas RAMP2 or 3 with CL generates adrenomedullin receptors. In this case, the RAMPs substantially alter the pharmacology and trafficking properties of this GPCR. Amylin receptor subtypes are formed from calcitonin receptor (CTR) interactions with RAMPs. Although the RAMPs themselves are not responsive to calcitonin peptide family ligands, there is clear evidence that they participate in ligand binding, although it is still unclear whether this is by directly participating in binding or through allosteric modulation of CL or CTR. A considerable amount of mutagenesis data have now been generated on RAMPs to try and identify the residues that play a role in ligand interactions, and to also identify which residues in RAMPs interact with CL and CTR. This review will focus on RAMP mutagenesis studies with CL, summarizing and discussing the available data in association with current RAMP models and structures. The data reveal key regions in RAMPs that are important for ligand binding and receptor interactions.

Novel Peptide Antagonists of Adrenomedullin and Calcitonin Gene-Related Peptide Receptors: Identification, Pharmacological Characterization, and Interactions with Position 74 in Receptor Activity-Modifying Protein 1/3

Human adrenomedullin (AM) is a 52-amino acid peptide belonging to the calcitonin peptide family, which also includes calcitonin gene-related peptide (CGRP) and AM2. The two AM receptors, AM(1) and AM(2), are calcitonin receptor-like receptor (CL)/receptor activity-modifying protein (RAMP) (RAMP2 and RAMP3, respectively) heterodimers. CGRP receptors comprise CL/RAMP1. The only human AM receptor antagonist (AM(22-52)) is a truncated form of AM; it has low affinity and is only weakly selective for AM(1) over AM(2) receptors. To develop novel AM receptor antagonists, we explored the importance of different regions of AM in interactions with AM(1), AM(2), and CGRP receptors. AM(22-52) was the framework for generating further AM fragments (AM(26-52) and AM(30-52)), novel AM/alphaCGRP chimeras (C1-C5 and C9), and AM/AM(2) chimeras (C6-C8). cAMP assays were used to screen the antagonists at all receptors to determine their affinity and selectivity. Circular dichroism spectroscopy was used to investigate the secondary structures of AM and its related peptides. The data indicate that the structures of AM, AM2, and alphaCGRP differ from one another. Our chimeric approach enabled the identification of two nonselective high-affinity antagonists of AM(1), AM(2), and CGRP receptors (C2 and C6), one high-affinity antagonist of AM(2) receptors (C7), and a weak antagonist selective for the CGRP receptor (C5). By use of receptor mutagenesis, we also determined that the C-terminal nine amino acids of AM seem to be responsible for its interaction with Glu74 of RAMP3. We provide new information on the structure-activity relationship of AM, alphaCGRP, and AM2 and how AM interacts with CGRP and AM(2) receptors.