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Introduction: Adrenomedullin (AM) is a peptide responsible for many physiological processes including vascular health and hormone regulation. Dysregulation of AM signaling can stimulate cancers by promoting proliferation, angiogenesis and metastasis. Two AM receptors contribute to tumor progression in different ways. Adrenomedullin-1 receptor (AM 1 R) regulates blood pressure and blocking AM signaling via AM 1 R would be clinically unacceptable. Therefore, antagonizing adrenomedullin-2 receptor (AM 2 R) presents as an avenue for anti-cancer drug development., Areas Covered: We review the literature to highlight AM's role in cancer as well as delineating the specific roles AM 1 R and AM 2 R mediate in the development of a pro-tumoral microenvironment. We highlight the importance of exploring the residue differences between the receptors that led to the development of first-in-class selective AM 2 R small molecule antagonists. We also summarize the current approaches targeting AM and its receptors, their anti-tumor effects and their limitations., Expert Opinion: As tool compounds, AM 2 R antagonists will allow the dissection of the functions of CGRPR (calcitonin gene-related peptide receptor), AM 1 R and AM 2 R, and has considerable potential as a first-in-class oncology therapy. Furthermore, the lack of detectable side effects and good drug-like pharmacokinetic properties of these AM 2 R antagonists support the promise of this class of compounds as potential anti-cancer therapeutics.

A series of macrocyclic calcitonin gene-related peptide (CGRP) receptor antagonists identified using structure-based design principles, exemplified by HTL0028016 ( 1 ) and HTL0028125 ( 2 ), is described. Structural characterization by X-ray crystallography of the interaction of two of the macrocycle antagonists with the CGRP receptor ectodomain is described, along with structure-activity relationships associated with point changes to the macrocyclic antagonists. The identification of non-peptidic/natural product-derived, macrocyclic ligands for a G protein coupled receptor (GPCR) is noteworthy.

Calcitonin gene-related peptide receptor (CGRPR) is a heterodimer protein complex consisting of a class-B G protein-coupled receptor (GPCR) named calcitonin receptor-like receptor (CLR) and an accessory protein, receptor activity modifying protein type 1 (RAMP1). Here in this study, with several molecular modeling approaches and molecular dynamics (MD) simulations, the structural and dynamical effects of RAMP1 on the binding of small molecule CGRPR inhibitors (namely rimegepant and telcagepant) to the CGRPR extracellular ectodomain complex site (site 1) and transmembrane binding site (site 2) are investigated. Results showed that although these molecules stay stable at site 1, they can also bind to site 2, which may be interpreted as non-specificity of the ligands, however, most of these interactions at transmembrane binding site are not sustainable or are weak. Furthermore, to examine the site 2 for gepant binding, different in silico experiments (i.e., alanine scanning mutagenesis, SiteMap, ligand decomposition binding free energy analyses) are also conducted and the results confirmed the putative binding pocket (site 2) of the gepants at the CGRPRs., (Copyright © 2021. Published by Elsevier Ltd.)

Background and Purpose: The calcitonin (CT) receptor family is complex, comprising two receptors (the CT receptor [CTR] and the CTR-like receptor [CLR]), three accessory proteins (RAMPs) and multiple endogenous peptides. This family contains several important drug targets, including CGRP, which is targeted by migraine therapeutics. The pharmacology of this receptor family is poorly characterised in species other than rats and humans. To facilitate understanding of translational and preclinical data, we need to know the receptor pharmacology of this family in mice., Experimental Approach: Plasmids encoding mouse CLR/CTR and RAMPs were transiently transfected into Cos-7 cells. cAMP production was measured in response to agonists in the absence or presence of antagonists., Key Results: We report the first synthesis and characterisation of mouse adrenomedullin, adrenomedullin 2 and βCGRP and of mouse CTR without or with mouse RAMPs. Receptors containing m-CTR had subtly different pharmacology than human receptors; they were promiscuous in their pharmacology, both with and without RAMPs. Several peptides, including mouse αCGRP and mouse adrenomedullin 2, were potent agonists of the m-CTR:m-RAMP3 complex. Pharmacological profiles of receptors comprising m-CLR:m-RAMPs were generally similar to those of their human counterparts, albeit with reduced specificity., Conclusion and Implications: Mouse receptor pharmacology differed from that in humans, with mouse receptors displaying reduced discrimination between ligands. This creates challenges for interpreting which receptor may underlie an effect in preclinical models and thus translation of findings from mice to humans. It also highlights the need for new ligands to differentiate between these complexes., 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., (© 2021 The British Pharmacological Society.)

G protein-coupled receptors (GPCRs) are key regulators of information transmission between cells and organs. Despite this, we have only a limited understanding of the behavior of GPCRs in the apo state and the conformational changes upon agonist binding that lead to G protein recruitment and activation. We expressed and purified unmodified apo and peptide-bound calcitonin gene-related peptide (CGRP) receptors from insect cells to determine their cryo-electron microscopy (cryo-EM) structures, and we complemented these with analysis of protein conformational dynamics using hydrogen-deuterium exchange mass spectrometry and three-dimensional variance analysis of the cryo-EM data. Together with our previously published structure of the active, Gs-bound CGRP receptor complex, our work provides insight into the mechanisms of class B1 GPCR activation., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Receptor component protein (RCP) is a 148 amino acid intracellular peripheral membrane protein, previously identified as promoting the coupling of CGRP to cAMP production at the CGRP receptor, a heterodimer of calcitonin receptor like-receptor (CLR), a family B G protein-coupled receptor (GPCR) and receptor activity modifying protein 1 (RAMP1). We extend these observations to show that it selectively enhances CGRP receptor coupling to Gs but not Gq or pERK activation. At other family B GPCRs, it enhances cAMP production at the calcitonin, corticotrophin releasing factor type 1a and glucagon-like peptide type 2 receptors with their cognate ligands but not at the adrenomedullin type 1 (AM 1 ), gastric inhibitory peptide and glucagon-like peptide type 1 receptors, all expressed in transfected HEK293S cells. However, there is also cell-line variability as RCP did not enhance cAMP production at the endogenous calcitonin receptor in HEK293T cells and it has previously been reported that it is active on the AM 1 receptor expressed on NIH3T3 cells. RCP appears to behave as a positive allosteric modulator at coupling a number of family B GPCRs to Gs, albeit in a manner that is regulated by cell-specific factors. It may exert its effects at the interface between the 2nd intracellular loop of the GPCR and Gs, although there is likely to be some overlap between this location and that occupied by the C-terminus of RAMPs if they bind to the GPCRs., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Conflict of interest A.A.P. is inventor on a patent describing enhanced affinity and altered selectivity AM and CGRP variant peptides., (© 2020 Elsevier Inc. All rights reserved.)

Working at the border between innate and adaptive immunity, natural killer (NK) cells play a key role in the immune system by protecting healthy cells and by eliminating malignantly transformed, stressed or virally infected cells. NK cell recognition of a target cell is mediated by a receptor "zipper" consisting of various activating and inhibitory receptors, including C-type lectin-like receptors. Among this major group of receptors, two of the largest rodent receptor families are the NKR-P1 and the Clr receptor families. Although these families have been shown to encode receptor-ligand pairs involved in MHC-independent self-nonself discrimination and are a target for immune evasion by tumour cells and viruses, structural mechanisms of their mutual recognition remain less well characterized. Therefore, we developed a non-viral eukaryotic expression system based on transient transfection of suspension-adapted human embryonic kidney 293 cells to produce soluble native disulphide dimers of NK cell C-type lectin-like receptor ectodomains. The expression system was optimized using green fluorescent protein and secreted alkaline phosphatase, easily quantifiable markers of recombinant protein production. We describe an application of this approach to the recombinant protein production and characterization of native rat NKR-P1B and Clr-11 proteins suitable for further structural and functional studies.

Calcitonin gene-related peptide (CGRP) is a widely expressed neuropeptide that has a major role in sensory neurotransmission. The CGRP receptor is a heterodimer of the calcitonin receptor-like receptor (CLR) class B G-protein-coupled receptor and a type 1 transmembrane domain protein, receptor activity-modifying protein 1 (RAMP1). Here we report the structure of the human CGRP receptor in complex with CGRP and the G s -protein heterotrimer at 3.3 Å global resolution, determined by Volta phase-plate cryo-electron microscopy. The receptor activity-modifying protein transmembrane domain sits at the interface between transmembrane domains 3, 4 and 5 of CLR, and stabilizes CLR extracellular loop 2. RAMP1 makes only limited direct contact with CGRP, consistent with its function in allosteric modulation of CLR. Molecular dynamics simulations indicate that RAMP1 provides stability to the receptor complex, particularly in the positioning of the extracellular domain of CLR. This work provides insights into the control of G-protein-coupled receptor function.

Calcitonin gene-related peptide (CGRP) binds to the complex of the calcitonin receptor-like receptor (CLR) with receptor activity-modifying protein 1 (RAMP1). How CGRP interacts with the transmembrane domain (including the extracellular loops) of this family B receptor remains unclear. In this study, a photoaffinity cross-linker, p-azido l-phenylalanine (azF), was incorporated into CLR, chiefly in the second extracellular loop (ECL2) using genetic code expansion and unnatural amino acid mutagenesis. The method was optimized to ensure efficient photolysis of azF residues near the transmembrane bundle of the receptor. A CGRP analogue modified with fluorescein at position 15 was used for detection of ultraviolet-induced cross-linking. The methodology was verified by confirming the known contacts of CGRP to the extracellular domain of CLR. Within ECL2, the chief contacts were I284 on the loop itself and L291, at the top of the fifth transmembrane helix (TM5). Minor contacts were noted along the lip of ECL2 between S286 and L290 and also with M223 in TM3 and F349 in TM6. Full length molecular models of the bound receptor complex suggest that CGRP sits at the top of the TM bundle, with Thr 6 of the peptide making contacts with L291 and H295. I284 is likely to contact Leu 12 and Ala 13 of CGRP, and Leu 16 of CGRP is at the ECL/extracellular domain boundary of CLR. The reduced potency, E max , and affinity of [Leu 16 Ala]-human α CGRP are consistent with this model. Contacts between Thr 6 of CGRP and H295 may be particularly important for receptor activation.

The calcitonin/CGRP family of peptides includes calcitonin, α and β CGRP, amylin, adrenomedullin (AM) and adrenomedullin 2/intermedin (AM2/IMD). Their receptors consist of one of two GPCRs, the calcitonin receptor (CTR) or the calcitonin receptor-like receptor (CLR). Further diversity arises from heterodimerization of these GPCRs with one of three receptor activity-modifying proteins (RAMPs). This gives the CGRP receptor (CLR/RAMP1), the AM 1 and AM 2 receptors (CLR/RAMP2 or RAMP3) and the AMY 1, AMY 2 and AMY 3 receptors (CTR/RAMPs1-3 complexes, respectively). Apart from the CGRP receptor, there are only peptide antagonists widely available for these receptors, and these have limited selectivity, thus defining the function of each receptor in vivo remains challenging. Further challenges arise from the probable co-expression of CTR with the CTR/RAMP complexes and species-dependent splice variants of the CTR (CT (a) and CT (b) ). Furthermore, the AMY 1(a) receptor is activated equally well by both amylin and CGRP, and the preferred receptor for AM2/IMD has been unclear. However, there are clear therapeutic rationales for developing agents against the various receptors for these peptides. For example, many agents targeting the CGRP system are in clinical trials, and pramlintide, an amylin analogue, is an approved therapy for insulin-requiring diabetes. This review provides an update on the pharmacology of the calcitonin family of peptides by members of the corresponding subcommittee of the International Union of Basic and Clinical Pharmacology and colleagues., (© 2017 The British Pharmacological Society.)

Calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors are heteromers of the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor, and one of three receptor activity-modifying proteins (RAMPs). How CGRP and AM activate CLR and how this process is modulated by RAMPs is unclear. We have defined how CGRP and AM induce Gs-coupling in CLR-RAMP heteromers by measuring the effect of targeted mutagenesis in the CLR transmembrane domain on cAMP production, modeling the active state conformations of CGRP and AM receptors in complex with the Gs C-terminus and conducting molecular dynamics simulations in an explicitly hydrated lipidic bilayer. The largest effects on receptor signaling were seen with H295A 5.40b , I298A 5.43b , L302A 5.47b , N305A 5.50b , L345A 6.49b and E348A 6.52b , F349A 6.53b and H374A 7.47b (class B numbering in superscript). Many of these residues are likely to form part of a group in close proximity to the peptide binding site and link to a network of hydrophilic and hydrophobic residues, which undergo rearrangements to facilitate Gs binding. Residues closer to the extracellular loops displayed more pronounced RAMP or ligand-dependent effects. Mutation of H374 7.47b to alanine increased AM potency 100-fold in the CGRP receptor. The molecular dynamics simulation showed that TM5 and TM6 pivoted around TM3. The data suggest that hydrophobic interactions are more important for CLR activation than other class B GPCRs, providing new insights into the mechanisms of activation of this class of receptor. Furthermore the data may aid in the understanding of how RAMPs modulate the signaling of other class B GPCRs., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The second extracellular loop (ECL2) of the G protein-coupled receptor (GPCR) family is important for ligand interaction and drug discovery. ECL2 of the family B cardioprotective calcitonin gene-related peptide (CGRP) receptor is required for cell signaling. Family B GPCR ligands have two regions; the N-terminus mediates receptor activation, and the remainder confers high-affinity binding. Comparing antagonism of CGRP 8-37 at a number of point mutations of ECL2 of the CGRP receptor, we show that the ECL2 potentially facilitates interaction with up to the 18 N-terminal residues of CGRP. This has implications for understanding family B GPCR activation and for drug design at the CGRP receptor.

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., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The human adrenomedullin (ADM) is a 52 amino acid peptide hormone belonging to the calcitonin family of peptides, which plays a major role in the development and regulation of cardiovascular and lymphatic systems. For potential use in clinical applications, we aimed to investigate the fate of the peptide ligand after binding and activation of the adrenomedullin receptor (AM1), a heterodimer consisting of the calcitonin receptor-like receptor (CLR), a G protein-coupled receptor, associated with the receptor activity-modifying protein 2 (RAMP2). Full length and N-terminally shortened ADM peptides were synthesized using Fmoc/tBu solid phase peptide synthesis and site-specifically labeled with the fluorophore carboxytetramethylrhodamine (Tam) either by amide bond formation or copper(I)-catalyzed azide alkyne cycloaddition. For the first time, Tam-labeled ligands allowed the observation of co-internalization of the whole ligand-receptor complex in living cells co-transfected with fluorescent fusion proteins of CLR and RAMP2. Application of a fluorescent probe to track lysosomal compartments revealed that ADM together with the CLR/RAMP2-complex is routed to the degradative pathway. Moreover, we found that the N-terminus of ADM is not a crucial component of the peptide sequence in terms of AM1 internalization behavior., (Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.)

Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Purpose: Receptor activity-modifying proteins (RAMPs) define the pharmacology of the calcitonin receptor-like receptor (CLR). The interactions of the different RAMPs with this class B GPCR yield high-affinity calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors. However, the mechanism for this is unclear., Experimental Approach: Guided by receptor models, we mutated residues in the N-terminal helix of CLR, RAMP2 and RAMP3 hypothesized to be involved in peptide interactions. These were assayed for cAMP production with AM, AM2 and CGRP together with their cell surface expression. Binding studies were also conducted for selected mutants., Key Results: An important domain for peptide interactions on CLR from I32 to I52 was defined. Although I41 was universally important for binding and receptor function, the role of other residues depended on both ligand and RAMP. Peptide binding to CLR/RAMP3 involved a more restricted range of residues than that to CLR/RAMP1 or CLR/RAMP2. E101 of RAMP2 had a major role in AM interactions, and F111/W84 of RAMP2/3 was important with each peptide., Conclusions and Implications: RAMP-dependent effects of CLR mutations suggest that the different RAMPs control accessibility of peptides to binding residues situated on the CLR N-terminus. RAMP3 appears to alter the role of specific residues at the CLR-RAMP interface compared with RAMP1 and RAMP2., (© 2013 The Authors. British Journal of Pharmacology published by John Wiley &. Sons Ltd on behalf of The British Pharmacological Society.)

Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are related peptides that are potent vasodilators. The CGRP and AM receptors are heteromeric protein complexes comprised of a shared calcitonin receptor-like receptor (CLR) subunit and a variable receptor activity modifying protein (RAMP) subunit. RAMP1 enables CGRP binding whereas RAMP2 confers AM specificity. How RAMPs determine peptide selectivity is unclear and the receptor stoichiometries are a topic of debate with evidence for 1:1, 2:2, and 2:1 CLR:RAMP stoichiometries. Here, we describe bacterial production of recombinant tethered RAMP-CLR extracellular domain (ECD) fusion proteins and biochemical characterization of their peptide binding properties. Tethering the two ECDs ensures complex stability and enforces defined stoichiometry. The RAMP1-CLR ECD fusion purified as a monomer, whereas the RAMP2-CLR ECD fusion purified as a dimer. Both proteins selectively bound their respective peptides with affinities in the low micromolar range. Truncated CGRP(27-37) and AM(37-52) fragments were identified as the minimal ECD complex binding regions. The CGRP C-terminal amide group contributed to, but was not required for, ECD binding, whereas the AM C-terminal amide group was essential for ECD binding. Alanine-scan experiments identified CGRP residues T30, V32, and F37 and AM residues P43, K46, I47, and Y52 as critical for ECD binding. Our results identify CGRP and AM determinants for receptor ECD complex binding and suggest that the CGRP receptor functions as a 1:1 heterodimer. In contrast, the AM receptor may function as a 2:2 dimer of heterodimers, although our results cannot rule out 2:1 or 1:1 stoichiometries., (© 2013 The Protein Society.)

The calcitonin gene-related peptide (CGRP) receptor is a complex of a calcitonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modelling. An alanine scan of residues 271-294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed.

Adrenomedullin (AM) is a peptide hormone that is a potent vasodilator and is essential for vascular development. The AM receptor is a heterodimeric cell surface receptor composed of the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor, in association with either of two receptor activity modifying protein (RAMP) coreceptors, RAMP2 or -3. The extracellular domains (ECDs) of CLR and the RAMPs form the primary AM binding site. Here, we present novel methodology for expression and purification of a heterodimeric AM receptor ECD complex as an MBP-CLR ECD fusion protein in association with the RAMP2 ECD. Co-expression of the RAMP2 ECD with the disulfide bond isomerase DsbC in the oxidizing cytoplasm of E. coli trxB gor enabled proper disulfide formation in vivo. The isolated RAMP2 ECD was purified to homogeneity. Co-expression of a soluble MBP-CLR ECD fusion protein with DsbC in E. coli trxB gor yielded a heterogeneous mixture of species with misfolded ECD. Incubation of affinity-purified MBP-CLR ECD in vitro with purified RAMP2 ECD, DsbC, and glutathione redox buffer promoted proper folding of the CLR ECD and formation of a stable MBP-CLR ECD:RAMP2 ECD complex that was purified by size-exclusion chromatography and which exhibited specific AM binding. Approximately 40mg of highly purified complex was obtained starting with 6L bacterial cultures for each protein. The methodology reported here will facilitate structure/function studies of the AM receptor., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The calcitonin receptor-like receptor (CRLR), a class B GPCR, forms a heterodimer with receptor activity-modifying protein 2 (RAMP2), and serves as the adrenomedullin (AM) receptor to control neovascularization, while CRLR and RAMP1 form the calcitonin gene-related peptide (CGRP) receptor. Here, we report the crystal structures of the RAMP2 extracellular domain alone and in the complex with the CRLR extracellular domain. The CRLR-RAMP2 complex exhibits several intermolecular interactions that were not observed in the previously reported CRLR-RAMP1 complex, and thus the shape of the putative ligand-binding pocket of CRLR-RAMP2 is distinct from that of CRLR-RAMP1. The CRLR-RAMP2 interactions were confirmed for the full-length proteins on the cell surface by site-specific photo-crosslinking. Mutagenesis revealed that AM binding requires RAMP2 residues that are not conserved in RAMP1. Therefore, the differences in both the shapes and the key residues of the binding pocket are essential for the ligand specificity., (Copyright © 2011 The Protein Society.)

The first and third extracellular loops (ECL) of G protein-coupled receptors (GPCRs) have been implicated in ligand binding and receptor function. This study describes the results of an alanine/leucine scan of ECLs 1 and 3 and loop-associated transmembrane (TM) domains of the secretin-like GPCR calcitonin receptor-like receptor which associates with receptor activity modifying protein 1 to form the CGRP receptor. Leu195Ala, Val198Ala and Ala199Leu at the top of TM2 all reduced αCGRP-mediated cAMP production and internalization; Leu195Ala and Ala199Leu also reduced αCGRP binding. These residues form a hydrophobic cluster within an area defined as the "minor groove" of rhodopsin-like GPCRs. Within ECL1, Ala203Leu and Ala206Leu influenced the ability of αCGRP to stimulate adenylate cyclase. In TM3, His219Ala, Leu220Ala and Leu222Ala have influences on αCGRP binding and cAMP production; they are likely to indirectly influence the binding site for αCGRP as well as having an involvement in signal transduction. On the exofacial surfaces of TMs 6 and 7, a number of residues were identified that reduced cell surface receptor expression, most noticeably Leu351Ala and Glu357Ala in TM6. The residues may contribute to the RAMP1 binding interface. Ile360Ala impaired αCGRP-mediated cAMP production. Ile360 is predicted to be located close to ECL2 and may facilitate receptor activation. Identification of several crucial functional loci gives further insight into the activation mechanism of this complex receptor system and may aid rational drug design., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Adrenomedullin (AM) is a novel hypotensive peptide that exerts a variety of strongly protective effects against multiorgan damage. AM-specific receptors were first identified as heterodimers composed of calcitonin-receptor-like receptor (CLR), a G protein coupled receptor, and one of two receptor activity-modifying proteins (RAMP2 or RAMP3), which are accessory proteins containing a single transmembrane domain. RAMPs are required for the surface delivery of CLR and the determination of its phenotype. CLR/RAMP2 (AM₁ receptor) is more highly AM-specific than CLR/RAMP3 (AM₂ receptor). Although there have been no reports showing differences in intracellular signaling via the two AM receptors, in vitro studies have shed light on their distinct trafficking and functionality. In addition, the tissue distributions of RAMP2 and RAMP3 differ, and their gene expression is differentially altered under pathophysiological conditions, which is suggestive of the separate roles played by AM₁ and AM₂ receptors in vivo. Both AM and the AM₁ receptor, but not the AM₂ receptor, are crucial for the development of the fetal cardiovascular system and are able to effectively protect against various vascular diseases. However, AM₂ receptors reportedly play an important role in maintaining a normal body weight in old age and may be involved in immune function. In this review article, we focus on the shared and separate functions of the AM receptor subtypes and also discuss the potential for related drug discovery. In addition, we mention their possible function as receptors for AM2 (or intermedin), an AM-related peptide whose biological functions are similar to those of AM., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Adrenomedullin 1 (AM(1)) receptor is a heterodimer composed of calcitonin receptor-like receptor (CLR) - a family B G protein-coupled receptor (GPCR) - and receptor activity-modifying protein 2 (RAMP2). Both family A and family B GPCRs possess an eighth helix (helix 8) in the proximal portion of their C-terminal tails; however, little is known about the function of helix 8 in family B GPCRs. We therefore investigated the structure-function relationship of human (h)CLR helix 8, which extends from Glu430 to Trp439, by separately transfecting nine point mutants into HEK-293 cells stably expressing hRAMP2. Glu430, Val431, Arg437 and Trp439 are all conserved among family B GPCRs. Flow cytometric analysis revealed that Arg437Ala or Trp438Ala mutation significantly reduced cell surface expression of the receptor complex, leading to a ∼20% reduction in specific (125)I-AM binding but little change in their IC(50) values. Both mutants showed 6-8-fold higher EC(50) values for AM-induced cAMP production and ∼50% reductions in their maximum responses. Glu430Ala mutation also reduced AM signaling by ∼45%, but surface expression and (125)I-AM binding were nearly the same as with wild-type CLR. Surprisingly, Glu430Ala and Val431Ala mutations significantly enhanced AM-induced internalization of the mutant receptor complexes. Taken together, these findings suggest that within hCLR helix 8, Glu430 is crucial for Gs coupling, and Arg437 and Trp439 are involved in both cell surface expression of the hAM(1) receptor and Gs coupling. Moreover, the Glu430-Val431 sequence may participate in the negative regulation of hAM(1) receptor internalization, which is not dependent on Gs coupling., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Dysregulation of the calcitonin gene-related peptide (CGRP), a potent vasodilator, is directly implicated in the pathogenesis of migraine. CGRP binds to and signals through the CGRP receptor (CGRP-R), a heterodimer containing the calcitonin receptor-like receptor (CLR), a class B GPCR, and RAMP1, a receptor activity-modifying protein. We have solved the crystal structure of the CLR/RAMP1 N-terminal ectodomain heterodimer, revealing how RAMPs bind to and potentially modulate the activities of the CLR GPCR subfamily. We also report the structures of CLR/RAMP1 in complex with the clinical receptor antagonists olcegepant (BIBN4096BS) and telcagepant (MK0974). Both drugs act by blocking access to the peptide-binding cleft at the interface of CLR and RAMP1. These structures illustrate, for the first time, how small molecules bind to and modulate the activity of a class B GPCR, and highlight the challenges of designing potent receptor antagonists for the treatment of migraine and other class B GPCR-related diseases., (Copyright © 2010 Elsevier Ltd. All rights reserved.)