Obesity is a major and increasingly prevalent chronic metabolic disease with numerous comorbidities. While recent incretin-based therapies have provided pharmaceutical inroads into treatment of obesity, there remains an ongoing need for additional medicines with distinct modes of action as independent or complementary therapeutics. Among the most promising candidates, supported by phase 1 and 2 clinical trials, is cagrilintide, a long-acting amylin and calcitonin receptor agonist. As such, understanding how cagrilintide functionally engages target receptors is critical for future development of this target class. Here, we determine structures of cagrilintide bound to Gs-coupled, active, amylin receptors (AMY1R, AMY2R, AMY3R) and calcitonin receptor (CTR) and compare cagrilintide interactions and the dynamics of receptor complexes with previously reported structures of receptors bound to rat amylin, salmon calcitonin or recently developed amylin-based peptides. These data reveal that cagrilintide has an amylin-like binding mode but, compared to other peptides, induces distinct conformational dynamics at calcitonin-family receptors that could contribute to its clinical efficacy.

The pituitary adenylate cyclase-activating polypeptide (PACAP) 1 receptor (PAC1R) is a class B1 G protein–coupled receptor activated by the endogenous peptide agonists PACAP and vasoactive intestinal peptide (VIP). Alternate splicing within the receptor extracellular domain (ECD) generates the PAC1R short variant (PAC1sR) that has selectively enhanced VIP function compared to the full-length, PAC1R null variant (PAC1nR). However, to date, a comprehensive pharmacological assessment of the downstream signaling outcomes of PAC1sR activation compared to PAC1nR has not been performed, and little information is available to mechanistically understand how ECD splicing may alter ligand engagement. Here, we demonstrated that VIP, but not PACAP, has globally enhanced activity across a broad range of functional endpoints at PAC1sR compared to PAC1nR. Cryo-EM structures of VIP-bound, stimulatory G protein (G s )-coupled PAC1sR and PAC1nR, supported by molecular dynamics (MD) simulations, demonstrate transient engagement of the null loop in PAC1nR, which is absent in PAC1sR, with residues in extracellular loop 2 (ECL2) and the N-terminal helix of the ECD. These interactions result in differential engagement of VIP with these domains and the top of TM2/ECL1 with PAC1sR and PAC1nR. Moreover, MD simulations predicted differential interactions of the G s protein with the two PAC1R variants when bound by VIP that correlate with a greater allosteric influence of the G s protein on VIP affinity at the PAC1sR, relative to PAC1nR. Our study provides insights into the structural basis and functional consequences of PAC1R ECD splicing, increasing understanding of PAC1R ligand selectivity and signaling.

Dual amylin and calcitonin receptor agonists (DACRAs) show promise as efficacious therapeutics for treatment of metabolic disease, including obesity. However, differences in efficacy in vivo have been observed for individual DACRAs, indicating that detailed understanding of the pharmacology of these agents across target receptors is required for rational drug development. To date, such understanding has been hampered by lack of direct, subtype-selective, functional assays for the amylin receptors (AMYRs). Here, we describe the generation of receptor-specific assays for recruitment of Venus-tagged Gs protein through fusion of luciferase to either the human calcitonin receptor (CTR), human receptor activity-modifying protein (RAMP)-1, RAMP1 (AMY1R), human RAMP2 (AMY2R), or human RAMP3 (AMY3R). These assays revealed a complex pattern of receptor activation by calcitonin, amylin, or DACRA peptides that was distinct at each receptor subtype. Of particular note, although both of the CT-based DACRAs, sCT and AM1784, displayed relatively similar behaviors at CTR and AMY1R, they generated distinct responses at AMY2R and AMY3R. These data aid the rationalization of in vivo differences in response to DACRA peptides in rodent models of obesity. Direct assessment of the pharmacology of novel DACRAs at AMYR subtypes is likely to be important for development of optimized therapeutics for treatment of metabolic diseases. SIGNIFICANCE STATEMENT: Amylin receptors (AMYRs) are important obesity targets. Here we describe a novel assay that allows selective functional assessment of individual amylin receptor subtypes that provides unique insight into the pharmacology of potential therapeutic ligands. Direct assessment of the pharmacology of novel agonists at AMYR subtypes is likely to be important for development of optimized therapeutics for treatment of metabolic diseases.

The class A orphan G protein-coupled receptor (GPCR), GPR3, has been implicated in a variety of conditions, including Alzheimer's and premature ovarian failure. GPR3 constitutively couples with Gαs, resulting in the production of cAMP in cells. While tool compounds and several putative endogenous ligands have emerged for the receptor, its endogenous ligand, if it exists, remains a mystery. As novel potential drug targets, the structures of orphan GPCRs have been of increasing interest, revealing distinct modes of activation, including autoactivation, presence of constitutively activating mutations, or via cryptic ligands. Here, we present a cryo-electron microscopy (cryo-EM) structure of the orphan GPCR, GPR3 in complex with DNGαs and Gβ1γ2. The structure revealed clear density for a lipid-like ligand that bound within an extended hydrophobic groove, suggesting that the observed "constitutive activity" was likely due to activation via a lipid that may be ubiquitously present. Analysis of conformational variance within the cryo-EM data set revealed twisting motions of the GPR3 transmembrane helices that appeared coordinated with changes in the lipid-like density. We propose a mechanism for the binding of a lipid to its putative orthosteric binding pocket linked to the GPR3 dynamics.

Class B G protein-coupled receptors (GPCRs) are important therapeutic targets for major diseases. Here, we present structures of peptide and Gs-bound pituitary adenylate cyclase-activating peptide, PAC1 receptor, and corticotropin-releasing factor (CRF), (CRF1) receptor. Together with recently solved structures, these provide coverage of the major class B GPCR subfamilies. Diverse orientations of the extracellular domain to the receptor core in different receptors are at least partially dependent on evolutionary conservation in the structure and nature of peptide interactions. Differences in peptide interactions to the receptor core also influence the interlinked TM2-TM1-TM6/ECL3/TM7 domain, and this is likely important in their diverse signaling. However, common conformational reorganization of ECL2, linked to reorganization of ICL2, modulates G protein contacts. Comparison between receptors reveals ICL2 as a key domain forming dynamic G protein interactions in a receptor- and ligand-specific manner. This work advances our understanding of class B GPCR activation and Gs coupling.

Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, such as osteoporosis, diabetes and obesity. Here we report the structure of a full-length class B receptor, the calcitonin receptor, in complex with peptide ligand and heterotrimeric Gα s βγ protein determined by Volta phase-plate single-particle cryo-electron microscopy. The peptide agonist engages the receptor by binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helices 6 and 7. This conformation is accompanied by a 60° kink in helix 6 and a large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the α5-helix of Gα s . Also observed is an extended intracellular helix 8 that contributes to both receptor stability and functional G-protein coupling via an interaction with the Gβ subunit. This structure provides a new framework for understanding G-protein-coupled receptor function.

The receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are complexes of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMP). The CGRP receptor is a CLR/RAMP1 pairing whereas CLR/RAMP2 and CLR/RAMP3 constitute two subtypes of AM receptor: AM1 and AM2, respectively. Previous studies identified Glu74 in RAMP3 to be important for AM binding and potency. To further understand the importance of this residue and its equivalent in RAMP1 (Trp74) we substituted the native amino acids with several others. In RAMP3, these were Trp, Phe, Tyr, Ala, Ser, Thr, Arg and Asn; in RAMP1, Glu, Phe, Tyr, Ala and Asn substitutions were made. The mutant RAMPs were co-expressed with CLR in Cos7 cells; receptor function in response to AM, AM2/intermedin and CGRP was measured in a cAMP assay and cell surface expression was determined by ELISA. Phe reduced AM potency in RAMP3 but had no effect in RAMP1. In contrast, Tyr had no effect in RAMP3 but enhanced AM potency in RAMP1. Most other substitutions had a small effect on AM potency in both receptors whereas there was little impact on CGRP or AM2 potency. Overall, these data suggest that the geometry and charge of the residue at position 74 contribute to how AM interacts with the AM2 and CGRP receptors and confirms the role of this position in dictating differential AM pharmacology at the AM2 and CGRP receptors.

The three receptor activity-modifying proteins (RAMPS) have been recognized as being important for the trafficking and function of a subset of family B G protein-coupled receptors, although the structural basis for this has not been well established. In the current work, we use morphological fluorescence techniques, bioluminescence resonance energy transfer, and bimolecular fluorescence complementation to demonstrate that the secretin receptor associates specifically with RAMP3, but not with RAMP 1 or RAMP2. We use truncation constructs, peptide competition experiments, and chimeric secretin-GLP1 receptor constructs to establish that this association is structurally specific, dependent on the intramembranous region of the RAMP and TM6 and TM7 of this receptor. There were no observed changes in secretin-stimulated cAMP, intracellular calcium, ERK1/2 phosphorylation, or receptor internalization in receptor-bearing COS or CHO-K1 cells in the presence or absence of exogenous RAM P transfection, although the secretin receptor trafficks normally to the cell surface in these cells in a RAMP-independent manner, resulting in both free and RAMP-associated receptor on the cell surface. RAMP3 association with this receptor was shown to be capable of rescuing a receptor mutant (G241C) that is normally trapped intracellularly in the biosynthetic machinery. Similarly, secretin receptor expression had functional effects on adrenomedullin activity, with increasing secretin receptor expression competing for RAMP3 association with the calcitonin receptor-like receptor to yield a functional adrenomedullin receptor. These data provide important new insights into the structural basis for RAMP3 interaction with a family B G protein-coupled receptor, potentially providing a highly selective target for drug action. This may be representative of similar interactions between other members of this receptor family and RAMP proteins.

Receptor activity modifying proteins (RAMPs) interact with calcitonin receptors to produce novel amylin receptor phenotypes. We have recently demonstrated that the short intracellular C-terminus of RAMPs plays a key role in the function of amylin receptors derived from the CTa calcitonin receptor through the use of chimeric RAMPs and RAMPs that are truncated at the C-terminus [15, Udawela M, Christopoulos G, Morfis M, Christopoulos A, Ye S, Tilakaratne N, Sexton PM. A critical role for the short intracellular C terminus in receptor activity modifying protein function. Mol Pharmacol 2006;70:1750-60., 18, Udawela M, Christopoulos G, Tilakaratne N, Christopoulos A, Albiston A, Sexton PM. Distinct receptor activity-modifying protein domains differentially modulate interaction with calcitonin receptors. Mol Pharmacol 2006;69:1984-89.]. The calcitonin receptor in humans is expressed as two major alternatively spliced isoforms termed CTa and CTb. Relatively little is known about how alternate splicing of the receptor affects the interaction between calcitonin receptors and RAMPs. We have examined the effect of RAMP truncation, through use of mutant constructs that delete the last 8 amino acids of each of the 3 known human RAMPs, and characterised these for interaction with CTb receptors through co-expression in COS-7 cells. As seen with the CTa receptor isoform, RAMP truncation caused a marked loss in induction of AMYb receptor phenotypes as characterised by (125)I-rat amylin radioligand binding assays and cAMP accumulation assays; the latter as a marker of receptor signalling. The effect was most pronounced for RAMP1 and RAMP2 deletion mutants, but attenuated responses were also observed with co-expressed RAMP3 deletion mutants. These data support a direct role for the RAMP C-terminus in the interaction of RAMP/calcitonin receptor complexes with intracellular accessory proteins involved in signalling and/or receptor trafficking.

Calcitonin-family receptors comprise calcitonin receptor-like receptor (CL) or calcitonin receptor and receptor activity-modifying protein (RAMP) pairings. Calcitonin gene-related peptide (CGRP) receptors are CL/RAMP1, whereas adrenomedullin (AM) receptors are CL/RAMP2 (AM1 receptor) or CL/RAMP3 (AM2 receptor). Amylin (Amy) receptors are RAMP hetero-oligomers with the calcitonin receptor (AMY1, AMY2, and AMY3, respectively). How RAMPs change G protein-coupled receptor pharmacology is not fully understood. We exploited sequence differences between RAMP1 and RAMP3 to identify individual residues capable of altering receptor pharmacology. Alignment of human RAMPs revealed eight residues that are conserved in RAMP2 and RAMP3 but are different in RAMP1. We hypothesized that residues in RAMP2 and RAMP3, but not RAMP1, are responsible for making CL/RAMP2 and CL/RAMP3 AM receptors. Using site-directed mutagenesis, we introduced individual RAMP3 residues into RAMP1 and vice versa in these eight positions. Mutant or wild-type RAMPs were transfected into Cos7 cells with CL or the insert-negative form of the calcitonin receptor [CT(a)]. Agonist-stimulated cAMP production and cell-surface expression of constructs were measured. Position 74 in RAMP1 and RAMP3 was critical for determining AM potency and affinity, and Phe93 in RAMP1 was an important contributor to alphaCGRP potency at CGRP receptors. Mutant RAMP/CT(a) receptor complexes displayed different phenotypes. It is noteworthy that RAMP1 S103N and W74E mutations led to enhanced rAmy potency, probably related to increased cell-surface expression of these complexes. This differs from the effect on CL-based receptors where expression was unchanged. Targeted substitution has emphasized the importance of position 74 in RAMP1/RAMP3 as a key determinant of AM pharmacology.

Context: Sepsis is a major cause of death in the United States and accounts for ∼50% of the fatalities in intensive care units. Serum procalcitonin (ProCT) levels are markedly elevated in sepsis and correlate positively with severity of the illness and mortality, however, little is known about the biological activity of ProCT. Objective: To explore the biological activity of purified human ProCT at the calcitonin (CT) family of receptors. Design: Human ProCT was purified from the TT medullary thyroid carcinoma cell line. Human CTa receptor or human CT receptor-like receptor (CLR) was transiently expressed in COS-7 cells alone or together with individual receptor activity-modifying proteins (RAMPs) to generate the CTa (CT) receptor, the AMY1 (amylin) receptor, the CGRP1 (CT gene-related peptide) receptor, and the AM1 and AM2 (adrenomedullin) receptors. Biological activity of ProCT was assessed by measurement of cAMP accumulation. Results: ProCT was effectively inert at CTa, AM1, and AM2 receptors. In contrast, it was a potent partial agonist (50–60% of the CGRP efficacy) of the CGRP1 receptor with an EC50 as high as 0.56 nM, although the potency was batch dependent. ProCT also displayed weak partial agonist activity at the AMY1 receptor with an EC50 of ∼100 nM. Moreover, ProCT also robustly inhibited CGRP-dependent cyclic adenosine monophosphate responses at the CGRP1 receptor. Conclusions: Our data provide a potential molecular mechanism for the observation that ProCT appears to be toxic while CGRP treatment appears to be beneficial in animal models of sepsis.

Receptor activity-modifying proteins (RAMPs) 1, 2, and 3 are prototypic G protein-coupled receptor accessory proteins that can alter not only receptor trafficking but also receptor phenotype. Specific RAMP interaction with the calcitonin receptor (CTR) generates novel and distinct receptors for the peptide amylin; however, the role of RAMPs in receptor signaling is not understood. The current study demonstrates that RAMP interaction with the CTRa in COS-7 or HEK-293 cells leads to selective modulation of signaling pathways activated by the receptor complex. There was a 20- to 30-fold induction in amylin potency at CTR/RAMP1 (AMY1) and CTR/RAMP3 (AMY3) receptors, compared with CTR alone, for formation of the second-messenger cAMP that parallels an increase in amylin binding affinity. In contrast, only 2- to 5-fold induction of amylin potency was seen for mobilization of intracellular Ca++ or activation of ERK1/2. In addition, in COS-7 cells, the increase in amylin potency for Ca++ mobilization was 2-fold greater for AMY3 receptors, compared with AMY1 receptors and this paralleled the relative capacity of overexpression of Gαq proteins to augment induction of high affinity 125I-amylin binding. These data demonstrate that RAMP-complexed receptors have a different signaling profile to CTRs expressed in the absence of RAMPs, and this is likely due to direct effects of the RAMP on G protein-coupling efficiency.

Calcitonin receptors (CTRs) dimerize with receptor activity-modifying proteins (RAMPs) to generate high-affinity amylin (AMY) receptors; however, the relative contribution of individual RAMP domains to the formation of AMY receptors is poorly understood. We have used chimeras between RAMP1 and RAMP2 that specifically exchanged the N-terminal, transmembrane, or C-terminal domain and examined these in assays of [(125)I]amylin binding or peptide-induced cAMP signaling in COS-7 cells transiently transfected with wild-type or chimeric RAMPs and human CTRa. The specificity of peptides in competition for [(125)I]amylin binding was principally dictated by the N-terminal domain present in the chimeras; however, the maximal level of binding induced was dictated by the transmembrane domain present. This extended previous data (Zumpe et al., 2000) to provide a distinction between the transmembrane domain and the C terminus in this function. In contrast to the effects on binding, each of the RAMP domains played a role in the signaling phenotype of the receptors. In particular, the potency of calcitonin gene-related peptide (CGRP) was most influenced by the C-terminal domain present, in which the presence of the RAMP1 C-terminal domain led to increased potency over CTRa alone, whereas chimeras with the RAMP2 C-terminal domain did not induce increased CGRP potency. The data provide additional support for the importance of the N terminus in determining binding affinity but reveal a prominent role of the transmembrane domain in the strength of amylin binding and a unique role for the C terminus in signaling by peptides to stimulate cAMP production.

1-Piperidinecarboxamide, N-[2-[[5-amino-l-[[4-(4-pyridinyl)-lpiperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)quinazolinyl) (BIBN4096BS), a calcitonin gene-related peptide (CGRP) receptor antagonist, can alleviate the symptoms of migraine and is highly selective for CGRP over adrenomedullin (AM) receptors. These receptors are heterodimers of the calcitonin receptor-like receptor (CL) and receptor activity modifying proteins (RAMPs), with the pharmacological properties determined by the RAMP subunit. BIBN4096BS-sensitive CGRP1 receptors are CL/RAMP1, whereas BIBN4096BSinsensitive AM receptors are CL/RAMP2 or CL/RAMP3 (AM1 and AM2, respectively), implicating RAMP1 in conferring BIBN4096BS sensitivity. Because calcitonin receptors [CT(a)] also interact with RAMP1 [AMY1(a) receptors], BIBN4096BS could also have affinity for these receptors. To test this, receptors were transfected into COS-7 cells and agonist-stimulated cAMP levels measured in the presence and absence of antagonists. We found that AMY1(a) receptors were 150-fold less sensitive to BIBN4096BS antagonism than CGRP1 receptors. In contrast, AMY3(a) [CT(a)/RAMP3] or AM2 receptors were not sensitive to BIBN4096BS antagonism. We investigated Trp74 in RAMP1, a residue implicated in the species selectivity of BIBN4096BS. BIBN4096BS affinity was reduced at AMY1(a) and CGRP1 receptors when this residue was mutated to lysine or alanine. The equivalent residue in RAMP3, Glu74, when mutated to tryptophan (E74W), induced BIBN4096BS sensitivity at AM2 and AMY3(a) receptors. It is interesting that a selective reduction in AM potency was observed at E74W AM2 receptors, implicating this residue in AM interactions with this receptor. These data support the importance of Trp74 in RAMP1 in the interaction of BIBN4096BS with CGRP1 and AMY1(a) receptors and identified Glu74 in RAMP3 as the first amino acid in RAMP important for agonist interactions with calcitonin-family receptors. Calcitonin (CT) gene-related peptide (CGRP), a 37-amino acid neuropeptide, has potent effects in the vasculature and has been implicated in migraine (Brain and Grant, 2004). CGRP-like immunoreactivity is elevated in a migraine attack, and triptans normalize these levels (Edvinsson, 2001). Furthermore, infusion of CGRP into subjects prone to migraine can trigger an attack (Lassen et al., 2002). However, the most compelling evidence for the inThis work was supported by the National Health and Medical Research Council (NHMRC) of Australia (grants 145702 and 299810), the Ian Potter Foundation Neuropeptide Laboratory, the Auckland Medical Research Foundation, and Lottery Health Commission (New Zealand). P.M.S. is a Principal Research Fellow of the NHMRC. A.C. is a Senior Research Fellow of the NHMRC. D.L.H. and G.C. contributed equally to this work. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.027953. ABBREVIATIONS: CT, calcitonin; AM, adrenomedullin; AMY, amylin receptor phenotype; Amy, amylin; BIBN4096BS, 1-piperidinecarboxamide, N-[2[[5-amino-l-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxphenyl)methyl] -2-oxoethyl]-4-(1,4dihydro-2-oxo-3-(2H)-quinazolinyl; CGRP, calcitonin gene-related peptide; CL, calcitonin receptor-like receptor; CT(a), calcitonin receptor; HA, hemagglutinin; hCT, human calcitonin; RAMP, receptor activity modifying protein; rAmy, rat amylin; VPAC1, vasoactive intestinal polypeptide/ pituitary adenylate cyclase-activating peptide receptor 1; WT, wild type; ANOVA, analysis of variance; VIP, vasoactive intestinal peptide. 0026-895X/06/7006-1984–1991$20.00 MOLECULAR PHARMACOLOGY Vol. 70, No. 6 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 27953/3153373 Mol Pharmacol 70:1984–1991, 2006 Printed in U.S.A. 1984 at A PE T Jornals on N ovem er 7, 2017 m oharm .aspeurnals.org D ow nladed from volvement of the CGRP system in the pathogenesis of migraine comes from studies using the CGRP antagonist BIBN4096BS, which was able to normalize migraine pain (Olesen et al., 2004). Together, the data show the importance of CGRP in this condition and make the study of the mode of interaction of BIBN4096BS with CGRP receptors of particular interest. In previous studies, it has been demonstrated that BIBN4096BS is highly selective for CGRP binding sites over those for the related peptide, adrenomedullin (AM) (Doods et al., 2000; Hay et al., 2002, 2003). Likewise, this compound has been shown to have particularly high species selectivity, having at least 100-fold greater affinity for primate over rodent CGRP receptors (Doods et al., 2000; Mallee et al., 2002). CGRP receptors are heterodimers of the CT receptor-like receptor (CL), a family B G proteincoupled receptor, and receptor activity modifying protein (RAMP) 1 (McLatchie et al., 1998). Only together are fully functional CGRP1 receptors formed. On the other hand, AM receptors, for which BIBN4096BS has little affinity, are also heterodimers, this time of CL with RAMP2 or RAMP3 (AM1 and AM2, respectively; McLatchie et al., 1998; Fraser et al., 1999; Poyner et al., 2002). Given that CL is shared between these receptors but that BIBN4096BS only weakly interacts with AM receptors, RAMP1 is strongly implicated in conferring the high-affinity BIBN4096BS interaction with the CGRP1 receptor. A predominant role for RAMP1 in BIBN4096BS affinity has been confirmed through studies of chimeras of rat and human RAMP1 that identified tryptophan at position 74 of RAMP1 as a key amino acid for the affinity differences across species (Fig. 1) (Mallee et al., 2002). In addition to heterodimerizing with CL, RAMPs functionally complex with the related CT receptor to form the AMY family of receptors, each having high affinity for the peptide hormone amylin (Amy) but a distinct specificity of interaction with the related peptides CT, CGRP, and AM (Christopoulos et al., 1999; Muff et al., 1999; Hay et al., 2006). In particular, the AMY1(a) receptor, the heterodimer of the CT(a) receptor and RAMP1, has high affinity for CGRP and thus may also be a target for BIBN4096BS with potential implication for the side-effect profile of the drug. In this study, we explored the specificity of BIBN4096BS interaction at AMY receptors and, in particular, the contribution of Trp74 and the equivalent amino acid in other RAMPs to BIBN4096BS activity. We demonstrate that the RAMP1 containing AMY1(a) receptor has significant affinity for BIBN4096BS, but a high degree of selectivity is retained for the CGRP1 receptor. Trp74 was a key residue for BIBN4096BS affinity for all receptor phenotypes, but the equivalent Glu74 in the AM2 receptor also played an important role for AM potency at this receptor. Materials and Methods Materials. Human AM, human CGRP, human CGRP8–37, and human CGRP were purchased from Bachem (Bubendorf, Switzerland). Rat Amy (rAmy) was from Auspep (Parkville, Australia). BIBN4096BS was kindly provided by Henri Doods (Boehringer Ingelheim GmbH, Ingelheim, Germany) or David Smith (AstraZeneca, Pharmaceuticals LP, Wilmington, DE) and was prepared as described previously (Hay et al., 2002); drugs from both sources had equivalent activity. Bovine serum albumin and isobutylmethylxanthine were from Sigma (St. Louis, MO) and amplified luminescent proximity homogenous assay-screen cAMP kits were purchased from Perkin Elmer (Boston, MA). Dulbecco’s modified Eagle’s medium, fetal bovine serum, and HEPES were from Invitrogen (Carlsbad, CA). Cell culture plastic ware was manufactured by Nunc (Roskible, Denmark), and Metafectine was purchased from Scientifix (Cheltenham, VIC, Australia). I-labeled goat anti-mouse IgG (I-IgG) was obtained from Perkin Elmer. Na-I (100 mCi/ml) was supplied by ICN Biochemicals (Irvine, CA). N-Succinimidyl 3,4-hydroxy,5,[I]iodophenyl)propionate (Bolton-Hunter reagent; 2000 Ci/mmol) was from Amersham (Little Chalfont, Buckinghamshire, UK). IrAmy (specific activity, 2000 Ci/mmol) was iodinated by the BoltonHunter method and purified by reverse-phase high-performance liquid chromatography as described previously (Bhogal et al., 1992). All other reagents were of analytical grade. Expression Constructs and Site-Directed Mutagenesis. Double hemagglutinin (HA) epitope-tagged human CT(a) receptor was prepared as described previously (Pham et al., 2004). This receptor is the Leu447 polymorphic variant of the receptor (Kuestner et al., 1994). Human RAMP1, human N-terminally tagged mycRAMP1, RAMP3, and human CL receptor were gifts from Dr. Steven Foord (McLatchie et al., 1998). The vasoactive intestinal polypeptide/pituitary adenylate cyclase activating peptide receptor 1 (VPAC1 receptor) cDNA was a gift from Dr. Marc Laburthe (Couvineau et al., 1994). Single point mutations in the RAMPs were generated using the QuikChange method according to the manufacturer’s instructions (Stratagene, La Jolla, CA). Cell Culture and Transfection. In most experiments, COS-7 cells were subcultured and transfected as described previously (Zumpe et al., 2000; Hay et al., 2005). In experiments using the VPAC1 receptor, COS-7 cells were cultured in a similar manner, but cells were transfected with 0.25 g of DNA per well (0.125 g of VPAC1 with either 0.125 g of pcDNA3 or 0.125 g of mycRAMP1) in 96-well plates using polyethylenimine (Bailey and Hay, 2006). Measurement of cAMP Production. Cells transfected with various receptor components were harvested approximately 40 h after transfection, and cAMP assays were performed as described previously (Hay et al., 2005). Agonists with or without antagonists were added to 384-well plates, and then transfected cell suspensions (20,000 cells/well) were added to this mixture for 30 min at 37°C before lysis and assay of cAMP content by amplified luminescent proximity homogenous assay screen (Hay et al., 2005). In experiments using the VPAC1 re

Receptor activity-modifying proteins ( RAMPs) interact with and modify the behavior of the calcitonin receptor ( CTR) and calcitonin receptor-like receptor ( CLR). We have examined the contribution of the short intracellular C terminus, using constructs that delete the last eight amino acids of each RAMP. C-Terminal deletion of individual RAMPs had little effect on the signaling profile induced when complexed with CLR in COS-7 or human embryonic kidney ( HEK) 293 cells. Likewise, confocal microscopy revealed each of the mutant RAMPs translocated hemagglutinin-tagged CLR to the cell surface. In contrast, a pronounced effect of RAMP C-terminal truncation was seen for RAMP/CTRa complexes, studied in COS-7 cells, with significant attenuation of amylin receptor phenotype induction that was stronger for RAMP1 and -2 than RAMP3. The loss of amylin binding upon C-terminal deletion could be partially recovered with overexpression of G alpha(s), suggesting an impact of the RAMP C terminus on coupling of G proteins to the receptor complex. In HEK293 cells the c-Myc-RAMP1 C-terminal deletion mutant showed high receptor-independent cell surface expression; however, this construct showed low cell surface expression when expressed alone in COS-7 cells, indicating interaction of RAMPs with other cellular components via the C terminus. This mutant also had reduced cell surface expression when coexpressed with CTR. Thus, this study reveals important functionality of the RAMP C-terminal domain and identifies key differences in the role of the RAMP C terminus for CTR versus CLR-based receptors.

1-Piperidinecarboxamide, N-[2-[[5-amino-l-[[4-(4-pyridinyl)-l-piperazinyl] carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl) methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)quinazolinyl) (BIBN4096BS), a calcitonin gene-related peptide (CGRP) receptor antagonist, can alleviate the symptoms of migraine and is highly selective for CGRP over adrenomedullin (AM) receptors. These receptors are heterodimers of the calcitonin receptor-like receptor (CL) and receptor activity modifying proteins (RAMPs), with the pharmacological properties determined by the RAMP subunit. BIBN4096BS-sensitive CGRP1 receptors are CL/RAMP1, whereas BIBN4096BS-insensitive AM receptors are CL/RAMP2 or CL/RAMP3 (AM(1) and AM(2), respectively), implicating RAMP1 in conferring BIBN4096BS sensitivity. Because calcitonin receptors [CT(a)] also interact with RAMP1 [AMY(1(a)) receptors], BIBN4096BS could also have affinity for these receptors. To test this, receptors were transfected into COS-7 cells and agonist-stimulated cAMP levels measured in the presence and absence of antagonists. We found that AMY(1(a)) receptors were similar to 150-fold less sensitive to BIBN4096BS antagonism than CGRP1 receptors. In contrast, AMY(3(a)) [CT(a)/RAMP3] or AM(2) receptors were not sensitive to BIBN4096BS antagonism. We investigated Trp74 in RAMP1, a residue implicated in the species selectivity of BIBN4096BS. BIBN4096BS affinity was reduced at AMY(1(a)) and CGRP1 receptors when this residue was mutated to lysine or alanine. The equivalent residue in RAMP3, Glu74, when mutated to tryptophan (E74W), induced BIBN4096BS sensitivity at AM(2) and AMY(3(a)) receptors. It is interesting that a selective reduction in AM potency was observed at E74W AM(2) receptors, implicating this residue in AM interactions with this receptor. These data support the importance of Trp74 in RAMP1 in the interaction of BIBN4096BS with CGRP(1) and AMY (1(a)) receptors and identified Glu74 in RAMP3 as the first amino acid in RAMP important for agonist interactions with calcitonin-family receptors.