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

  • Harriet A. Watkins
    Footnotes
    Affiliations
    From the School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand

    the Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland 1010, New Zealand
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  • Madhuri Chakravarthy
    Affiliations
    From the School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand
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  • Rekhati S. Abhayawardana
    Affiliations
    From the School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand
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  • Joseph J. Gingell
    Affiliations
    From the School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand

    the Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland 1010, New Zealand
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  • Michael Garelja
    Affiliations
    From the School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand
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  • Meenakshi Pardamwar
    Affiliations
    the School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom,
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  • James M.W.R. McElhinney
    Affiliations
    the School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom,
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  • Alex Lathbridge
    Affiliations
    the School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom,
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  • Arran Constantine
    Affiliations
    the School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom,
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  • Paul W.R. Harris
    Affiliations
    the Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland 1010, New Zealand

    the School of Chemical Sciences, University of Auckland, Auckland 1010, New Zealand,
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  • Tsz-Ying Yuen
    Affiliations
    the Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland 1010, New Zealand

    the School of Chemical Sciences, University of Auckland, Auckland 1010, New Zealand,
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  • Margaret A. Brimble
    Affiliations
    the Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland 1010, New Zealand

    the School of Chemical Sciences, University of Auckland, Auckland 1010, New Zealand,
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  • James Barwell
    Footnotes
    Affiliations
    the School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, United Kingdom,
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  • David R. Poyner
    Affiliations
    the School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, United Kingdom,
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  • Michael J. Woolley
    Footnotes
    Affiliations
    the School of Clinical and Experimental Medicine, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom,
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  • Alex C. Conner
    Affiliations
    the School of Clinical and Experimental Medicine, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom,
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  • Augen A. Pioszak
    Affiliations
    the Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104
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  • Christopher A. Reynolds
    Correspondence
    To whom correspondence may be addressed: School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom.
    Affiliations
    the School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom,
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  • Debbie L. Hay
    Correspondence
    To whom correspondence may be addressed: School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand.
    Footnotes
    Affiliations
    From the School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand

    the Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland 1010, New Zealand
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  • Author Footnotes
    1 Supported by the National Heart Foundation of New Zealand, the Maurice and Phyllis Paykel Trust, and the Maurice Wilkins Centre for Molecular Biodiscovery.
    2 Supported by British Heart Foundation Grant FS/05/054.
    3 Supported by British Heart Foundation Grant PG/12/59/29795.
    6 The abbreviations used are: AMadrenomedullinCGRPcalcitonin gene-related peptideCLRcalcitonin-like receptorECDextracellular domainECLextracellular loopGPCRG protein-coupled receptorRAMPreceptor activity-modifying proteinTMtransmembrane domainFmocN-(9-fluorenyl)methoxycarbonylRArelative activityPTHparathyroid hormonePDBProtein Data BankDOPEdiscrete optimized protein energyGCGRglucagon receptorCRF1Rcorticotropin releasing factor 1 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.

      Introduction

      The endothelium-derived peptide hormone adrenomedullin (AM)
      The abbreviations used are: AM
      adrenomedullin
      CGRP
      calcitonin gene-related peptide
      CLR
      calcitonin-like receptor
      ECD
      extracellular domain
      ECL
      extracellular loop
      GPCR
      G protein-coupled receptor
      RAMP
      receptor activity-modifying protein
      TM
      transmembrane domain
      Fmoc
      N-(9-fluorenyl)methoxycarbonyl
      RA
      relative activity
      PTH
      parathyroid hormone
      PDB
      Protein Data Bank
      DOPE
      discrete optimized protein energy
      GCGR
      glucagon receptor
      CRF1R
      corticotropin releasing factor 1 receptor.
      is a protective factor in the cardiovascular system and a biomarker for cardiovascular disease (
      • Nishikimi T.
      • Kuwahara K.
      • Nakagawa Y.
      • Kangawa K.
      • Nakao K.
      Adrenomedullin in cardiovascular disease: a useful biomarker, its pathological roles and therapeutic application.
      • Manuela C.
      • Laura S.
      • Benedetta S.
      • Raffaele C.
      • Alessandro V.
      • Chiara C.
      • Tommaso P.
      • Daniela G.
      • Silvia D.R.
      Adrenomedullin and intermedin gene transcription is increased in leukocytes of patients with chronic heart failure at different stages of the disease.
      ,
      • Wong C.P.
      • Loh S.Y.
      • Loh K.K.
      • Ong P.J.
      • Foo D.
      • Ho H.H.
      Acute myocardial infarction: clinical features and outcomes in young adults in Singapore.
      ,
      • Burley D.S.
      • Hamid S.A.
      • Baxter G.F.
      Cardioprotective actions of peptide hormones in myocardial ischemia.
      • Nagaya N.
      • Nishikimi T.
      • Uematsu M.
      • Satoh T.
      • Oya H.
      • Kyotani S.
      • Sakamaki F.
      • Ueno K.
      • Nakanishi N.
      • Miyatake K.
      • Kangawa K.
      Haemodynamic and hormonal effects of adrenomedullin in patients with pulmonary hypertension.
      ). AM administration in human subjects has several positive outcomes, significantly improving patient recovery from myocardial infarction, inhibiting myocyte apoptosis, reducing mean pulmonary arterial pressure, and increasing cardiac output in heart failure patients (
      • Nishikimi T.
      • Kuwahara K.
      • Nakagawa Y.
      • Kangawa K.
      • Nakao K.
      Adrenomedullin in cardiovascular disease: a useful biomarker, its pathological roles and therapeutic application.
      ,
      • Nagaya N.
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      • Uematsu M.
      • Satoh T.
      • Oya H.
      • Kyotani S.
      • Sakamaki F.
      • Ueno K.
      • Nakanishi N.
      • Miyatake K.
      • Kangawa K.
      Haemodynamic and hormonal effects of adrenomedullin in patients with pulmonary hypertension.
      ,
      • Lainchbury J.G.
      • Nicholls M.G.
      • Espiner E.A.
      • Yandle T.G.
      • Lewis L.K.
      • Richards A.M.
      Bioactivity and interactions of adrenomedullin and brain natriuretic peptide in patients with heart failure.
      • Kataoka Y.
      • Miyazaki S.
      • Yasuda S.
      • Nagaya N.
      • Noguchi T.
      • Yamada N.
      • Morii I.
      • Kawamura A.
      • Doi K.
      • Miyatake K.
      • Tomoike H.
      • Kangawa K.
      The first clinical pilot study of intravenous adrenomedullin administration in patients with acute myocardial infarction.
      ). However, serious adverse hypotension in some patients, coupled with rapid metabolism of the peptide, means that optimal targeting of the AM system still needs to be achieved (
      • Kataoka Y.
      • Miyazaki S.
      • Yasuda S.
      • Nagaya N.
      • Noguchi T.
      • Yamada N.
      • Morii I.
      • Kawamura A.
      • Doi K.
      • Miyatake K.
      • Tomoike H.
      • Kangawa K.
      The first clinical pilot study of intravenous adrenomedullin administration in patients with acute myocardial infarction.
      ,
      • Dupuis J.
      • Caron A.
      • Ruël N.
      Biodistribution, plasma kinetics and quantification of single-pass pulmonary clearance of adrenomedullin.
      ). The pro-angiogenic effects of AM mean that receptor agonists or antagonists could be useful in a range of other conditions, such as lymphedema or cancer (
      • Kato J.
      • Kitamura K.
      Bench-to-bedside pharmacology of adrenomedullin.
      ). Realizing any of these therapeutic goals, however, requires a much greater understanding of AM receptor biology. Here we explore receptor architecture to lay the foundations for the design of selective AM receptor ligands.
      AM signals through two receptors. These both contain the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor (GPCR) that has an absolute requirement for association with a receptor activity-modifying protein (RAMP) for ligand binding and receptor activation to occur. Association of CLR with RAMP2 generates the AM1 receptor, whereas CLR with RAMP3 forms the AM2 receptor (
      • Poyner D.R.
      • Sexton P.M.
      • Marshall I.
      • Smith D.M.
      • Quirion R.
      • Born W.
      • Muff R.
      • Fischer J.A.
      • Foord S.M.
      International Union of Pharmacology. XXXII. The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors.
      ).
      The AM1 receptor has an important role in cardiovascular system development. Deletion of the genes for AM, CLR, or RAMP2 results in embryonic lethality due to the development of hydrops fetalis and cardiovascular abnormalities (
      • Dackor R.T.
      • Fritz-Six K.
      • Dunworth W.P.
      • Gibbons C.L.
      • Smithies O.
      • Caron K.M.
      Hydrops fetalis, cardiovascular defects, and embryonic lethality in mice lacking the calcitonin receptor-like receptor gene.
      ,
      • Dackor R.
      • Fritz-Six K.
      • Smithies O.
      • Caron K.
      Receptor activity-modifying proteins 2 and 3 have distinct physiological functions from embryogenesis to old age.
      • Caron K.M.
      • Smithies O.
      Extreme hydrops fetalis and cardiovascular abnormalities in mice lacking a functional adrenomedullin gene.
      ). For example, Adm−/− mice have small and disorganized hearts (
      • Caron K.M.
      • Smithies O.
      Extreme hydrops fetalis and cardiovascular abnormalities in mice lacking a functional adrenomedullin gene.
      ). Cardiomyocyte-specific RAMP2 knock-out disrupted cardiac metabolism and homeostasis by causing cardiac dilation and changes in mitochondrial structure (
      • Yoshizawa T.
      • Sakurai T.
      • Kamiyoshi A.
      • Ichikawa-Shindo Y.
      • Kawate H.
      • Iesato Y.
      • Koyama T.
      • Uetake R.
      • Yang L.
      • Yamauchi A.
      • Tanaka M.
      • Toriyama Y.
      • Igarashi K.
      • Nakada T.
      • Kashihara T.
      • et al.
      Novel regulation of cardiac metabolism and homeostasis by the adrenomedullin-receptor activity-modifying protein 2 system.
      ). Furthermore, targeted RAMP2 overexpression in vascular smooth muscle suggests that the AM1 receptor could protect against vascular remodeling invoked by prolonged hypertension (
      • Liang L.
      • Tam C.W.
      • Pozsgai G.
      • Siow R.
      • Clark N.
      • Keeble J.
      • Husmann K.
      • Born W.
      • Fischer J.A.
      • Poston R.
      • Shah A.
      • Brain S.D.
      Protection of angiotensin II-induced vascular hypertrophy in vascular smooth muscle-targeted receptor activity-modifying protein 2 transgenic mice.
      ).
      RAMP3 knock-out mice give important insight into the likely role of the AM2 receptor in cardiac biology. Unlike RAMP2 knock-out mice, these animals survive into old age and exhibit normal angiogenesis (
      • Dackor R.
      • Fritz-Six K.
      • Smithies O.
      • Caron K.
      Receptor activity-modifying proteins 2 and 3 have distinct physiological functions from embryogenesis to old age.
      ,
      • Barrick C.J.
      • Lenhart P.M.
      • Dackor R.T.
      • Nagle E.
      • Caron K.M.
      Loss of receptor activity-modifying protein 3 exacerbates cardiac hypertrophy and transition to heart failure in a sex-dependent manner.
      ). When challenged by crossing Ramp3−/− with RenTgMK mice (a genetic model of angiotensin II-mediated cardiovascular disease), sex-dependent cardiovascular phenotypic differences emerge (i.e. renal failure and cardiac hypertrophy occur only in male mice) (
      • Barrick C.J.
      • Lenhart P.M.
      • Dackor R.T.
      • Nagle E.
      • Caron K.M.
      Loss of receptor activity-modifying protein 3 exacerbates cardiac hypertrophy and transition to heart failure in a sex-dependent manner.
      ). A separate Ramp3−/− model exhibited narrowed lymphatic vessels, impaired lymphatic drainage, and thus post-operative lymphedema and prolonged inflammation (
      • Yamauchi A.
      • Sakurai T.
      • Kamiyoshi A.
      • Ichikawa-Shindo Y.
      • Kawate H.
      • Igarashi K.
      • Toriyama Y.
      • Tanaka M.
      • Liu T.
      • Xian X.
      • Imai A.
      • Zhai L.
      • Owa S.
      • Arai T.
      • Shindo T.
      Functional differentiation of RAMP2 and RAMP3 in their regulation of the vascular system.
      ).
      Thus, the AM1 and AM2 receptors have distinct roles. In animal models of cardiovascular disease, both the relative and absolute expression of the AM1 and AM2 receptor subunits change in different disease states. In the kidney of hypertensive rats, RAMP2 expression decreases, and RAMP3 expression increases (
      • Tadokoro K.
      • Nishikimi T.
      • Mori Y.
      • Wang X.
      • Akimoto K.
      • Matsuoka H.
      Altered gene expression of adrenomedullin and its receptor system and molecular forms of tissue adrenomedullin in left ventricular hypertrophy induced by malignant hypertension.
      ). Each AM receptor is a potential drug target, and it is important to develop selective molecules for each receptor that can tease out the most beneficial receptor activity. For example, AM1 receptor antagonists could be useful anti-angiogenic agents in cancer (
      • Kato J.
      • Kitamura K.
      Bench-to-bedside pharmacology of adrenomedullin.
      ). In cardiovascular disease, either receptor could be a drug target. Receptor-selective molecules are urgently needed to tease out the role of each receptor and enable drug development efforts.
      The AM receptors are compelling targets from a drug discovery perspective because of their biological effects and because they belong to the large GPCR superfamily of transmembrane proteins that are the cellular targets for 36% of all approved therapeutics (
      • Garland S.L.
      Are GPCRs still a source of new targets?.
      ). Peptide-binding class B GPCRs (including CLR) maintain the conserved heptahelical conformation observed across the wider superfamily with attendant intracellular loops, extracellular loops (ECLs), and a large extracellular domain (ECD) (
      • Hollenstein K.
      • de Graaf C.
      • Bortolato A.
      • Wang M.W.
      • Marshall F.H.
      • Stevens R.C.
      Insights into the structure of class B GPCRs.
      ). Class B GPCR peptide ligands are known to interact with the ECD through their C terminus, with a second interaction of their N terminus with the juxtamembrane domain (the ECLs and the upper region of the transmembrane (TM) helices) that initiates receptor activation. However, the fact that the two AM receptors share a common GPCR (CLR) and the natural ligand (AM) makes minimal direct contact with the RAMP ECD (
      • Booe J.M.
      • Walker C.S.
      • Barwell J.
      • Kuteyi G.
      • Simms J.
      • Jamaluddin M.A.
      • Warner M.L.
      • Bill R.M.
      • Harris P.W.
      • Brimble M.A.
      • Poyner D.R.
      • Hay D.L.
      • Pioszak A.A.
      Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor.
      ) makes the design of receptor-specific drugs a challenge. Rational design of specific ligands would therefore benefit from improved knowledge of the full impact of RAMPs upon AM1 and AM2 receptor structure and function. In the pursuit of AM receptor agonists, a focus on the regions of CLR that trigger signaling is critical.
      Here we explore how RAMPs affect the CLR juxtamembrane domain through extensive site-directed mutagenesis and molecular modeling. Our data suggest that RAMP2 and RAMP3 each create unique CLR conformations that may be exploitable for the development of small molecule ligands.

      Experimental Procedures

       Materials

      Human AM (AM(1–52)) was purchased from American Peptide (Sunnyvale, CA). Forskolin was from Tocris Bioscience (Wiltshire, UK). ALPHAscreen cAMP assay kits were from PerkinElmer Life Sciences. Poly-d-lysine-coated plates were from BD (Auckland, New Zealand). 125I-AM(13–52) was from PerkinElmer Life Sciences.

       Expression Constructs and Mutagenesis

      Wild type (WT) human CLR with an N-terminal hemagglutinin (HA) epitope tag, human RAMP2 with an N-terminal FLAG epitope tag, and untagged human RAMP3 were used in this study (
      • Watkins H.A.
      • Walker C.S.
      • Ly K.N.
      • Bailey R.J.
      • Barwell J.
      • Poyner D.R.
      • Hay D.L.
      Receptor activity-modifying protein-dependent effects of mutations in the calcitonin receptor-like receptor: implications for adrenomedullin and calcitonin gene-related peptide pharmacology.
      ,
      • Qi T.
      • Dong M.
      • Watkins H.A.
      • Wootten D.
      • Miller L.J.
      • Hay D.L.
      Receptor activity-modifying protein-dependent impairment of calcitonin receptor splice variant Δ(1–47)hCT (a) function.
      ). The HA-CLR mutants and RAMP constructs have been described previously (
      • Barwell J.
      • Conner A.
      • Poyner D.R.
      Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function.
      ,
      • Qi T.
      • Christopoulos G.
      • Bailey R.J.
      • Christopoulos A.
      • Sexton P.M.
      • Hay D.L.
      Identification of N-terminal receptor activity-modifying protein residues important for calcitonin gene-related peptide, adrenomedullin, and amylin receptor function.
      • Woolley M.J.
      • Watkins H.A.
      • Taddese B.
      • Karakullukcu Z.G.
      • Barwell J.
      • Smith K.J.
      • Hay D.L.
      • Poyner D.R.
      • Reynolds C.A.
      • Conner A.C.
      The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach.
      ).

       Cell Culture and Transfection

      Culture of HEK293S cells was performed as described previously (
      • Qi T.
      • Dong M.
      • Watkins H.A.
      • Wootten D.
      • Miller L.J.
      • Hay D.L.
      Receptor activity-modifying protein-dependent impairment of calcitonin receptor splice variant Δ(1–47)hCT (a) function.
      ). Cells were counted using a Countess CounterTM (Invitrogen) and seeded at a density of 15,000 cells/well into 96-well poly-d-lysine-coated plates. For binding assays, 24-well plates were used (
      • Watkins H.A.
      • Walker C.S.
      • Ly K.N.
      • Bailey R.J.
      • Barwell J.
      • Poyner D.R.
      • Hay D.L.
      Receptor activity-modifying protein-dependent effects of mutations in the calcitonin receptor-like receptor: implications for adrenomedullin and calcitonin gene-related peptide pharmacology.
      ). These were transiently transfected using polyethyleneimine as described previously (
      • Bailey R.J.
      • Hay D.L.
      Pharmacology of the human CGRP1 receptor in Cos 7 cells.
      ).

       Synthesis of Alanine-substituted AM(15–52) and Experiments with Phe18 AM

      For experiments investigating the role of Phe18 in the AM peptide, we used an F18A AM(15–52) peptide, alongside a WT AM(15–52) control. As is evident from the data for full-length AM(1–52) and AM(15–52) (TABLE 2, TABLE 4), these peptides have equivalent function. The AM(15–52) peptides were synthesized by solid phase peptide synthesis using the Fmoc/tert-butyl method on a 0.1-mmol scale. Briefly, Rink amide aminomethyl resin was prepared (
      • Harris P.W.R.
      • Yang S.H.
      • Brimble M.A.
      An improved procedure for the preparation of aminomethyl polystyrene resin and its use in solid phase (peptide) synthesis.
      ), and the peptide was elongated using a CEM Liberty microwave peptide synthesizer (CEM Corp., Matthews, NC) using 5% (w/v) piperazine containing 0.1 m 6-chlorobenzatriazole in N,N-dimethylformamide as Fmoc deblocking reagent and O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, and N,N-diisopropylethylamine as coupling reagents using microwave settings as described previously (
      • Harris P.W.R.
      • Williams G.M.
      • Shepherd P.
      • Brimble M.A.
      The synthesis of phosphopeptides using microwave-assisted solid phase peptide synthesis.
      ). The peptides were cleaved from the resin with concomitant removal of side chain protecting groups with 94.0% trifluoroacetic acid, 1.0% triisopropylsilane, 2.5% water, and 2.5% 2,2′-(ethylenedioxy)diethanethiol (v/v/v/v) for 2–3 h, precipitated with cold diethyl ether, recovered by centrifugation, dissolved in 50% aqueous acetonitrile containing 0.1% trifluoroacetic acid, and lyophilized. The crude peptides were dissolved in 0.1 m Tris (pH 8.1) at a concentration of 1 mg/ml, and the oxidation (disulfide formation) was allowed to proceed at room temperature open to air. Monitoring by reverse phase HPLC and/or LC-MS indicated that the reaction was typically complete within 1 day. The solution was acidified to pH 2 with 5 m HCl, purified directly by semipreparative reverse phase HPLC using a C18 Gemini (Phenomenex, Torrance, CA) column (10 × 250 mm) at a flow rate of 5 ml/min, and eluted using an appropriate gradient based on the analytical HPLC profile. Fractions containing the pure peptide were identified by electrospray mass spectrometry and/or HPLC, pooled, and lyophilized. All peptides were >95% pure as judged by integration of the HPLC chromatogram at 210 nm, and peptide masses were confirmed by electrospray mass spectrometry.
      TABLE 4Pharmacological parameters of cAMP accumulation for F18A substituted AM(15–52) versus wild type (WT) AM(15–52) stimulation of the WT AM1 and AM2 receptors
      WT AM(15–52) pEC50F18A AM(15–52) pEC50ΔLog EC50% Emax WT AM(15–52)n
      AM1 receptor8.89 ± 0.137.85 ± 0.11***1.0440.3 ± 9.73***4
      AM2 receptor8.91 ± 0.247.42 ± 0.24*1.49101.7 ± 5.263

       cAMP Assays

      We selected the mutants to study based on the boundaries of the ECLs according to our homology model of the calcitonin gene-related peptide (CGRP) receptor (CLR/RAMP1) (
      • Woolley M.J.
      • Watkins H.A.
      • Taddese B.
      • Karakullukcu Z.G.
      • Barwell J.
      • Smith K.J.
      • Hay D.L.
      • Poyner D.R.
      • Reynolds C.A.
      • Conner A.C.
      The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach.
      ,
      • Vohra S.
      • Taddese B.
      • Conner A.C.
      • Poyner D.R.
      • Hay D.L.
      • Barwell J.
      • Reeves P.J.
      • Upton G.J.
      • Reynolds C.A.
      Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies.
      ). CLR is predominantly Gs-coupled, so we characterized AM-stimulated cAMP signaling of alanine (or leucine, where natively alanine) mutants of CLR complexed with either RAMP2 or RAMP3. cAMP assays were performed as described previously using 1 mm isobutylmethylxanthine and a 15-min cell stimulation period (
      • Gingell J.J.
      • Qi T.
      • Bailey R.J.
      • Hay D.L.
      A key role for tryptophan 84 in receptor activity-modifying protein 1 in the amylin 1 receptor.
      ). cAMP was then quantified using ALPHAscreen on a JANUS automated work station (PerkinElmer Life Sciences).

       Analysis of Cell Surface Expression of Mutants by ELISA

      CLR, RAMP2, and RAMP3 are inefficiently expressed on their own at the cell surface (
      • McLatchie L.M.
      • Fraser N.J.
      • Main M.J.
      • Wise A.
      • Brown J.
      • Thompson N.
      • Solari R.
      • Lee M.G.
      • Foord S.M.
      RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor.
      ). However, when CLR is expressed with either RAMP, a functional AM1 or AM2 receptor is translocated to the cell surface. We determined expression levels of WT CLR/RAMP2 and CLR/RAMP3 heterodimers and cell surface expression of the mutant receptors as described previously, by measuring HA-CLR (
      • Bailey R.J.
      • Hay D.L.
      Agonist-dependent consequences of proline to alanine substitution in the transmembrane helices of the calcitonin receptor.
      ,
      • Conner A.C.
      • Hay D.L.
      • Simms J.
      • Howitt S.G.
      • Schindler M.
      • Smith D.M.
      • Wheatley M.
      • Poyner D.R.
      A key role for transmembrane prolines in calcitonin receptor-like receptor agonist binding and signalling: implications for family B G-protein-coupled receptors.
      ). Due to the RAMP-dependent effects observed, we first ensured that each RAMP was capable of producing equivalent HA-CLR translocation to the cell surface: HA-CLR cell surface expression with (A490A650/595) untagged RAMP1, 4.32 ± 0.31 (n = 3); Myc-RAMP1, 4.16 ± 0.22 (n = 3); untagged RAMP2, 2.81 ± 0.42 (n = 3); FLAG-RAMP2, 3.08 ± 0.38 (n = 3); or untagged RAMP3, 2.96 ± 0.36 (n = 3) (no significant differences by one-way analysis of variance). Thus, RAMP-specific effects of CLR mutations are unlikely to be due to an alteration in receptor density at the cell surface.

       Radioligand Binding

      AM binding assays were performed as described previously, displacing 125I-AM(13–52) with unlabeled AM (
      • Watkins H.A.
      • Walker C.S.
      • Ly K.N.
      • Bailey R.J.
      • Barwell J.
      • Poyner D.R.
      • Hay D.L.
      Receptor activity-modifying protein-dependent effects of mutations in the calcitonin receptor-like receptor: implications for adrenomedullin and calcitonin gene-related peptide pharmacology.
      ).

       Data Analysis

      All experiments were independently replicated at least three times, with two or three technical replicates in each experiment. Data analysis for cAMP assays was performed in GraphPad Prism version 6 (GraphPad Software, La Jolla, CA). Concentration-response curves were initially fitted to a four-parameter logistic equation; in all cases, the Hill slope was not significantly different from unity. Consequently, this was constrained to equal 1, the data were refitted to a three-parameter logistic equation, and pEC50 values were obtained. In order to determine Emax values for the mutant receptor curves, the data were normalized with respect to the fitted minimum and maximum of the WT curve. The combined normalized data sets were generated by combining the mean of the data points from the curves of each individual experiment. Variations in pEC50 between WT and mutant receptors were analyzed for statistical significance using an unpaired t test on the values obtained before curve normalization (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Emax values expressed as a percentage of WT were analyzed similarly. A Δlog pEC50 of ≥0.5 and a ≥30% Emax difference (compared with WT) coupled to significance at the p < 0.05 level were used to identify residues with an unambiguous effect.
      To further identify mutants that discriminated between AM1 and AM2 receptors, the differences in relative activity (RA) between the WT and mutant receptors were considered (
      • Kenakin T.P.
      A Pharmacology Primer: Techniques for More Effective and Strategic Drug Discovery.
      ). The log(RA) for each mutant and corresponding WT were calculated as log(mutant Emax/mutant EC50) and log(WT Emax/WT EC50). The mutant value was subtracted from the WT value to obtain Δlog(RA). Δlog(RA) values different from 0 were identified using multiple t tests with the false discovery rate set at 1%; differences between Δlog(RA)at the AM1 receptor and AM2 receptor were investigated by a two-way analysis of variance followed by Sidak’s multiple comparison test to compare individual means. Radioligand binding was analyzed in GraphPad Prism version 6 to a three-parameter logistic equation to obtain the pIC50 and maximum specific binding.
      For ELISA, values were normalized to WT HA-CLR/RAMP as 100% and empty vector-transfected cells as 0%. Statistical significance between WT and mutants was determined using the 95% confidence interval.

       AM Peptide Structure Model

      The AM peptide structure (Fig. 1) was modeled from the known structures of its component parts (the disulfide-bonded region, the helical region, and the ECD region). The key stages in this modeling involved (i) the use of an in-house multiple-reference sequence alignment method tailored for aligning helices with low sequence identity (
      • Lock A.
      • Forfar R.
      • Weston C.
      • Bowsher L.
      • Upton G.J.G.
      • Reynolds C.A.
      • Ladds G.
      • Dixon A.M.
      One motif to bind them: a small-XXX-small motif affects transmembrane domain 1 oligomerization, function, localization, and cross-talk between two yeast GPCRs.
      ) and (ii) the comparative modeling capabilities of PLOP (
      • Jacobson M.P.
      • Pincus D.L.
      • Rapp C.S.
      • Day T.J.
      • Honig B.
      • Shaw D.E.
      • Friesner R.A.
      A hierarchical approach to all-atom protein loop prediction.
      ). There is little structural information for full-length AM in its receptor-bound conformation, making structure-based sequence alignments difficult. Moreover, class B GPCR peptide ligands appear to lie in a number of distinct groups (
      • Watkins H.A.
      • Au M.
      • Hay D.L.
      The structure of secretin family GPCR peptide ligands: implications for receptor pharmacology and drug development.
      ), so sequence alignment is not trivial. Consequently, separate alignments of the glucagon, GLP-1, PTH, and AM sequences were generated by ClustalX (
      • Larkin M.A.
      • Blackshields G.
      • Brown N.P.
      • Chenna R.
      • McGettigan P.A.
      • McWilliam H.
      • Valentin F.
      • Wallace I.M.
      • Wilm A.
      • Lopez R.
      • Thompson J.D.
      • Gibson T.J.
      • Higgins D.G.
      Clustal W and Clustal X version 2.0.
      ). The helical region of the AM peptide homologs, as indicated by the NMR structure (PDB code 2L7S) (
      • Pérez-Castells J.
      • Martín-Santamaría S.
      • Nieto L.
      • Ramos A.
      • Martínez A.
      • Pascual-Teresa B.
      • Jiménez-Barbero J.
      Structure of micelle-bound adrenomedullin: a first step toward the analysis of its interactions with receptors and small molecules.
      ), was aligned to those of the equivalent helical region in the glucagon/GLP-1/PTH family of peptides using an in-house multiple-reference method tailored for aligning helices with low sequence identity (
      • Lock A.
      • Forfar R.
      • Weston C.
      • Bowsher L.
      • Upton G.J.G.
      • Reynolds C.A.
      • Ladds G.
      • Dixon A.M.
      One motif to bind them: a small-XXX-small motif affects transmembrane domain 1 oligomerization, function, localization, and cross-talk between two yeast GPCRs.
      ) that is a development of the methods of reference (
      • Taddese B.
      • Upton G.J.G.
      • Bailey G.R.
      • Jordan S.R.D.
      • Abdulla N.Y.
      • Reeves P.J.
      • Reynolds C.A.
      Do plants contain G protein-coupled receptors?.
      ). The alignment is given in Fig. 1A; the alignment scores shown in Fig. 1B (and Fig. 1C) give strong support for the proposed alignment over the only plausible alternative involving a shift left of the AM helix by 4 positions. The AM/CLR/RAMP2 (PDB code 4RWF) ECD (
      • Booe J.M.
      • Walker C.S.
      • Barwell J.
      • Kuteyi G.
      • Simms J.
      • Jamaluddin M.A.
      • Warner M.L.
      • Bill R.M.
      • Harris P.W.
      • Brimble M.A.
      • Poyner D.R.
      • Hay D.L.
      • Pioszak A.A.
      Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor.
      ), the GLP-1/exendin-4 structure (PDB code 3C59) (
      • Runge S.
      • Thøhgersen H.
      • Madsen K.
      • Lau J.
      • Rudolph R.
      Crystal structure of the ligand-bound glucagon-like peptide-1 receptor extracellular domain.
      ), and the glucagon model structure (
      • Siu F.Y.
      • He M.
      • de Graaf C.
      • Han G.W.
      • Yang D.
      • Zhang Z.
      • Zhou C.
      • Xu Q.
      • Wacker D.
      • Joseph J.S.
      • Liu W.
      • Lau J.
      • Cherezov V.
      • Katritch V.
      • Wang M.W.
      • Stevens R.C.
      Structure of the human glucagon class B G-protein-coupled receptor.
      ) were structurally aligned using the SALIGN module of MODELER (
      • Eswar N.
      • Webb B.
      • Marti-Renom M.A.
      • Madhusudhan M.S.
      • Eramian D.
      • Shen M.Y.
      • Pieper U.
      • Sali A.
      Comparative protein structure modeling using MODELLER.
      ) (Fig. 1D), from which a template was constructed using Asp35–Tyr52 from the AM x-ray structure and Thr7–Tyr13 of the glucagon model peptide structure, which was preferred over the corresponding (Thr7)-Asp9-Gln13 of exendin-4 because the angle was more appropriate for peptide binding to the TM bundle. The missing loop was inserted using the comparative modeling, loop modeling, and minimization capabilities of PLOP (
      • Jacobson M.P.
      • Pincus D.L.
      • Rapp C.S.
      • Day T.J.
      • Honig B.
      • Shaw D.E.
      • Friesner R.A.
      A hierarchical approach to all-atom protein loop prediction.
      ) based on the alignment in Fig. 1F. The N terminus, taken from Woolley et al. (
      • Woolley M.J.
      • Watkins H.A.
      • Taddese B.
      • Karakullukcu Z.G.
      • Barwell J.
      • Smith K.J.
      • Hay D.L.
      • Poyner D.R.
      • Reynolds C.A.
      • Conner A.C.
      The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach.
      ), was added by structural alignment of the common helical domain using VMD (
      • Humphrey W.
      • Dalke A.
      • Schulten K.
      VMD: visual molecular dynamics.
      ), again using the alignment in Fig. 1A. The resulting peptide structure of AM(15–52) (structurally aligned to the CLR ECD) is shown in Fig. 1E.
      Figure thumbnail gr1
      FIGURE 1.Modeling the AM peptide. A, selected class B peptide alignments. Homologs of each of PTH, glucagon, and GLP-1 were aligned against AM homologs in a multireference profile alignment, as described by Lock et al. (
      • Lock A.
      • Forfar R.
      • Weston C.
      • Bowsher L.
      • Upton G.J.G.
      • Reynolds C.A.
      • Ladds G.
      • Dixon A.M.
      One motif to bind them: a small-XXX-small motif affects transmembrane domain 1 oligomerization, function, localization, and cross-talk between two yeast GPCRs.
      ), over the helical region denoted X. B, the multireference alignment scores. Alignment 0, corresponding to the alignment in A, has the highest score; the next highest score (alignment −4) corresponds to moving the AM helix 4 residues to the left, but this alternative score is low. C, as for B but missing PTH (red), glucagon (green), or GLP-1 (cyan); the results are presented as a control. D, a structural alignment of CLR (light green surface, schematic)/AM(35–52) (dark green schematic), GLP-1R (orange schematic)/exendin-4 (wheat/orange schematic), and GCGR (yellow schematic)/glucagon Thr7–Tyr13 (yellow). The AM(23–52) comparative modeling template was taken from AM(35–52) and glucagon Thr7–Tyr13. The exendin-4 is largely wheat-colored, but the region corresponding to Thr7–Tyr13 of glucagon is orange. E, the final AM(16–52) structure (black schematic, used as one of the templates for modeling the AM receptor) structurally aligned to the CLR ECD. The various components of AM are shown as color-coded transparent spheres: yellow, carbon atoms (disulfide-bonded loop); green, carbon atoms (helix); cyan, carbon atoms (loop); blue, carbon atoms (from the original x-ray structure). The final structure is very similar to this initial template structure. F, the alignment for the comparative modeling of AM(23–52).

       AM1 and AM2 Receptor Models

      Comparative AM1 and AM2 receptor models were generated using MODELER version 9.12 (
      • Eswar N.
      • Webb B.
      • Marti-Renom M.A.
      • Madhusudhan M.S.
      • Eramian D.
      • Shen M.Y.
      • Pieper U.
      • Sali A.
      Comparative protein structure modeling using MODELLER.
      ), essentially from two x-ray structures, namely the AM CLR-RAMP2 ECD complex (
      • Booe J.M.
      • Walker C.S.
      • Barwell J.
      • Kuteyi G.
      • Simms J.
      • Jamaluddin M.A.
      • Warner M.L.
      • Bill R.M.
      • Harris P.W.
      • Brimble M.A.
      • Poyner D.R.
      • Hay D.L.
      • Pioszak A.A.
      Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor.
      ) (PDB code 4RWF) and the glucagon receptor (GCGR) TM domain (
      • Siu F.Y.
      • He M.
      • de Graaf C.
      • Han G.W.
      • Yang D.
      • Zhang Z.
      • Zhou C.
      • Xu Q.
      • Wacker D.
      • Joseph J.S.
      • Liu W.
      • Lau J.
      • Cherezov V.
      • Katritch V.
      • Wang M.W.
      • Stevens R.C.
      Structure of the human glucagon class B G-protein-coupled receptor.
      ) (PDB code 4L6R). The GCGR was preferred over the corticotropin-releasing factor 1 receptor (CRF1R) TM structure because of its overall conformation and compatibility with the full GCGR model (
      • Siu F.Y.
      • He M.
      • de Graaf C.
      • Han G.W.
      • Yang D.
      • Zhang Z.
      • Zhou C.
      • Xu Q.
      • Wacker D.
      • Joseph J.S.
      • Liu W.
      • Lau J.
      • Cherezov V.
      • Katritch V.
      • Wang M.W.
      • Stevens R.C.
      Structure of the human glucagon class B G-protein-coupled receptor.
      ), but part of the superior quality CRF1R structure (as denoted by ERRAT (
      • Colovos C.
      • Yeates T.O.
      Verification of protein structures: patterns of nonbonded atomic interactions.
      )) was used in subsequent refinement. In addition, model structures for the full GCGR model (
      • Siu F.Y.
      • He M.
      • de Graaf C.
      • Han G.W.
      • Yang D.
      • Zhang Z.
      • Zhou C.
      • Xu Q.
      • Wacker D.
      • Joseph J.S.
      • Liu W.
      • Lau J.
      • Cherezov V.
      • Katritch V.
      • Wang M.W.
      • Stevens R.C.
      Structure of the human glucagon class B G-protein-coupled receptor.
      ) containing only Ser8–Asp15 of glucagon (c.f. Fig. 1D), the full-length AM peptide (Fig. 1E), CGRP(1–7) docked to an active model of CLR (
      • Woolley M.J.
      • Watkins H.A.
      • Taddese B.
      • Karakullukcu Z.G.
      • Barwell J.
      • Smith K.J.
      • Hay D.L.
      • Poyner D.R.
      • Reynolds C.A.
      • Conner A.C.
      The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach.
      ), and a model of the RAMP1 TM helix docked to TM7 were used (Fig. 2). The active character of the model was also imposed by including TM5-6 of an active CLR model derived from the β2-adrenergic receptor active complex (
      • Rasmussen S.G.F.
      • DeVree B.T.
      • Zou Y.Z.
      • Kruse A.C.
      • Chung K.Y.
      • Kobilka T.S.
      • Thian F.S.
      • Chae P.S.
      • Pardon E.
      • Calinski D.
      • Mathiesen J.M.
      • Shah S.T.A.
      • Lyons J.A.
      • Caffrey M.
      • Gellman S.H.
      • et al.
      Crystal structure of the β2 adrenergic receptor-Gs protein complex.
      ); this template also contained the C-terminal peptide of the G protein, Gs (Arg373–Leu394). Each of these structural templates contained information on part but not all of the desired structure and was linked via a global alignment (Fig. 3). In addition, we also included short N- and C-terminal extensions (6 and 5 residues, respectively) to the RAMP TM helix and the RAMP ECD to prevent the linker between them from becoming entangled in the bulk of the receptor. Within this alignment, the position of the gap in the CLR sequence between the ECD and TM1 relative to the longer human glucagon receptor sequence was determined by analysis of gaps in similar subsets within the glucagon multiple-sequence alignment (
      • Roy A.
      • Taddese B.
      • Vohra S.
      • Thimmaraju P.K.
      • Illingworth C.J.R.
      • Simpson L.M.
      • Mukherjee K.
      • Reynolds C.A.
      • Chintapalli S.V.
      Identifying subset errors in multiple sequence alignments.
      ). Two thousand models were generated, and the model having the lowest (best) DOPE score was chosen for further refinement. ECL1 was refined using MODELER from TM1–4 of a CLR model derived from the CRF1R structure in which variability (
      • Vohra S.
      • Taddese B.
      • Conner A.C.
      • Poyner D.R.
      • Hay D.L.
      • Barwell J.
      • Reeves P.J.
      • Upton G.J.
      • Reynolds C.A.
      Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies.
      ,
      • Baldwin J.M.
      • Schertler G.F.X.
      • Unger V.M.
      An α-carbon template for the transmembrane helices in the rhodopsin family of G-protein-coupled receptors.
      ,
      • Hollenstein K.
      • Kean J.
      • Bortolato A.
      • Cheng R.K.
      • Doré A.S.
      • Jazayeri A.
      • Cooke R.M.
      • Weir M.
      • Marshall F.H.
      Structure of class B GPCR corticotropin-releasing factor receptor 1.
      ) was used to orient the CLR ECL1 helix, as in a recent GLP-1 receptor model (
      • Wootten D.
      • Reynolds C.A.
      • Koole C.
      • Smith K.J.
      • Mobarec J.C.
      • Simms J.
      • Quon T.
      • Furness S.G.
      • Miller L.J.
      • Christopoulos A.
      • Sexton P.M.
      A hydrogen-bonded polar network in the core of the glucagon-like peptide-1 receptor is a fulcrum for biased agonism: lessons from class B crystal structures.
      ). The ECLs and the RAMP linker (here defined as the region connecting the extracellular helical domain and the TM helix (i.e. residues Val134–Leu147 for RAMP2 and Val106–Leu119 for RAMP3)) were refined using PLOP, which has been shown to perform well in GPCR loop modeling (
      • Jacobson M.P.
      • Pincus D.L.
      • Rapp C.S.
      • Day T.J.
      • Honig B.
      • Shaw D.E.
      • Friesner R.A.
      A hierarchical approach to all-atom protein loop prediction.
      ); this refinement removed any bias introduced by the extensions. The final models were minimized using PLOP (
      • Jacobson M.P.
      • Pincus D.L.
      • Rapp C.S.
      • Day T.J.
      • Honig B.
      • Shaw D.E.
      • Friesner R.A.
      A hierarchical approach to all-atom protein loop prediction.
      ).
      Figure thumbnail gr2
      FIGURE 2.The template structure of RAMP docked to an active model of CLR. The template structure (gray) was generated as follows. The length of the TM helix for RAMP1 is given as 21 residues by UniProt, but this is too short for a tilted helix to span the membrane. Consequently, for RAMP1, helices of lengths 26, 28, and 30 residues were constructed using Maestro, commencing at Ser117, Pro115, and Asp113, respectively. For RAMP2, helices of length 24, 26, and 28 residues were constructed, commencing at Asp144, Pro142, and Asp140, respectively. For RAMP3, helices of lengths 25, 26, and 28 residues were constructed, commencing at Asp116, Pro115, and Asp113, respectively. The helices were docked using the Cluspro, PatchDock, and SwarmDock servers to two active models of the CLR transmembrane helical bundle (six docking experiments) (
      • Vohra S.
      • Taddese B.
      • Conner A.C.
      • Poyner D.R.
      • Hay D.L.
      • Barwell J.
      • Reeves P.J.
      • Upton G.J.
      • Reynolds C.A.
      Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies.
      ,
      • Schneidman-Duhovny D.
      • Inbar Y.
      • Nussinov R.
      • Wolfson H.J.
      PatchDock and SymmDock: servers for rigid and symmetric docking.
      ,
      • Kozakov D.
      • Hall D.R.
      • Beglov D.
      • Brenke R.
      • Comeau S.R.
      • Shen Y.
      • Li K.
      • Zheng J.
      • Vakili P.
      • Paschalidis I.C.
      • Vajda S.
      Achieving reliability and high accuracy in automated protein docking: ClusPro, PIPER, SOU, and stability analysis in CAPRI rounds 13–19.
      • Li X.
      • Moal I.H.
      • Bates P.A.
      Detection and refinement of encounter complexes for protein-protein docking: taking account of macromolecular crowding.
      ); the active explicit membrane CLR model has been shown to be in very good agreement with the x-ray crystal structures of the GCGR and CRF1R (
      • Woolley M.J.
      • Watkins H.A.
      • Taddese B.
      • Karakullukcu Z.G.
      • Barwell J.
      • Smith K.J.
      • Hay D.L.
      • Poyner D.R.
      • Reynolds C.A.
      • Conner A.C.
      The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach.
      ). Results from each server that were not compatible with the membrane topology were eliminated, and the remaining viable solutions were clustered. Representative solutions were then refined and rescored using the FireDock server (so that poses generated by the different servers are treated equally) (
      • Andrusier N.
      • Nussinov R.
      • Wolfson H.J.
      FireDock: fast interaction refinement in molecular docking.
      ,
      • Mashiach E.
      • Schneidman-Duhovny D.
      • Andrusier N.
      • Nussinov R.
      • Wolfson H.J.
      FireDock: a web server for fast interaction refinement in molecular docking.
      ). The three best poses (on the basis of lowest energy and geometry consensus) were then docked using RosettaDock (
      • Gray J.J.
      • Moughon S.
      • Wang C.
      • Schueler-Furman O.
      • Kuhlman B.
      • Rohl C.A.
      • Baker D.
      Protein-protein docking with simultaneous optimization of rigid-body displacement and side-chain conformations.
      ,
      • Wang D.F.
      • Wiest O.G.
      • Helquist P.
      Homology modeling, docking and molecular dynamics simulation of class IHDACs.
      • Wang J.G.
      • Xiao Y.J.
      • Li Y.H.
      • Ma Y.
      • Li Z.M.
      Identification of some novel AHAS inhibitors via molecular docking and virtual screening approach.
      ). The consensus result showed a preference for the helix to dock to TM7 of the active receptor, in agreement with experimental results that indicate an interaction with TM6/7 (
      • Harikumar K.G.
      • Simms J.
      • Christopoulos G.
      • Sexton P.M.
      • Miller L.J.
      Molecular basis of association of receptor activity-modifying protein 3 with the family B G protein-coupled secretin receptor.
      ). The active AM1 (light blue) and AM2 (wheat) model TM domains, the inactive GCGR (yellow), and TM1–TM4 of inactive CRF1R (orange) structures, superimposed over TM1, TM2, TM3, the top of TM4 (because of irregularities in the GCGR x-ray structure; c.f. CRF1R), and TM7, are also shown. TM5, TM6, and the top of TM7 were omitted from the fitting because of differences in active and inactive structures in this region. All root mean square deviations were <2 Å.
      Figure thumbnail gr3
      FIGURE 3.The alignment for comparative modeling. The alignment was generated by structural alignment of the templates using the SALIGN module of MODELER and refined using Jalview (
      • Clamp M.
      • Cuff J.
      • Searle S.M.
      • Barton G.J.
      The Jalview Java alignment editor.
      ). The residues are color-coded according to their properties as follows: blue, positive; red, negative or small polar; purple, polar; green large hydrophobic; yellow, small hydrophobic; cyan, polar, aromatic. This corresponds to the “Taylor” scheme, as implemented in Jalview. The extracellular loops are denoted by gray shading and the loop number.
      Druggability was assessed using the PockDrug (
      • Hussein H.A.
      • Borrel A.
      • Geneix C.
      • Petitjean M.
      • Regad L.
      • Camproux A.C.
      PockDrug-Server: a new web server for predicting pocket druggability on holo and apo proteins.
      ,
      • Borrel A.
      • Regad L.
      • Xhaard H.
      • Petitjean M.
      • Camproux A.C.
      Pock drug: a model for predicting pocket druggability that overcomes pocket estimation uncertainties.
      ) and DoGSiteScorer Web servers (
      • Volkamer A.
      • Kuhn D.
      • Rippmann F.
      • Rarey M.
      DoGSiteScorer: a web server for automatic binding site prediction, analysis and druggability assessment.
      ); pocket hull volumes (which include atoms within the druggable binding pockets) were also determined using PockDrug; distances were measured using the PyMOL Molecular Graphics System (version 1.7.4; Schrödinger, LLC, New York), which was also used for image generation. The models are available as supplemental models 1 and 2.

      Results

       Receptor Cell Surface Expression

      The cell surface expression levels of the WT AM1 and AM2 receptors were not significantly different (see “Experimental Procedures”). The cell surface expression of all mutant receptors showed very few significant differences compared with WT (Table 1). L351A and E357A CLR showed an ≥80% reduction of cell surface expression with both RAMPs, suggesting that these mutations caused the receptors to fail quality control processes prior to reaching the cell surface. Further data for these mutants is not discussed.
      TABLE 1Cell surface expression of AM1 and AM2 receptors expressed as a percentage of the wild type receptor
      TM2-ECL1-TM3TM4-ECL2-TM5TM6-ECL3-TM7
      AM1 receptorAM2 receptorAM1 receptorAM2 receptorAM1 receptorAM2 receptor
      %%%%%%
      L195A91.2 ± 2.0881.4 ± 25.9A271L118.8 ± 13.2118.8 ± 20.6F349A98.5 ± 11.393.9 ± 22.5
      T196A94.9 ± 17.890.5 ± 20.1I272A115.9 ± 15.664.1 ± 23.5V350A104.2 ± 8.06106.5 ± 33.4
      A197L111.4 ± 7.2885.9 ± 21.2A273L112.3 ± 11.6107.7 ± 6.74L351A9.75 ± 3.17*21.9 ± 12.3*
      V198A105.8 ± 14.184.3 ± 19.0R274A98.1 ± 17.3107.2 ± 14.4I352A68.9 ± 10.2*121.2 ± 13.7
      A199L99.8 ± 7.56136.7 ± 76.6S275A77.3 ± 10.1113.0 ± 25.3P353A94.2 ± 8.95104.0 ± 23.8
      N200A100.1 ± 6.6948.4 ± 8.62L276A73.8 ± 10.2110.3 ± 21.7W354A85.9 ± 9.30103.7 ± 24.0
      N201A81.9 ± 15.938.9 ± 13.4Y277A94.0 ± 7.26132.4 ± 23.0R355A114.7 ± 13.086.5 ± 11.8
      Q202A94.8 ± 1.9283.2 ± 10.2Y278A75.8 ± 27.6154.1 ± 27.1P356A101.3 ± 3.5987.6 ± 13.3
      A203L95.5 ± 9.6772.1 ± 18.4N279A97.0 ± 6.3096.8 ± 17.0E357A17.2 ± 4.85*15.1 ± 9.97*
      L204A98.3 ± 2.83114.0 ± 15.8D280A155.8 ± 68.9126.1 ± 16.8G358A108.2 ± 7.7285.7 ± 9.81
      V205A117.3 ± 6.4988.7 ± 13.1N281A108.1 ± 2.90119.9 ± 2.97K359A99.6 ± 2.6376.3 ± 11.9
      A206L92.9 ± 8.4591.4 ± 13.1C282A64.9 ± 16.8100.7 ± 11.7I360A85.8 ± 3.8182.5 ± 7.48
      T207A99.9 ± 1.5282.2 ± 15.5W283A107.7 ± 11.9100.4 ± 13.4A361L102.2 ± 3.4583.1 ± 8.75
      N208A98.6 ± 1.02111.5 ± 18.4I284A67.1 ± 8.80107.6 ± 26.2E362A103.8 ± 6.1475.4 ± 4.93
      P209A100.3 ± 5.96118.6 ± 34.5S285A86.1 ± 20.2124.7 ± 31.6E363A103.0 ± 2.7475.6 ± 4.22
      V210A100.4 ± 5.6388.2 ± 15.0S286A93.2 ± 12.9104.7 ± 15.0V364A94.0 ± 2.74100.5 ± 12.5
      S211A97.5 ± 3.7395.4 ± 18.7D287A77.4 ± 13.4107.3 ± 7.33Y365A92.5 ± 6.21121.3 ± 24.5
      C212A119.0 ± 25.193.6 ± 22.1I288A94.8 ± 23.590.0 ± 7.14D366A107.5 ± 4.33103.1 ± 19.4
      K213A131.3 ± 33.184.3 ± 21.3H289A93.1 ± 16.295.7 ± 12.9Y367A110.0 ± 2.03106.2 ± 16.4
      V214A87.5 ± 9.7186.6 ± 17.9L290A102.5 ± 2.4598.6 ± 7.36I368A90.8 ± 15.2118.6 ± 24.7
      S215A86.2 ± 10.784.5 ± 17.3L291A103.5 ± 7.54130.8 ± 10.3M369A82.8 ± 13.9108.2 ± 25.1
      Q216A95.7 ± 2.47124.9 ± 23.7Y292A83.5 ± 7.47176.5 ± 40.6
      F217A101.8 ± 7.4599.0 ± 12.0I293A99.2 ± 1.44107.9 ± 12.5
      I294A103.2 ± 14.7102.8 ± 29.8

       Functional Analysis of Receptor Mutations

      We assayed a total of 68 CLR mutants with RAMP2 and with RAMP3. All results are reported in TABLE 2, TABLE 3. cAMP data for selected mutants, which illustrate a breadth of effects, are shown in FIGURE 4., FIGURE 5.. The mutations could in principle change either the affinity of binding of AM or its ability to activate the receptor (efficacy). Efficacy can be estimated to some extent from Emax, but this is limited by receptor reserve. Furthermore, for many mutants, we cannot measure affinity directly because the only radioligand available to us is the agonist, 125I-AM, which will not give detectable binding once its affinity goes below around 10 nm. The EC50 describes potency but does not provide a ready means for identifying mutants that alter efficacy as well as affinity. Accordingly, we have used Δlog(RA) (see “Experimental Procedures”) as a simple parameter to characterize the effect of the mutations in functional assays; where appropriate, we supplement this with observations on Emax or EC50. Using this, we describe below our major observations, categorized according to the effect of the mutation. We have also conducted radioligand binding assays using 125I-AM on selected mutants to provide additional information (Fig. 6).
      TABLE 2Pharmacological parameters of cAMP accumulation for the AM receptors when stimulated by AM
      AM1 receptorAM2 receptor
      WT pEC50Mutant pEC50ΔLog pEC50Emax (%WT)ΔLog(RA)nWT pEC50Mutant pEC50ΔLog pEC50Emax (%WT)ΔLog(RA)n
      TM2
      L195A9.28 ± 0.10<6>2.00No curve
      a No curve, cAMP response was too low for a concentration-response curve to be fitted (pEC50 and Δlog pEC50 are denoted as <6 and >2).
      -59.11 ± 0.167.40 ± 0.17***1.7165.3 ± 11.3*1.90 ± 0.25
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      ,
      c ΔLog(RA) values where only the AM2 receptor was active.
      5
      T196A8.94 ± 0.138.95 ± 0.03−0.01119.5 ± 23.5−0.09 ± 0.1638.90 ± 0.198.66 ± 0.190.24116.5 ± 20.40.17 ± 0.283
      A197L8.91 ± 0.258.61 ± 0.130.3090.4 ± 6.320.34 ± 0.2849.19 ± 0.148.90 ± 0.110.29136.9 ± 30.10.15 ± 0.203
      V198A9.13 ± 0.118.32 ± 0.15***0.8174.5 ± 12.20.94 ± 0.20
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      79.41 ± 0.148.64 ± 0.19*0.77160.6 ± 26.16*0.56 ± 0.253
      ECL1
      A199L9.14 ± 0.128.10 ± 0.23**1.0455.4 ± 8.56**1.29 ± 0.28
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      69.29 ± 0.167.98 ± 0.20**1.3188.6 ± 9.461.36 ± 0.264
      N200A9.11 ± 0.169.55 ± 0.100.4480.9 ± 17.4−0.35 ± 0.2139.16 ± 0.149.57 ± 0.22−0.4169.3 ± 3.75***−0.25 ± 0.263
      N201A9.02 ± 0.168.70 ± 0.180.3289.8 ± 4.700.37 ± 0.2449.14 ± 0.108.50 ± 0.220.64133.3 ± 36.30.52 ± 0.273
      Q202A9.00 ± 0.079.15 ± 0.09−0.1582.9 ± 11.1−0.069 ± 0.1349.14 ± 0.109.30 ± 0.13−0.16184.4 ± 40.3−0.43 ± 0.193
      A203L9.27 ± 0.089.14 ± 0.080.1382.5 ± 6.92*0.21 ± 0.1259.14 ± 0.109.14 ± 0.080.00207.5 ± 57.6−0.32 ± 0.173
      L204A8.93 ± 0.048.62 ± 0.03**0.3198.9 ± 5.360.31 ± 0.0639.42 ± 0.298.89 ± 0.160.53145.7 ± 24.90.37 ± 0.343
      V205A9.10 ± 0.068.67 ± 0.14*0.4379.9 ± 16.80.53 ± 30.1849.42 ± 0.299.10 ± 0.100.32135.2 ± 34.70.19 ± 0.323
      A206L9.30 ± 0.088.93 ± 0.09*0.37113.3 ± 17.80.32 ± 0.1449.42 ± 0.299.33 ± 0.140.09118.2 ± 43.40.02 ± 0.363
      T207A9.10 ± 0.069.08 ± 0.160.0287.6 ± 2.59*0.077 ± 0.1749.41 ± 0.059.21 ± 0.050.2081.7 ± 14.20.29 ± 0.104
      N208A8.97 ± 0.068.73 ± 0.100.2484.9 ± 10.70.31 ± 0.1359.41 ± 0.058.82 ± 0.04***0.59105.4 ± 39.80.57 ± 0.184
      P209A8.99 ± 0.028.63 ± 0.09**0.36109.0 ± 13.40.32 ± 0.1149.41 ± 0.058.88 ± 0.15*0.53107.2 ± 40.20.50 ± 0.234
      V210A9.19 ± 0.069.03 ± 0.140.1674.9 ± 5.17**0.29 ± 0.1649.25 ± 0.109.03 ± 0.080.22133.3 ± 18.90.10 ± 0.143
      S211A9.09 ± 0.108.97 ± 0.110.12103.5 ± 12.10.11 ± 0.1559.25 ± 0.109.10 ± 0.030.15143.8 ± 66.6−0.01 ± 0.233
      TM3
      C212A9.04 ± 0.21<6>2.00No curve-69.20 ± 0.078.59 ± 0.15**0.6191.51 ± 18.70.65 ± 0.19
      c ΔLog(RA) values where only the AM2 receptor was active.
      6
      K213A9.22 ± 0.098.05 ± 0.09***1.1721.2 ± 7.38***1.84 ± 0.204
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      59.14 ± 0.088.31 ± 0.07***0.8397.7 ± 8.430.84 ± 0.11
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      5
      V214A9.11 ± 0.129.01 ± 0.170.1089.4 ± 7.110.15 ± 0.2159.14 ± 0.119.14 ± 0.170.0098.7 ± 9.120.01 ± 0.214
      S215A8.90 ± 0.128.81 ± 0.060.0999.3 ± 1.870.093 ± 0.1349.21 ± 0.129.22 ± 0.210.01111.4 ± 13.8−0.06 ± 0.253
      Q216A9.00 ± 0.129.60 ± 0.24−0.6100.2 ± 20.1−0.60 ± 0.2859.45 ± 0.149.42 ± 0.150.0384.9 ± 7.410.10 ± 0.213
      F217A9.19 ± 0.038.82 ± 0.11**0.3797.3 ± 18.80.38 ± 0.1479.41 ± 0.118.99 ± 0.150.44100.3 ± 17.00.42 ± 0.205
      TM4
      A271L9.27 ± 0.148.33 ± 0.23**0.9471.4 ± 9.83*1.09 ± 0.2859.41 ± 0.099.16 ± 0.180.2598.8 ± 33.70.26 ± 0.255
      I272A9.31 ± 0.189.65 ± 0.18−0.34151.6 ± 87.8−0.52 ± 0.3669.41 ± 0.099.42 ± 0.080.0194.3 ± 18.10.02 ± 0.155
      A273L8.96 ± 0.148.91 ± 0.090.05147.4 ± 42.7−0.12 ± 0.21549.26 ± 0.109.15 ± 0.090.11100.4 ± 9.900.11 ± 0.144
      R274A9.51 ± 0.187.32 ± 0.14***2.1917.7 ± 5.01***2.94 ± 0.26
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      59.24 ± 0.088.24 ± 0.11***1.0031.6 ± 10.1***1.50 ± 0.19
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      ***
      5
      ECL2
      S275A9.29 ± 0.139.39 ± 0.21−0.10103.3 ± 18.2−0.11 ± 0.2659.30 ± 0.159.23 ± 0.22−0.10114.9 ± 22.60.01 ± 0.285
      L276A9.29 ± 0.179.23 ± 0.150.0696.8 ± 18.00.074 ± 0.2449.30 ± 0.159.34 ± 0.16−0.0470.2 ± 12.40.11 ± 0.234
      Y277A9.51 ± 0.138.79 ± 0.19*0.7233.3 ± 8.98***1.20 ± 0.26
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      79.35 ± 0.099.23 ± 0.050.12113.7 ± 21.20.06 ± 0.13**5
      Y278A9.54 ± 0.138.45 ± 0.13***1.1355.9 ± 9.33***1.34 ± 0.20
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      79.34 ± 0.128.92 ± 0.090.42106.0 ± 29.50.39 ± 0.195
      N279A9.04 ± 0.108.53 ± 0.310.2521.7 ± 25.91.17 ± 0.61449.45 ± 0.109.17 ± 0.100.2895.6 ± 11.80.30 ± 0.154
      D280A9.29 ± 0.088.39 ± 0.08***0.9073.6 ± 7.61**1.03 ± 0.12
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      69.56 ± 0.078.31 ± 0.26**1.2593.7 ± 19.01.28 ± 0.28
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      5
      N281A9.32 ± 0.089.26 ± 0.100.06128.7 ± 16.9−0.05 ± 0.1459.64 ± 0.109.44 ± 0.190.20143.9 ± 16.70.04 ± 0.224
      C282A9.09 ± 0.058.15 ± 0.21**0.9448.5 ± 10.2***1.25 ± 0.23
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      69.10 ± 0.118.88 ± 0.180.22117.3 ± 16.70.15 ± 0.22*5
      W283A9.19 ± 0.136.96 ± 0.17***2.2325.9 ± 11.7***2.82 ± 0.29
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      59.54 ± 0.087.96 ± 0.10***1.5871.7 ± 27.51.72 ± 0.21
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      *
      5
      I284A8.97 ± 0.116.75 ± 0.35**2.0235.2 ± 7.25***2.67 ± 0.38
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      59.08 ± 0.127.36 ± 0.21***1.7251.8 ± 18.4**2.01 ± 0.29
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      5
      S285A9.33 ± 0.079.02 ± 0.080.31196.7 ± 113.90.02 ± 0.2749.60 ± 0.099.37 ± 0.110.23157.7 ± 52.80.03 ± 0.205
      S286A9.16 ± 0.279.31 ± 0.20−0.1599.2 ± 16.8−0.15 ± 0.3449.45 ± 0.189.66 ± 0.11−0.1971.4 ± 26.4−0.06 ± 0.263
      D287A9.26 ± 0.059.51 ± 0.47−0.25105.3 ± 34.5−0.27 ± 0.4949.31 ± 0.059.12 ± 0.120.19104.5 ± 18.80.17 ± 0.154
      T288A9.15 ± 0.138.37 ± 0.05***0.7851.4 ± 3.32***1.07 ± 0.14
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      49.60 ± 0.098.97 ± 0.11***0.6378.46 ± 14.60.74 ± 0.164
      H289A9.09 ± 0.049.12 ± 0.13−0.03118.7 ± 8.74−0.10 ± 0.1449.58 ± 0.269.74 ± 0.25−0.16298.8 ± 143.9−0.64 ± 0.425
      L290A8.96 ± 0.148.83 ± 0.090.13190.9 ± 84.2−0.15 ± 0.2549.26 ± 0.098.97 ± 0.080.2994.4 ± 13.80.32 ± 0.143
      L291A9.09 ± 0.048.62 ± 0.14*0.47136.7 ± 45.10.33 ± 0.2049.18 ± 0.128.83 ± 0.150.35125.1 ± 32.20.25 ± 0.226
      TM5
      Y292A8.96 ± 0.148.54 ± 0.06*0.4291.2 ± 18.90.46 ± 0.1849.32 ± 0.108.67 ± 0.17*0.65135.1 ± 42.20.52 ± 0.244
      I293A8.97 ± 0.049.20 ± 0.07*−0.2893.2 ± 29.6−0.20 ± 0.1649.01 ± 0.079.13 ± 0.02−0.12121.8 ± 23.1−0.21 ± 0.113
      I294A9.09 ± 0.048.83 ± 0.210.26147.5 ± 53.50.09 ± 0.2649.18 ± 0.129.12 ± 0.140.06128.3 ± 19.1−0.05 ± 0.195
      TM6
      F349A9.01 ± 0.118.72 ± 0.110.2921.5 ± 1.81***0.96 ± 0.16
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      59.14 ± 0.058.61 ± 0.07**0.5324.0 ± 9.13***1.15 ± 0.19
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      5
      V350A8.86 ± 0.288.72 ± 0.070.1463.7 ± 4.94***0.34 ± 0.2949.14 ± 0.058.87 ± 0.340.2765.8 ± 17.30.45 ± 0.364
      L351A9.15 ± 0.10No curve>3No Curve-49.09 ± 0.06No Curve>3No Curve-3
      I352A9.01 ± 0.118.28 ± 0.27*0.7337.7 ± 9.87***1.15 ± 0.3159.40 ± 0.088.46 ± 0.10***0.9486.9 ± 30.11.00 ± 0.20
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      5
      P353A9.07 ± 0.13No Curve>3No Curve-59.42 ± 0.098.49 ± 0.06***0.9147.1 ± 9.19***1.26 ± 0.14
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      ,
      c ΔLog(RA) values where only the AM2 receptor was active.
      5
      W354A9.29 ± 0.068.50 ± 0.12***0.7937.2 ± 7.38***1.22 ± 0.16
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      59.40 ± 0.088.59 ± 0.13***0.81112.4 ± 17.50.76 ± 0.17
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      5
      R355A9.13 ± 0.089.50 ± 0.12*−0.3780.4 ± 7.97*−0.27 ± 0.1549.27 ± 0.269.89 ± 0.03−0.6291.0 ± 18.6−0.58 ± 0.283
      P356A9.15 ± 0.108.99 ± 0.100.1640.4 ± 3.66***0.55 ± 0.1549.34 ± 0.209.12 ± 0.290.2260.0 ± 23.10.44 ± 0.394
      ECL3
      E357A9.00 ± 0.08No Curve>3No Curve-49.23 ± 0.26No Curve>3No Curve-3
      G358A9.09 ± 0.119.06 ± 0.110.03104.5 ± 5.460.01 ± 0.1648.79 ± 0.148.84 ± 0.27−0.0580.9 ± 55.30.04 ± 0.423
      K359A9.09 ± 0.119.19 ± 0.10−0.10107.5 ± 4.28−0.13 ± 0.1549.27 ± 0.269.30 ± 0.14−0.0375.9 ± 4.14***0.09 ± 0.293
      I360A9.13 ± 0.188.99 ± 0.080.1483.3 ± 9.470.22 ± 0.2048.79 ± 0.148.79 ± 0.290.0082.8 ± 35.90.08 ± 0.373
      A361L9.10 ± 0.189.52 ± 0.15−0.4277.8 ± 6.34*−0.31 ± 0.2449.15 ± 0.138.62 ± 0.400.5367.4 ± 12.4*0.70 ± 0.434
      E362A9.10 ± 0.189.00 ± 0.070.1098.0 ± 6.530.11 ± 0.1949.22 ± 0.108.91 ± 0.100.21241.7 ± 76.1−0.07 ± 0.203
      E363A9.00 ± 0.088.87 ± 0.150.1393.9 ± 13.50.16 ± 0.1849.22 ± 0.108.99 ± 0.110.23217.7 ± 72.3−0.11 ± 0.213
      V364A9.00 ± 0.088.84 ± 0.130.1684.3 ± 8.490.23 ± 0.1649.16 ± 0.099.14 ± 0.140.0283.2 ± 3.570.10 ± 0.174
      TM7
      Y365A9.09 ± 0.118.83 ± 0.110.2668.9 ± 6.54**0.42 ± 0.1649.02 ± 0.128.51 ± 0.330.5165.2 ± 0.85**0.70 ± 0.353
      D366A8.97 ± 0.088.76 ± 0.150.2169.9 ± 15.70.37 ± 0.1949.17 ± 0.119.19 ± 0.30−0.02105.7 ± 23.8−0.04 ± 0.333
      Y367A9.16 ± 0.109.11 ± 0.030.0470.0 ± 6.690.20 ± 0.1139.28 ± 0.178.61 ± 0.16*0.67163.7 ± 32.80.46 ± 0.253
      I368A9.06 ± 0.129.06 ± 0.080.0077.3 ± 12.60.11 ± 0.1649.28 ± 0.179.39 ± 0.25−0.11251.1 ± 120−0.51 ± 0.373
      M369A9.06 ± 0.129.06 ± 0.130.00185.1 ± 28.0*−0.27 ± 0.1949.28 ± 0.178.82 ± 0.270.46295.7 ± 103.1−0.01 ± 0.353
      a No curve, cAMP response was too low for a concentration-response curve to be fitted (pEC50 and Δlog pEC50 are denoted as <6 and >2).
      b Different from 0, as assessed by multiple t tests with the false discovery rate set to 1%.
      c ΔLog(RA) values where only the AM2 receptor was active.
      TABLE 3Pharmacological parameters for 125I-AM(13–52) binding for WT or mutant AM receptors
      AM1 receptorAM2 receptor
      pIC50Maximum specific binding (%WT)npIC50Maximum specific binding (%WT)n
      WT8.56 ± 0.0448.64 ± 0.074
      C212A8.06 ± 0.1671.7 ± 20.738.35 ± 0.18145.5 ± 23.43
      Y277A8.46 ± 0.3534.3 ± 11.1
      a 95% confidence interval does not include 100%.
      38.52 ± 0.1597.4 ± 21.63
      C282A8.40 ± 0.1585.4 ± 24.938.31 ± 0.29180.4 ± 46.63
      I352A8.56 ± 0.1043.5 ± 7.0
      a 95% confidence interval does not include 100%.
      38.65 ± 0.2142.2 ± 13.2
      a 95% confidence interval does not include 100%.
      3
      a 95% confidence interval does not include 100%.
      Figure thumbnail gr4
      FIGURE 4.Examples of mutants with common effects on cAMP production in both the AM1 and AM2 receptors. Concentration-response curves are combined normalized data ± S.E. (error bars) for at least three individual experiments.
      Figure thumbnail gr5
      FIGURE 5.Examples of mutants with common-differential and differential (C282A and Y277A) effects on cAMP production between the AM receptors. WT curves were included in every experiment but are only shown as examples for L195A so that mutant differences between the receptors are not obscured by these curves in the other panels. The horizontal line represents maximal (100%) cAMP accumulation for the WT receptors. Concentration-response curves are combined normalized data ± S.E. (error bars) for at least three individual experiments.
      Figure thumbnail gr6
      FIGURE 6.125I-AM(13–52) binding at selected mutants with common-differential and differential (Y277A and C282A) effects in cAMP assays. The curves are combined normalized data ± S.E. (error bars) for three individual experiments.
      There was a core subset of six residues that were important for the function of the AM1 and AM2 receptors (Ala199, Asp280, Ile284, Thr288, Phe349, and Tyr365), producing shared changes in pEC50, Emax or Δlog(RA). We define all of these six as having common effects (Fig. 4). These residues are situated within ECL2 and the TM6-ECL3-TM7 juxtamembrane region, along with A199L in TM2.
      A further 10 mutations had an effect at both AM receptors, but the nature of the effect differed between the two receptors (Leu195, Val198, Cys212, Lys213, Arg274, Trp283, Ile352, Pro353, Trp354, and Ala361). These are defined as residues with common but differential effects (Fig. 5). L195A in TM2, C212A at the ECL1-TM3 boundary, and P353A at the TM6-ECL3 boundary abolished AM-mediated cAMP production at the AM1 receptor, whereas K213A reduced this by 80%. For C212A, there was a trend for the radioligand binding to be modestly reduced at the AM1 receptor but enhanced at the AM2 receptor, consistent with a differential effect of this mutation at both receptors (Fig. 6). The corresponding mutations in the AM2 receptor were less deleterious. I352A and W354A mutations gave very similar changes in Δlog(RA), and radioligand binding shows a similar reduction in specific binding for I352A at both receptors (Fig. 6). However, in both cases, the effects on Emax were more marked at the AM1 receptor, so these have been included as common but differential residues. Whereas V198A showed only a small difference in Δlog(RA), it significantly increased Emax at the AM2 receptor but not the AM1 receptor. A361L was a difficult mutant to characterize; whereas the Emax is reduced at both the AM1 and AM2 receptors, the changes in Δlog(RA) were of opposing directions.
      Five of the 68 mutants had more pronounced differential effects between the receptors. These are referred to as differential residues (Fig. 5). A271L, Y277A, Y278A, N279A, and C282A all increased Δlog(RA) at the AM1 receptor but had no significant effect at the AM2 receptor. For Y277A, radioligand binding was substantially reduced at the AM1 receptor but retained at the AM2 receptor, consistent with a differential effect of this mutation at both receptors. C282A binding was unchanged at the AM1 receptor but showed a trend to be enhanced at the AM2 receptor (Fig. 6). In addition, for Y367A, we observed a decrease in pEC50 at the AM2 receptor but no effect at the AM1 receptor. Although the differences in Emax at either receptor did not reach statistical significance, the effect was opposite with an increase at the AM2 receptor and a decrease at the AM1 receptor. This is an atypical mutation because the effect is greater at the AM2 receptor.

       Overall Description of the AM1 and AM2 Receptor Models

      To assist in data interpretation, we generated AM1 and AM2 receptor models, which we understand to be the first models of a full-length GPCR in complex with a RAMP (Fig. 7, A and B). The RAMP TM helix lies between TM6 and TM7 of CLR without inducing strain in the sequence joining the RAMP ECD to the TM (the RAMP linker). The predicted arrangement of the TM helices forms a conical pocket (the peptide binding site) into which the disulfide loop of the AM peptide docks (Fig. 7, C and D). ECL boundaries are very similar to those in the CGRP receptor (
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      The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach.
      ) and those of other class B GPCR x-ray structures (
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      • Katritch V.
      • Wang M.W.
      • Stevens R.C.
      Structure of the human glucagon class B G-protein-coupled receptor.
      ,
      • Hollenstein K.
      • Kean J.
      • Bortolato A.
      • Cheng R.K.
      • Doré A.S.
      • Jazayeri A.
      • Cooke R.M.
      • Weir M.
      • Marshall F.H.
      Structure of class B GPCR corticotropin-releasing factor receptor 1.
      ).
      Figure thumbnail gr7
      FIGURE 7.Models of the full-length AM receptors. A, AM1 receptor; B, AM2 receptor. Images were generated from an overlay aligning CLR residues 138–394 for both models (root mean square deviation = 2.0 Å). Relative sizes and orientations are thus not an artifact of figure generation. C, surface representation of the peptide binding pocket of the AM1 and AM2 receptors illustrating the changes in receptor conformation and the peptide binding pocket. D, close-up surface representation of the peptide binding pocket showing the docked AM peptide and its five close receptor neighbors, determined by the models in blue sticks (AM1 receptor) and yellow sticks (AM2 receptor). Other colors in C and D are as described for A and B.
      In the AM peptide model, residues 15–21 form a disulfide loop, residues 22–31 are helical (
      • Pérez-Castells J.
      • Martín-Santamaría S.
      • Nieto L.
      • Ramos A.
      • Martínez A.
      • Pascual-Teresa B.
      • Jiménez-Barbero J.
      Structure of micelle-bound adrenomedullin: a first step toward the analysis of its interactions with receptors and small molecules.
      ), and residues 35–52 adopt the largely non-helical structure bound to the ECD of the AM1 receptor (
      • Booe J.M.
      • Walker C.S.
      • Barwell J.
      • Kuteyi G.
      • Simms J.
      • Jamaluddin M.A.
      • Warner M.L.
      • Bill R.M.
      • Harris P.W.
      • Brimble M.A.
      • Poyner D.R.
      • Hay D.L.
      • Pioszak A.A.
      Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor.
      ); the remaining residues (positions 33–41) form a loop, creating the AM structure. The model therefore rationalizes previous work on the degree of helicity within AM (Fig. 1E). The RAMP2 linker (residues Val134–Leu147 between the ECD and the TM region) is displaced relative to that of RAMP3, lies closer to the peptide binding pocket than does RAMP3, and is predicted by the models to interact with ECL3 and the top of TM7 of CLR (FIGURE 7., FIGURE 9.).
      Figure thumbnail gr9
      FIGURE 9.Receptor model overlay. Residues with common (A), common-differential (B), or differential (C) effects are shown as sticks, with oxygen atoms in red and nitrogen in blue. A, residues Ala199, Asp280, Ile284, and Phe349 have similar side chain and main chain orientations; Tyr365 has side chain rotation of ∼180° between the two AM receptors. B, residues Lys213, Ile352, and Trp354 have similar main chain but differing side chain orientations. C, Tyr277 shows substantial movement between the two receptors, whereas Tyr278 shows some movement but maintains similar interactions. D, close-up view of TM6-ECL3-TM7 showing the main residues involved in the change of the ECL3 position (red arrow denotes change in position). The increased proximity of RAMP2 to the CLR ECL3 in the AM1 receptor is clearly visible. AM1 and AM2 receptors are colored as per the figure; the movement of residues between the receptors is shown with arrows. E and F, juxtamembrane region of the two receptors with distances between residues (dotted lines) in Å. Distances were measured between the same set of Cα atoms in both receptors.
      The electrostatic potential of AM in its proposed bound conformation (Fig. 8, A and B) is largely positive because AM carries a charge of +4. The electrostatic potential of CLR in the absence of RAMP and AM is largely positive or neutral (Fig. 8, C and D). Both RAMP2 and RAMP3 convey an advantage in binding the positive AM because they switch this potential in the conical TM pocket and particularly on the ECD to more negative values, which will aid in binding the positively charged AM (Fig. 8, E and F). RAMP3 gives rise to the most negative ECD electrostatic potential.
      Figure thumbnail gr8
      FIGURE 8.The electrostatic potential of AM, CLR, and the AM1 and AM2 receptors. Blue, positive; red, negative; the potential has been contoured between −5 and +5 onto the solvent-accessible surface. A, the electrostatic potential on the face of AM that is exposed as it binds to the CLR ECD; the electrostatic potential is weakly positive, and the general orientation is as shown in C. B, the electrostatic potential on the ECD-binding surface, as defined by C; the electrostatic potential is strongly positive. The +4 charge on AM includes the N-terminal amine. C, a representation of AM binding to the AM1 receptor that can be used to identify the peptide-binding region in D–F: the CLR solvent-accessible surface is blue, the RAMP surface is red, and the C-terminal peptide of Gs is cyan. The solvent radius was expanded to 2.4 Å to mimic that in D–F. AM is shown in white. D, CLR electrostatic potential. E, AM1 electrostatic potential. F, AM2 electrostatic potential. The electrostatic potential of the AM1 and AM2 receptors was evaluated in an implicit membrane using APBS (the Adaptive Poisson-Boltzmann Solver) coupled with apbs_mem version 2.0 and the pdb2PQR server (
      • Dolinsky T.J.
      • Czodrowski P.
      • Li H.
      • Nielsen J.E.
      • Jensen J.H.
      • Klebe G.
      • Baker N.A.
      PDB2PQR: expanding and upgrading automated preparation of biomolecular structures for molecular simulations.
      ,
      • Dolinsky T.J.
      • Nielsen J.E.
      • McCammon J.A.
      • Baker N.A.
      PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations.
      • Callenberg K.M.
      • Choudhary O.P.
      • de Forest G.L.
      • Gohara D.W.
      • Baker N.A.
      • Grabe M.
      APBSmem: a graphical interface for electrostatic calculations at the membrane.
      ). The parameters for the APBSmem calculations were as follows: PARSE atomic charges (
      • Sitkoff D.
      • Sharp K.A.
      • Honig B.
      Correlating solvation free energies and surface tensions of hydrocarbon solutes.
      ); temperature, 298.15 K; ionic strength, 0.15 mm; protein and membrane relative dieletric constant, 2.0; relative solvent dielectric, 80; membrane thickness, 40 Å. The grid lengths were 300 × 300 × 300 Å with two levels of focusing; the grid dimensions were 97 × 97 × 97 for A and B and 129 × 129 × 225 for D–F. The CHARMM-gui was used to assist in placing the receptor within the membrane (
      • Jo S.
      • Kim T.
      • Iyer V.G.
      • Im W.
      CHARMM-GUI: a Web-based graphical user interface for CHARMM.
      ,
      • Miller L.J.
      • Chen Q.
      • Lam P.C.
      • Pinon D.I.
      • Sexton P.M.
      • Abagyan R.
      • Dong M.
      Refinement of glucagon-like peptide 1 docking to its intact receptor using mid-region photolabile probes and molecular modeling.
      ).

       Detailed Comparison between AM1 and AM2 Receptor Models

      Overall, the ECL2 conformation is similar between the two models, consistent with the observation that many of the residues with common cAMP effects are located in this invariant region and may contact the peptide (Fig. 9A). Residues with common but differential effects at each receptor also have largely similar orientations within the models (Fig. 9B). These residues are also mostly situated at the tops of the TM2 (Leu195 and Val198), TM3 (Cys212 and Lys213), and TM6 (Ile352, Pro353, and Trp354). Along with the common residues Ala199 and Phe349 and common but differential Arg274, these form a network around the top of the TM helices. Differential residues Tyr277 and Cys282 are situated in ECL2 (Fig. 9C). Cys212, Tyr278, Cys282, and Lys213 do not appear to change their orientation significantly between the two AM receptors (Fig. 9, B and C). Lys213 remains parallel to the Cys212–Cys282 bond, facing Tyr278 in both structures. Tyr277 moves outward in the AM2 receptor and points away from the peptide binding pocket, thus changing its environment (Fig. 9C).
      The most striking conformational difference between the AM1 and AM2 receptor models is the dramatic change in the position of ECL3 (Fig. 9D). The extracellular end of TM6 forms a distorted helix as a result of the influence of Pro353, Pro356, and Gly358. The conformation of ECL3 begins to diverge between the two models after the common differential residue Pro353. Trp354 stacks with ECL3 in the AM1 receptor, whereas in the AM2 receptor it is rotated by 90°, moving it away from the loop to face the lipid membrane. In the AM2 receptor model, ECL3 makes extensive contacts with AM, whereas in the AM1 receptor, these contacts are minimal. The cumulative result of these differences is that distances relevant to the binding site vary in size (Fig. 9, E and F).

       Probing the Model; Differential Peptide Contacts within the AM1 and AM2 Receptor TM Pockets

      The divergence between the models translates into different transmembrane AM binding pocket hull volumes of 4874 Å3 for the AM1 receptor versus 3313 Å3 for the AM2 receptor; the shapes of the two pockets also differ. The disulfide loop (Cys16–Cys21) of the docked AM peptide is located in the wide mouth of the peptide binding pocket with the side chain of Phe18 occupying the lower part of the pocket (Fig. 7D). Visual analysis and loop modeling indicated that Phe18, unlike its neighbors, occupied a more constrained pocket in the AM2 receptor than in the AM1 receptor. Consequently, we examined R17A, F18A, G19A, and T20A mutations in both the AM1 and AM2 receptors using MODELER; 100 models were generated, and the model with the best DOPE score was analyzed. In each case, apart from F18A, there was an equivalent decrease in the number of contacts (<4 Å) in both AM1 and AM2, but for F18A, there was a bigger decrease in the number of side chain contacts in the AM1 receptor (from eight to two) rather than in the AM2 receptor (from six to two). We therefore proposed that substitution of Phe18 with alanine would have a greater impact in the AM1 receptor, compared with the AM2 receptor. Consistent with our hypothesis, an F18A AM peptide stimulated cAMP production to a lesser degree at the AM1 receptor (60% decrease in Emax) than at the AM2 receptor (no change in Emax) (Table 4 and Fig. 10). This demonstrates that it is possible to engineer ligand-specific effects at these two receptors.
      Figure thumbnail gr10
      FIGURE 10.Concentration-response curves for the alanine-substituted AM peptide, F18A AM(15–52). Curves are combined normalized data from at least three individual experiments ± S.E. (error bars).

       Small Molecule Druggability of the AM Receptors

      We next analyzed the two receptor binding pockets for their druggability for small molecule, orally bioavailable ligands using the PockDrug and DoGSiteScorer druggability servers (
      • Hussein H.A.
      • Borrel A.
      • Geneix C.
      • Petitjean M.
      • Regad L.
      • Camproux A.C.
      PockDrug-Server: a new web server for predicting pocket druggability on holo and apo proteins.
      ,
      • Borrel A.
      • Regad L.
      • Xhaard H.
      • Petitjean M.
      • Camproux A.C.
      Pock drug: a model for predicting pocket druggability that overcomes pocket estimation uncertainties.
      • Volkamer A.
      • Kuhn D.
      • Rippmann F.
      • Rarey M.
      DoGSiteScorer: a web server for automatic binding site prediction, analysis and druggability assessment.
      ), which were trained to predict pockets with promising properties for the design of small molecule druglike ligands. Because druggability analysis is highly dependent on the cavity detection (
      • Borrel A.
      • Regad L.
      • Xhaard H.
      • Petitjean M.
      • Camproux A.C.
      Pock drug: a model for predicting pocket druggability that overcomes pocket estimation uncertainties.
      ), we only discuss residues predicted by both servers to reside in the main helical binding pocket, namely 43 residues common to the AM1 receptor pocket and 31 for the smaller AM2 receptor pocket; these consensus residues largely coincide with the largest subpocket given by DoGSiteScorer. This analysis showed that the main druggable pocket in the helical domain of each AM receptor partially overlapped with the peptide binding pocket identified by our models (Fig. 11, A and B). In both receptors, the druggable pocket includes the hydrophobic patch at the top of TM2 (e.g. Leu195), the distal residues of ECL2 (Trp283–Thr288), and residues on TM3 (e.g. Asp366, Tyr367, and His370). The druggable pockets extend below the limits of the peptide binding pocket and include Met223 and Tyr227 on TM3 for both receptors, but the AM1 pocket includes other TM3 residues (e.g. Leu220). The druggable pockets also extend lower on TM6 to include Ile370 and Ile371 for the AM1 receptor. The AM1 pocket includes more residues on TM1 (e.g. Thr145 and His149). Twenty-four residues were unique to the AM1 receptor, and seven were unique to the AM2 receptor, indicating that selectivity is possible. Some of the residues listed as part of the druggable pocket are more accessible than others (e.g. Phe228 in the AM1 receptor is not obviously accessible in the absence of induced fit, because it is partially shielded by Tyr227), but such residues may nevertheless be important in drug design. The AM1 receptor pocket reaches 14 Å below the top of ECL3 with drug scores of 0.97 and 0.81, from PockDrug and DoGSiteScorer, respectively. The AM2 receptor druggable pocket forms a narrow channel and is deeper (partly because of the ECL3 conformation), with PockDrug and DoGSiteScorer drug scores of 0.91 and 0.81, respectively; because the scores are above 0.5, both receptors are predicted to be druggable.
      Figure thumbnail gr11
      FIGURE 11.Small molecule druggable sites predicted using PockDrug and viewed from above. A, the AM1 site is shown in light blue, and the site residues that contact AM are shown in blue. B, the AM2 site is shown in magenta, and the site residues that contact AM are shown in red. This site is narrower and deeper than the AM1 site; the PockDrug druggability scores for the AM1 and AM2 sites are 0.97 and 0.91, respectively. C and D, surface cutaway views of the receptors; the different size, conformation, and situation of the pockets are evident from the shading. Selected residues are labeled.

      Discussion

      Pharmacological tools to help tease out the relative importance of each of the two AM receptors are needed, but it has not been apparent how to develop these because both receptors share the common GPCR, CLR. We report that RAMP2 and RAMP3 confer conformational variation in the CLR juxtamembrane region, yielding distinct binding pockets that may be tractable for the development of selective pharmacological tools and future drugs.
      Our study combined extensive mutagenesis of CLR with independent modeling studies (i.e. not adjusted to enhance agreement with data tables) that allowed us to effectively interpret our complex data set. Recent crystallographic and modeling studies have generated a consensus conformation for the TM bundle of the class B GPCRs (
      • Woolley M.J.
      • Watkins H.A.
      • Taddese B.
      • Karakullukcu Z.G.
      • Barwell J.
      • Smith K.J.
      • Hay D.L.
      • Poyner D.R.
      • Reynolds C.A.
      • Conner A.C.
      The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach.
      ,
      • Siu F.Y.
      • He M.
      • de Graaf C.
      • Han G.W.
      • Yang D.
      • Zhang Z.
      • Zhou C.
      • Xu Q.
      • Wacker D.
      • Joseph J.S.
      • Liu W.
      • Lau J.
      • Cherezov V.
      • Katritch V.
      • Wang M.W.
      • Stevens R.C.
      Structure of the human glucagon class B G-protein-coupled receptor.
      ,
      • Hollenstein K.
      • Kean J.
      • Bortolato A.
      • Cheng R.K.
      • Doré A.S.
      • Jazayeri A.
      • Cooke R.M.
      • Weir M.
      • Marshall F.H.
      Structure of class B GPCR corticotropin-releasing factor receptor 1.
      ,
      • Wootten D.
      • Simms J.
      • Miller L.J.
      • Christopoulos A.
      • Sexton P.M.
      Polar transmembrane interactions drive formation of ligand-specific and signal pathway-biased family B G protein-coupled receptor conformations.
      ). Crystallographic studies have so far, however, proved unsatisfactory for determining the structure for a complete class B GPCR or for the class B ECL conformation due to the inherent mobility of the loops. The only structural data on the arrangement of the ECD of a class B GPCR with respect to the TM bundle comes from an electron microscopy study of the GCGR, and this is necessarily low resolution (
      • Yang L.
      • Yang D.
      • de Graaf C.
      • Moeller A.
      • West G.M.
      • Dharmarajan V.
      • Wang C.
      • Siu F.Y.
      • Song G.
      • Reedtz-Runge S.
      • Pascal B.D.
      • Wu B.
      • Potter C.S.
      • Zhou H.
      • Griffin P.R.
      • et al.
      Conformational states of the full-length glucagon receptor.
      ). Although molecular models do not have the accuracy of x-ray structures, they are nevertheless useful for providing a framework against which experimental results can be considered. While it would be unwise to overinterpret any model, ours is largely consistent with the effects of the mutagenesis (Table 5) and also successfully predicted the activity of F18A AM.
      TABLE 5Comments on the mutation data of residues discussed in this work and shown in TABLE 1, TABLE 2 in light of the receptor models
      We initially compare our AM receptor models with that of the GCGR model structure (
      • Siu F.Y.
      • He M.
      • de Graaf C.
      • Han G.W.
      • Yang D.
      • Zhang Z.
      • Zhou C.
      • Xu Q.
      • Wacker D.
      • Joseph J.S.
      • Liu W.
      • Lau J.
      • Cherezov V.
      • Katritch V.
      • Wang M.W.
      • Stevens R.C.
      Structure of the human glucagon class B G-protein-coupled receptor.
      ), a canonical class B GPCR that does not require a RAMP. The main difference in the receptor is a ∼30° change in the orientation of the ECD to a more open conformation as a result of the constraint created by the RAMP on the structure of the AM receptor. The orientation of the ECD is more consistent with the open (i.e. active, agonist-bound) conformational ensemble of the GCGR (
      • Yang L.
      • Yang D.
      • de Graaf C.
      • Moeller A.
      • West G.M.
      • Dharmarajan V.
      • Wang C.
      • Siu F.Y.
      • Song G.
      • Reedtz-Runge S.
      • Pascal B.D.
      • Wu B.
      • Potter C.S.
      • Zhou H.
      • Griffin P.R.
      • et al.
      Conformational states of the full-length glucagon receptor.
      ) than the closed ensemble because the difference between the centers of mass of the TM and ECD domains is ∼57 Å, the polar angle θ is similarly ∼23°, and the projection of the ECD center of mass onto the membrane plane lies outside of the helical bundle. In the GCGR, the simulated closed state described by Yang et al. (
      • Yang L.
      • Yang D.
      • de Graaf C.
      • Moeller A.
      • West G.M.
      • Dharmarajan V.
      • Wang C.
      • Siu F.Y.
      • Song G.
      • Reedtz-Runge S.
      • Pascal B.D.
      • Wu B.
      • Potter C.S.
      • Zhou H.
      • Griffin P.R.
      • et al.
      Conformational states of the full-length glucagon receptor.
      ) may well be the inactive conformation, satisfying the proposed ECD-ECL3 interaction proposed by Koth et al. (
      • Koth C.M.
      • Murray J.M.
      • Mukund S.
      • Madjidi A.
      • Minn A.
      • Clarke H.J.
      • Wong T.
      • Chiang V.
      • Luis E.
      • Estevez A.
      • Rondon J.
      • Zhang Y.
      • Hötzel I.
      • Allan B.B.
      Molecular basis for negative regulation of the glucagon receptor.
      ), but in the AM receptors, because the RAMP binds to the peptide-binding face of the ECD, it is likely to inhibit formation of the fully closed conformation. The peptide shows more marked differences: the glucagon model peptide adopts a helical structure from Ser8 through to Met27, spanning from the juxtamembrane region through to the ECD, in agreement with most x-ray crystal structures on isolated class B ECDs. In contrast, AM has a more complex structure, with a non-helical ECD region, in agreement with the x-ray crystal structure of the isolated ECD and a helical region that binds to the juxtamembrane region, as in previous related models and the AM NMR structure (
      • Pérez-Castells J.
      • Martín-Santamaría S.
      • Nieto L.
      • Ramos A.
      • Martínez A.
      • Pascual-Teresa B.
      • Jiménez-Barbero J.
      Structure of micelle-bound adrenomedullin: a first step toward the analysis of its interactions with receptors and small molecules.
      ). The AM peptide helix binds to the same depth as the glucagon peptide, as judged by the alignment of the helical region (Fig. 1A), but the glucagon peptide N terminus binds to a greater depth (consistent with cross-linking data on the related PTH system (
      • Monaghan P.
      • Thomas B.E.
      • Woznica I.
      • Wittelsberger A.
      • Mierke D.F.
      • Rosenblatt M.
      Mapping peptide hormone-receptor interactions using a disulfide-trapping approach.
      ), whereas AM forms a disulfide-bonded loop consistent not only with the binding of the usual AM(16–52) (
      • Woolley M.J.
      • Watkins H.A.
      • Taddese B.
      • Karakullukcu Z.G.
      • Barwell J.
      • Smith K.J.
      • Hay D.L.
      • Poyner D.R.
      • Reynolds C.A.
      • Conner A.C.
      The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach.
      ) but also AM(1–52) (i.e. the N terminus is orientated so that AM(1–15) can “escape” from the TM bundle). This N-terminal extension of AM does not seem important for AM activity, and the AM(15–52) fragment is more consistent with the length of other peptides in the AM family.
      We have pharmacological evidence of RAMP-induced changes in the function of CLR at the AM1 and AM2 receptors, which are reflected in conformational differences between our full-length AM1 and AM2 receptor models. The most striking difference between the two models is the ECL3 conformation; interestingly, this is a region that also shows large differences between the GCGR and CRF1R x-ray crystal structures. Although only Ala361 in ECL3 shows any kind of differential activity, the residues flanking ECL3 do show this. Moreover, at the CGRP receptor, the CLR-RAMP1 complex, Ile360 is involved in receptor activation as opposed to Ala361 in the AM receptors (
      • Barwell J.
      • Conner A.
      • Poyner D.R.
      Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function.
      ), giving additional evidence of differential activity in ECL3. The extracellular region of TM6 in the AM receptors does contain residues with common-differential activity, namely Ile352, Pro353, and Trp354. The predicted stacking of Trp354 with ECL3 in the AM1 receptor combined with changes in the positions of Ile352 and Pro353 may stabilize the altered orientation of ECL3. In the AM2 receptor, Trp354 lies perpendicular to its AM1 receptor position, allowing ECL3 to lie further toward the center of the peptide binding pocket. Movement of the upper regions of TM3, TM6, and TM7 is involved in activation of class A GPCRs (
      • Wheatley M.
      • Wootten D.
      • Conner M.T.
      • Simms J.
      • Kendrick R.
      • Logan R.T.
      • Poyner D.R.
      • Barwell J.
      Lifting the lid on GPCRs: the role of extracellular loops.
      ). Some of the differences observed between the two AM receptors could therefore be reflected in differential activity of residues in ECL3/TM7 (Ala361 and Tyr367) and TM3 (Cys212 and Lys213) in the two AM receptors and in the CGRP receptor, where Cys212 is the only one of these residues involved in receptor activation (
      • Barwell J.
      • Conner A.
      • Poyner D.R.
      Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function.
      ).
      The AM model peptide interacts differently with ECL3/TM7 in the two AM receptors (FIGURE 1., FIGURE 7., A and B) in response to the effect of the different RAMPs; our models place the RAMP TM helix between TM6 and TM7 as in the class B secretin receptor (
      • Harikumar K.G.
      • Simms J.
      • Christopoulos G.
      • Sexton P.M.
      • Miller L.J.
      Molecular basis of association of receptor activity-modifying protein 3 with the family B G protein-coupled secretin receptor.
      ). The greater proximity of the RAMP2 ECD-TM linker to ECL3 is probably the main factor that contributes to the reorientation of ECL3 (Fig. 9D). RAMP2 and RAMP3 diverge in sequence in this region, and equivalent RAMP residues take up different positions relative to AM in the two models.
      The majority of the residues with a common or a common but differential effect on receptor activation vary little in their orientation and cluster around the upper TMs of our models (e.g. the hydrophobic cluster at the top of TM2 (Leu195, Val198, and Ala199), which is also essential to the function of the CGRP receptor (
      • Barwell J.
      • Conner A.
      • Poyner D.R.
      Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function.
      ). There are also common and common-differential residues situated in ECL2 (Asp280, Trp283, and Ile284); due to the position of the disulfide bond in our AM receptor models, these lie in close proximity to the upper TMs. Indeed, many of these common and common-differential residues are also essential for the activation of the CGRP receptor by both CGRP and AM (
      • Woolley M.J.
      • Watkins H.A.
      • Taddese B.
      • Karakullukcu Z.G.
      • Barwell J.
      • Smith K.J.
      • Hay D.L.
      • Poyner D.R.
      • Reynolds C.A.
      • Conner A.C.
      The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach.
      ). ECL2 is particularly important in activation in class A and B GPCRs (
      • Woolley M.J.
      • Watkins H.A.
      • Taddese B.
      • Karakullukcu Z.G.
      • Barwell J.
      • Smith K.J.
      • Hay D.L.
      • Poyner D.R.
      • Reynolds C.A.
      • Conner A.C.
      The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach.
      ,
      • Wheatley M.
      • Wootten D.
      • Conner M.T.
      • Simms J.
      • Kendrick R.
      • Logan R.T.
      • Poyner D.R.
      • Barwell J.
      Lifting the lid on GPCRs: the role of extracellular loops.
      ,
      • Koole C.
      • Wootten D.
      • Simms J.
      • Miller L.J.
      • Christopoulos A.
      • Sexton P.M.
      Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) has a critical role in GLP-1 peptide binding and receptor activation.
      ).
      Cys282 in ECL2 forms an essential conserved disulfide bond with Cys212 in TM3 in both the AM receptors and in the CGRP receptor (
      • Woolley M.J.
      • Watkins H.A.
      • Taddese B.
      • Karakullukcu Z.G.
      • Barwell J.
      • Smith K.J.
      • Hay D.L.
      • Poyner D.R.
      • Reynolds C.A.
      • Conner A.C.
      The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach.
      ). However, this bond does not appear to be critical to activation of the AM2 receptor (or the CGRP receptor). The smaller pocket in the AM2 receptor causes tighter packing of the common and common-differential residue network around the top of the TMs; this more restrained environment may limit the movement of the side chain of either Cys212 or Cys282 and allow the AM2 receptor to tolerate an unpaired cysteine residue without detrimental perturbation of its structural integrity and thus activation of the AM2 receptor. Significantly, ECL3 in the CGRP receptor adopts a similar conformation to the AM2 receptor (results not shown). In the more open AM1 receptor, this C212A or C282A mutation is fatal to receptor activation, but precise verification of the mechanism is beyond the scope of our models. However, we propose that the greater effect of mutation at the common but differential residues in the AM1 receptor is related to its degree of openness and hence stability. Thus, we note that other residues, such as Lys213, Tyr277, and Tyr278, that are predicted to stabilize ECL2 also show more pronounced effects on mutation in the AM1 receptor despite generally adopting similar interactions (Lys213 and Tyr278) in both structures, presumably because the mutated AM1 receptor structure is less stable than the mutated AM2 structure.
      These changes, especially those in ECL3, serve to alter the depth, volume, shape, and composition of the model binding pocket. Whereas the overall position of the docked peptide and in particular the Phe18 side chain in the peptide binding pocket does not change significantly, the number of close neighbors to the Phe18 side chain does. These changes have significant implications for the design of therapeutics that are either specific to the AM1 or AM2 receptors to treat receptor-specific pathophysiologies or conversely to harness the common effects of both receptors. Druggability screening highlighted two different druggable pockets for small molecules in the AM1 and AM2 receptors. This indicates scope for specific ligand design by targeting the additional and differential druggable residues of the two pockets, which lie within the TM domains.
      The drug scores of 0.81–0.97 and 0.81–0.91 for the AM1 and AM2 receptors, respectively, are clearly above the 0.5 threshold, indicating that they are druggable. Significantly, both sites display an appropriate balance of hydrophobic and polar residues, as required for a druggable site (
      • Bortolato A.
      • Doré A.S.
      • Hollenstein K.
      • Tehan B.G.
      • Mason J.S.
      • Marshall F.H.
      Structure of class B GPCRs: new horizons for drug discovery.
      ). Moreover, the difference in electrostatic potential for these receptors adds to the rationale for the design of selective AM1 or AM2 ligands. In addition, the structural model of the AM peptide structure (Fig. 1E) is distinctly different from that of glucagon and probably many other class B peptide ligands and so may also be useful in substrate-based drug design, especially because there are differences in the two loop regions. The CRF1R structure shows a narrow drug-bound channel that sits below the level of our peptide binding site. Interestingly, both druggability servers indicate additional druggable sites in this region (
      • Hollenstein K.
      • de Graaf C.
      • Bortolato A.
      • Wang M.W.
      • Marshall F.H.
      • Stevens R.C.
      Insights into the structure of class B GPCRs.
      ).
      We have based our current study on the measurement of cAMP as the canonical signaling pathway for CLR. It is important to note that GPCRs, such as this, also have the capacity to signal through alternative pathways, and it will be important to consider these in future studies (
      • Wootten D.
      • Simms J.
      • Miller L.J.
      • Christopoulos A.
      • Sexton P.M.
      Polar transmembrane interactions drive formation of ligand-specific and signal pathway-biased family B G protein-coupled receptor conformations.
      ). It is possible that some residues will have a greater or lesser role, depending on the pathway measured, indicating further conformational differences in the receptors.
      In summary, we suggest that the change in the predicted conformation of ECL3 and hence the different TM binding pockets in the AM1 and AM2 receptors is due to association with different RAMPs, as described above. The existence of distinct peptide and small molecule binding pockets with different properties has implications for the design of selective therapeutics, whether they be small molecules or peptides. This could facilitate the design of ligands to harness the individual physiological roles of the two AM receptors, validating the receptors as drug targets.
      Our data support the idea that RAMPs act allosterically to modify the conformation of CLR. This could lead to a range of possible outcomes, including biasing the receptor toward different ligands or signaling pathways. Two recent reports have suggested this mechanism for RAMP effects on the related calcitonin receptor (
      • Gingell J.J.
      • Simms J.
      • Barwell J.
      • Poyner D.R.
      • Watkins H.A.
      • Pioszak A.A.
      • Sexton P.M.
      • Hay D.L.
      An allosteric role for receptor activity-modifying proteins in defining GPCR pharmacology.
      ,
      • Lee S.M.
      • Hay D.L.
      • Pioszak A.A.
      Calcitonin and amylin receptor peptide interaction mechanisms: insights into peptide-binding modes and allosteric modulation of the calcitonin receptor by receptor activity-modifying proteins.
      ). Allostery between protomers in receptor oligomers could be a broad mechanism for generating diversity in GPCR function.

      Author Contributions

      H. A. W., J. J. G., M. C., R. S. A., and M. G. conducted experiments. C. A. R., M. P., J. M. W. R. M., A. L., and A. C. contributed to the modeling. P. W. R. H., T.-Y. Y., and M. A. B. were responsible for peptide synthesis. J. B., D. R. P., M. J. W., and A. C. C. contributed receptor mutants to the study. A. A. P. provided data that were used for the modeling. C. A. R., H. A. W., D. R. P., and D. L. H. interpreted the experiments and wrote the paper.

      Acknowledgments

      The GCGR-glucagon model (
      • Siu F.Y.
      • He M.
      • de Graaf C.
      • Han G.W.
      • Yang D.
      • Zhang Z.
      • Zhou C.
      • Xu Q.
      • Wacker D.
      • Joseph J.S.
      • Liu W.
      • Lau J.
      • Cherezov V.
      • Katritch V.
      • Wang M.W.
      • Stevens R.C.
      Structure of the human glucagon class B G-protein-coupled receptor.
      ) was kindly provided by Stevens and co-workers.

      Supplementary Material

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