Downloaded from
`
`molpharm.aspetjournals.org
`
` at ASPET Journals on September 25, 2016
`
`0026-895X/05/6705-1655–1665$20.00
`MOLECULAR PHARMACOLOGY
`Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics
`Mol Pharmacol 67:1655–1665, 2005
`
`Vol. 67, No. 5
`8615/1198150
`Printed in U.S.A.
`
`Pharmacological Discrimination of Calcitonin Receptor:
`Receptor Activity-Modifying Protein Complexes
`
`Debbie L. Hay, George Christopoulos, Arthur Christopoulos, David R. Poyner, and
`Patrick M. Sexton
`School of Biological Sciences, University of Auckland, Auckland, New Zealand (D.L.H.); Howard Florey Institute (G.C., P.M.S.)
`and Department of Pharmacology (A.C.), University of Melbourne, Victoria, Australia; and School of Life and Health Sciences,
`Aston University, Birmingham, United Kingdom (D.R.P.)
`Received October 21, 2004; accepted February 3, 2005
`
`ABSTRACT
`Calcitonin (CT) receptors dimerize with receptor activity-modi-
`fying proteins (RAMPs) to create high-affinity amylin (AMY)
`receptors, but there is no reliable means of pharmacologically
`distinguishing these receptors. We used agonists and antago-
`nists to define their pharmacology, expressing the CT(a) recep-
`tor alone or with RAMPs in COS-7 cells and measuring cAMP
`accumulation.
`Intermedin short, otherwise known as ad-
`renomedullin 2, mirrored the action of ␣CGRP, being a weak
`agonist at CT(a), AMY2(a), and AMY3(a) receptors but con-
`siderably more potent at AMY1(a) receptors. Likewise,
`the
`linear
`calcitonin gene-related peptide
`(CGRP)
`analogs
`(Cys(ACM)2,7)h␣CGRP and (Cys(Et)2,7)h␣CGRP were only ef-
`fective at AMY1(a) receptors, but they were partial agonists. As
`previously observed in COS-7 cells, there was little induction of
`the AMY2(a) receptor phenotype; thus, AMY2(a) was not exam-
`
`ined further in this study. The antagonist peptide salmon calci-
`tonin8-32 (sCT8-32) did not discriminate strongly between CT
`and AMY receptors; however, AC187 was a more effective
`antagonist of AMY responses at AMY receptors, and AC413
`additionally showed modest selectivity for AMY1(a) over AMY3(a)
`receptors. CGRP8-37 also demonstrated receptor-dependent
`effects. CGRP8-37 more effectively antagonized AMY at AMY1(a)
`than AMY3(a) receptors, although it was only a weak antagonist
`of both, but it did not inhibit responses at the CT(a) receptor.
`Low CGRP8-37 affinity and agonism by linear CGRP analogs at
`AMY1(a) are the classic signature of a CGRP2 receptor. Our data
`indicate that careful use of combinations of agonists and
`antagonists may allow pharmacological discrimination of CT(a),
`AMY1(a), and AMY3(a) receptors, providing a means to delineate
`the physiological significance of these receptors.
`
`The peptides typically designated as calcitonin (CT) pep-
`tide family members include CT gene-related peptide
`(CGRP), amylin (AMY), and adrenomedullin (AM) (Poyner
`et al., 2002), although an assortment of related peptides
`has recently been identified, including intermedin (IMD),
`also known as AM2 (Katafuchi et al., 2003; Roh et al.,
`2004; Takei et al., 2004). Although only weakly homolo-
`gous in terms of amino acid sequence, several common
`
`This work was supported by the National Health and Medical Research
`Council of Australia (grants 145702 and 299810), the Ian Potter Foundation
`Neuropeptide Laboratory and the University of Auckland Staff Research
`Fund, the Auckland Medical Research Foundation, and Lottery Health Com-
`mission (New Zealand). P.M.S. and A.C. are Senior Research Fellows of the
`National Health and Medical Research Council. D.R.P. was supported by the
`British Heart Foundation (Grant PG/03/079/15278).
`Article, publication date, and citation information can be found at
`http://molpharm.aspetjournals.org.
`doi:10.1124/mol.104.008615.
`
`features are shared, including an N-terminal ring struc-
`ture that is the key to agonist activity. Nonetheless, the
`similarity in peptide structure leads to promiscuity for
`many of these peptides across their cognate receptors.
`Numerous biological activities have been attributed to
`these peptides. CT, for example, is involved in bone ho-
`meostasis (Sexton et al., 1999). AMY is likely to be in-
`volved in nutrient intake and regulating blood glucose
`levels (Cooper, 1994). CGRP and AM are both potent va-
`sodilators, with AM necessary for vascular integrity (Hin-
`son et al., 2000; Shindo et al., 2001; Brain and Grant,
`2004). As with many other peptides, significant advances
`in understanding the physiological, pathophysiological,
`and clinical potential of CT family members are hampered
`by a lack of selective pharmacological agents that can be
`used to define function. Progress has been particularly
`slow for the CT peptide family because, until recently, the
`
`ABBREVIATIONS: CT, calcitonin; CGRP, calcitonin gene-related peptide; AMY, amylin; AM, adrenomedullin; IMD, intermedin; GPCR, G protein
`coupled receptor; CL, calcitonin receptor-like receptor; RAMP, receptor activity modifying protein; CT(a), calcitonin receptor; rAMY, rat amylin;
`IMDS, intermedin short; BSA, bovine serum albumin; ALPHA, amplified luminescent proximity homogenous assay; DMEM, Dulbecco’s modified
`Eagle’s medium; FBS, fetal bovine serum; HA, hemagglutinin; PBS, phosphate-buffered saline; hCT, human calcitonin; AC187, SC[acetyl-
`(Asn30,Tyr32)-calcitonin8-32].
`
`1655
`
`EX2060
`Eli Lilly & Co. v. Teva Pharms. Int'l GMBH
`IPR2018-01427
`
`1
`
`

`

`Downloaded from
`
`molpharm.aspetjournals.org
`
` at ASPET Journals on September 25, 2016
`
`La Jolla, CA). Human CT was obtained from the American Peptide
`Co., Inc. (Sunnyvale, CA). IMD short (IMDS) was a generous gift
`from Dr. Teddy Hsu (Stanford University School of Medicine, Stan-
`ford, CA; Roh et al., 2004). Peptide sequences are detailed in Fig. 1.
`Bovine serum albumin (BSA) and 3-isobutyl-1-methylxanthine were
`from Sigma-Aldrich (St. Louis, MO), and amplified luminescent
`proximity homogenous assay (ALPHA)-screen cAMP kits were pur-
`chased from PerkinElmer Life and Analytical Sciences (Boston, MA).
`Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum
`(FBS), and HEPES were from Invitrogen (Carlsbad, CA). Cell culture
`plastic ware was manufactured by NUNC A/S (Roskilde, Denmark),
`and Metafectene was purchased from Scientifix (Cheltenham, VIC,
`Australia). 125I-Labeled goat anti-mouse IgG was obtained from
`PerkinElmer Life and Analytical Sciences. Na-125I (100 mCi/ml) was
`supplied by MP Biomedicals (Irvine, CA). 125I-Salmon CT (specific
`activity, 700 Ci/mmol) was iodinated in-house as described previ-
`ously (Findlay et al., 1980). N-Succinimidyl 3-94-hydroxy,5,-[125I]io-
`dophenyl propionate (Bolton-Hunter reagent; 2000 Ci/mmol) was
`from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckingham-
`shire, UK). 125I-Rat amylin (specific activity, 2000 Ci/mmol) was
`iodinated by the Bolton-Hunter method and purified by reverse
`phase high-performance liquid chromatography as described previ-
`ously (Bhogal et al., 1992). All other reagents were of analytical
`grade.
`Expression Constructs. Double hemagglutinin (HA) epitope-
`tagged human CT(a) receptor was prepared as described previously
`(Pham et al., 2004). This receptor is the Leu447 polymorphic variant
`of the receptor (Kuestner et al., 1994). Human RAMP1, RAMP2, and
`RAMP3 and human CL receptor were a gift from Dr. Steven Foord
`(McLatchie et al., 1998).
`Cell Culture and Transfection. COS-7 cells were subcultured
`as described previously (Zumpe et al., 2000). One day before trans-
`fection, COS-7 cells were seeded into 25- or 75-cm2 cell culture flasks
`at high density to achieve 90 to 100% confluence for transfection the
`next day. The cells were then transfected using Metafectene accord-
`ing to the manufacturer’s instructions, with the following amounts of
`DNA: For 25-cm2 flasks, 1.25 ␮g of receptor DNA [CT(a) or CL] and
`1.9 ␮g of RAMP or pcDNA3 DNA; for 75-cm2 flasks, 3.8 ␮g of receptor
`DNA, and 5.7 ␮g of RAMP or pcDNA3 DNA. The transfection mix
`was removed after 16-h incubation, and the cells were recovered in
`complete media (DMEM with 5% FBS) for 8 h. The cells were then
`serum-starved for a further 16 h to minimize basal cAMP levels.
`Measurement of cAMP Production. Cells transfected with
`CT(a) or CL plus pcDNA3, RAMP1, -2, or -3 were harvested approx-
`imately 40 h after transfection. The cells were counted and diluted to
`20,000 cells per 10 ␮l and incubated, mixing for at least 30 min in
`serum and phenol red-free DMEM containing 0.1% (w/v) BSA and 1
`mM 3-isobutyl-1-methylxanthine (stimulation buffer). Agonist and
`antagonist dilutions were prepared in stimulation buffer and added
`to white 384-well plates, either alone or in combination, to a total
`volume of 10 ␮l. After incubation of cells with stimulation buffer,
`20,000 cells were added per well in a volume of 10 ␮l. The plates were
`centrifuged very briefly to ensure thorough mixing of these small
`volumes. The plates were then incubated for 30 min at 37°C. Drug-
`stimulated receptor activity was terminated by the addition of 20 ␮l
`of lysis buffer [0.3% (v/v) Tween 20, 5 mM HEPES, 0.1% (w/v) BSA
`in water, pH 7.4]. After addition of lysis buffer, the plates were again
`centrifuged briefly to ensure thorough mixing. The cAMP in the
`lysed cells was assayed in the same wells using ALPHA-screen assay
`kits. A cAMP standard curve was included in each assay. In brief,
`cAMP was measured with acceptor and donor beads that were pre-
`pared in lysis buffer and added to the plates according to the man-
`ufacturer’s instructions. After overnight incubation in the dark, the
`plates were read with an ALPHA-screen protocol on a Fusion plate
`reader (PerkinElmer Life and Analytical Sciences).
`Radioligand Binding. When harvested for cAMP assay (see
`above), the same transfected COS-7 cells were also seeded into 24-
`well culture plates at a density of approximately 250,000 cells per
`
`1656
`
`Hay et al.
`
`molecular nature of the cognate receptors for AMY, CGRP,
`and adrenomedullin was unknown.
`There is now some clarity regarding the nature of the
`receptor that probably mediates many of the effects of CGRP.
`It consists of a complex between a seven-transmembrane
`protein belonging to the secretin family of G protein-coupled
`receptors (GPCRs), the CT receptor-like receptor (CL), with
`receptor activity modifying protein (RAMP)1 (McLatchie et
`al., 1998). When these proteins are coexpressed, classic
`CGRP1-like pharmacology is observed (McLatchie et al.,
`1998; Hay et al., 2004). However, if CL is instead coexpressed
`with either of the two other RAMP family members, RAMP2
`or RAMP3, adrenomedullin is recognized most effectively
`(McLatchie et al., 1998). Thus, RAMPs act as pharmacologi-
`cal switches. It was soon realized that the function of RAMPs
`may be much broader, and there are now several examples of
`secretin family GPCRs with which these proteins are likely
`to interact (Christopoulos et al., 1999, 2003; Leuthauser
`et al., 2000).
`It is noteworthy that RAMPs have a strong interaction
`with the CT receptor, the closest relative to CL (Christopoulos
`et al., 1999). Together, RAMPs and the CT receptor generate
`receptors with high affinity for AMY, with the precise nature of
`these receptors depending on the CT receptor splice variant and
`cellular background (Tilakaratne et al., 2000). To our knowl-
`edge, there have been no other reports of a distinct molecular
`entity capable of responding to AMY with such high affinity. It
`is noteworthy that early attempts to clone the AMY receptor
`usually produced the CT receptor; thus, it is likely that CT
`receptor/RAMP complexes mediate at least some of the effects
`of AMY in vivo, although this has yet to be directly tested. It is
`crucial to note that there is no reliable means of distinguishing
`CT from AMY receptors or AMY receptor subtypes pharmaco-
`logically in functional systems. Although comprehensive bind-
`ing and agonist-interaction analyses have been performed,
`there has been no critical analysis of the way that antagonists
`interact with these receptors. This type of information may
`allow the different biological effects of AMY and related pep-
`tides to be attributed to distinct receptor subtypes. It can also
`provide a basis for the rational design of more selective agents.
`This is important because an AMY analog (Pramlintide) has
`now reached late-stage development for glycemic control in
`diabetic patients, illustrating the clinical importance of this
`peptide.
`Therefore, in this study, we have sought to address this
`issue by transfecting the CT receptor [CT(a); Poyner et al.,
`2002] with or without RAMPs into COS-7 cells that do not
`endogenously express phenotypically significant levels of
`RAMPs, CT receptors, or CL. We have identified several key
`aspects of pharmacology that relate to the way that AMY and
`its related peptides have historically been reported to act in
`tissues.
`
`Materials and Methods
`Materials. Human AM, human adrenomedullin22-52 (AM22-52),
`rat AMY8-37, human ␣CGRP, human ␣CGRP8-37, human ␤CGRP,
`and acetyl-(Asn30,Tyr32)-calcitonin8-32 (AC187) were purchased from
`Bachem (Bubendorf, Switzerland). Salmon calcitonin8-32 [sCT8-32]
`was from Peninsula Laboratories (Belmont, CA), and human
`Tyr0␣CGRP,
`(Cys(Et)2,7)-␣CGRP,
`(Cys(Acm)2,7)-␣CGRP, and rat
`AMY (rAMY) were from Auspep (Parkville, Australia). AC413 was a
`generous gift from Dr. Andrew Young (Amylin Pharmaceuticals Inc.,
`
`2
`
`

`

`Downloaded from
`
`molpharm.aspetjournals.org
`
` at ASPET Journals on September 25, 2016
`
`Molecularly and Pharmacologically Distinct Amylin Receptors
`
`1657
`
`well. These cells were then assayed for receptor binding to either
`125I-rAMY or 125I-sCT the next day (16 h later). Cells were initially
`washed with 500 ␮l of phosphate-buffered saline (PBS) and incu-
`bated for 30 min at 37°C in 500 ␮l of binding buffer [FBS-free DMEM
`with 0.1% (w/v) BSA]. Wells contained either 50 pM 125I-sCT or 100
`pM 125I-rAMY. Nonspecific binding levels were determined by com-
`peting with 10⫺7 M sCT or 10⫺6 M rAMY, respectively. Cells were
`then washed twice with 500 ␮l of PBS and were solubilized with 0.5
`ml of 0.5 M NaOH with the cell lysate counted for ␥-radiation using
`a PerkinElmer ␥-counter (COBRA Auto-gamma, Model B5010; 75%
`efficiency).
`For full-curve competition binding experiments, cells in 75-cm2
`flasks were transfected for 5 h using Metafectene, with 3.7 ␮g of
`CT(a) and either 5.2 ␮g of pcDNA3, RAMP1, or RAMP3 DNA. The
`cells were allowed to recover for 16 h and then harvested and seeded
`at around 80 to 90% confluence into 48-well plates. These were then
`allowed to adhere and recover for a further 16 h. Competition bind-
`ing was performed for 2 h atroom temperature. Each well contained
`225 ␮l of DMEM ⫹ 0.1% BSA, 200 pM 125I-rAMY, and 25 ␮l of
`competing peptide (10⫺12–10⫺7 M) or buffer control. Cells were
`washed once with PBS, lysed, and counted as described above.
`Measurement of Cell Surface Expression by Antibody Bind-
`ing. As for binding assays, at the time of harvesting for cAMP assay,
`transfected COS-7 cells were plated into 24-well plates and later
`assayed for cell-surface expression of the HA-tagged receptor. Cells
`were rinsed twice with 0.5 ml of binding buffer [50 mM Tris-HCl, pH
`7.7, 100 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 1% (w/v) BSA,
`adjusted to pH 7.7 with HCl] followed by addition of 2 ␮g of HA-
`specific mouse antibody in 250 ␮l of binding buffer to each well. Cells
`were incubated for 3 h at4°C, with gentle agitation. Cells were then
`
`rinsed three times with binding buffer, and 125I-labeled goat anti-
`mouse IgG (diluted to give 200 pM/250 ␮l per well) was added to the
`cells. The cells were incubated for a further 3 h at 4°C andthen
`rinsed three times with binding buffer. Cells were solubilized with
`0.5 ml of 0.5 M NaOH, and the cell lysate was counted for ␥-radia-
`tion. Nonspecific binding was determined from the wells that re-
`ceived 125I-labeled goat anti-mouse IgG but not the anti-HA primary
`antibody.
`Data Analysis and Statistics. Data were analyzed using Prism
`4.02 (GraphPad Software Inc., San Diego, CA). In each assay, the
`quantity of cAMP generated was calculated from the raw data using
`a cAMP standard curve. For agonist responses, concentration-effect
`curves were fitted to a four-parameter logistic equation (Motulsky
`and Christopoulos, 2003).
`For calculation of antagonist potency, agonist concentration-
`response curves in the absence and presence of antagonist were
`globally fitted to the following equation using Prism (Motulsky and
`Christopoulos, 2004):
`
`Response ⫽ Emin ⫹
`
`共Emax ⫺ Emin兲关A兴nH
`
`
`
`关A兴nH ⫹冉10⫺pEC50冋1 ⫹冉 关B兴10⫺pA2冊s册冊nH
`
`where Emax represents the maximal asymptote of the concentration-
`response curves, Emin represents the lowest asymptote of the con-
`centration-response curves, pEC50 represents the negative logarithm
`of the agonist EC50 in the absence of antagonist, [A] represents the
`concentration of the agonist, [B] represents the concentration of the
`antagonist, nH represents the Hill slope of the agonist curve, s
`represents the Schild slope for the antagonist, and pA2 represents
`
`Fig. 1. Peptide sequences and alignment. Sequences were aligned according to the ClustalV methods (PAM250) using the MegAlign program from
`DNAstar Inc. (Madison, WI). For agonist peptides, residues that match the consensus CGRP sequence are boxed (top). For antagonist peptides,
`residues that match the overall consensus are boxed (bottom). The location of the disulfide-linked cysteines in agonist peptides is also indicated. The
`exception to this are the analogs Cys(Et)2,7-␣CGRP and Cys(Acm)2,7-␣CGRP where the disulfide linkage has been blocked. Modification to these
`cysteines is indicated by bold boxes.
`
`3
`
`

`

`Downloaded from
`
`molpharm.aspetjournals.org
`
` at ASPET Journals on September 25, 2016
`
`endogenous RAMPs, CT receptors, and CL (Hay et al., 2003).
`Without significant background expression of such receptor
`components, defined receptor subtypes can be accurately
`compared.
`Agonist Pharmacology. The approach taken to generate
`a detailed pharmacological analysis of the molecularly de-
`fined AMY receptors was to compare the effects of all avail-
`able antagonists against the major agonists that were capa-
`ble of eliciting reliable receptor activation. Therefore, we
`initially examined agonist-induced cAMP responses in cells
`transfected with CT(a) alone or in combination with individ-
`ual RAMPs to assess the relative agonist activation profiles
`of the receptors defined as CT(a), AMY1(a), AMY2(a), and
`AMY3(a), respectively. In most experiments, cell surface ex-
`pression of the CT(a) was confirmed by binding of an anti-HA
`antibody to the epitope tag incorporated into the N terminus
`of the receptor (Fig. 2). In addition, in some experiments
`125I-sCT binding was also performed and confirmed that
`similar levels of the receptor protein were expressed at the
`cell surface (data not shown). Expression of the AMY recep-
`tor phenotype was confirmed by concomitant 125I-rAMY
`binding (data not shown).
`As shown in Table 1 and in accordance with previous
`results, hCT displayed equivalent high potency in cells trans-
`fected with CT(a) or AMY1(a) receptors but had ⬃10-fold lower
`potency at AMY3(a) receptors (p ⬍ 0.05; n ⫽ 6). In contrast,
`rAMY and the CGRPs had low potency at the CT(a) receptor
`and exhibited ⬃100-fold increased potency at the AMY1(a)
`receptor. As seen previously in this cellular background, pre-
`liminary analysis of radioligand binding and cAMP response
`indicated very little induction of AMY2(a) phenotype with
`pEC50 values for rAMY at this receptor equivalent to that
`seen with CT(a) alone (data not shown; Christopoulos et al.,
`1999; Tilakaratne et al., 2000). rAMY had high potency at the
`AMY3(a) receptor, but the CGRPs showed only modest in-
`creases in potency (⬍10-fold) at this receptor. At all receptor
`phenotypes, Tyr0-h␣CGRP was weaker than unmodified
`h␣CGRP, but it exhibited similar modulation of potency to ␣-
`and ␤-CGRP at AMY1(a) receptors.
`IMD displays efficacy at CL/RAMP-based receptors (Roh et
`al., 2004; Takei et al., 2004). We examined the interaction of
`the short form of this peptide, IMDS, with CT and AMY
`receptors and compared it with the behavior of the peptide at
`CGRP and AM receptors. IMDS had low potency at CT(a) and
`AMY2(a) receptors and displayed a similar increase in po-
`tency at AMY1(a) (⬃40-fold) and AMY3(a) (⬍10 fold) receptors,
`
`1658
`
`Hay et al.
`
`the negative logarithm of the concentration of antagonist that shifts
`the agonist EC50 by a factor of 2. Parallelism of agonist concentra-
`tion-response curves in the presence of antagonist relative to the
`absence of antagonist was assessed by F-test, which compared curve
`fits where the nH parameter was shared across each family of curves
`to fits where each curve within a family was allowed its own Hill
`slope factor. The F-test was similarly used to determine whether the
`Schild slope was significantly different from unity within a given
`data set. In the majority of instances, this was not the case, and thus
`all curves were refitted with the Schild slope constrained to a value
`of 1; under these conditions, the resulting estimate of pA2 represents
`the pKB.
`In all cases, potency and affinity values were estimated as
`logarithms (Christopoulos, 1998). Data shown are the mean ⫾
`S.E.M. Comparisons between mean values were performed by
`unpaired t tests or one-way analysis of variance, as appropriate.
`Unless otherwise stated, values of p ⬍ 0.05 were taken as statis-
`tically significant.
`
`Results
`COS-7 cells were chosen for transfection studies as they
`have been shown to lack phenotypically significant levels of
`
`Fig. 2. Cell surface expression of CT(a) protein, in COS-7 cells transiently
`transfected with CT(a) alone or CT(a) in the presence of either RAMP1
`[AMY1(a)], RAMP2 [AMY2(a)], or RAMP3 [AMY3(a)], measured by binding
`of anti-HA antibody to the 2xHA epitope incorporated at the N terminus
`of the receptor. Primary antibody binding is detected by incubation of a
`125I-labeled goat anti-mouse IgG antibody as described under Materials
`and Methods. In untransfected or mock-transfected cells the level of
`binding was ⬍15% of binding seen in CT(a)-transfected cells. Data are
`expressed as a percentage of the binding of 125I-antibody to cells express-
`ing the CT(a) protein in the absence of RAMP cotransfection. Data are
`from 10 independent experiments with duplicate repeats.
`
`TABLE 1
`Agonist potencies (pEC50 values) for stimulation of cAMP accumulation at human CT and AMY receptors
`Data are presented as mean ⫾ S.E.M. Values in parentheses represent the number of individual experiments analyzed.
`
`AMY3(a)
`AMY1(a)
`CT(a)
`8.02 ⫾ 0.22 (7)
`8.93 ⫾ 0.09 (7)
`8.99 ⫾ 0.1 (8)
`hCT
`8.63 ⫾ 0.09 (7)
`9.12 ⫾ 0.16 (10)
`6.95 ⫾ 0.18 (8)
`rAMY
`7.60 ⫾ 0.17 (6)
`8.70 ⫾ 0.17 (6)
`6.80 ⫾ 0.05 (5)
`h␣CGRP
`⬍6 (3)
`7.55 ⫾ 0.17 (7)
`⬍6 (2)
`Tyr0-h␣CGRP
`7.67 ⫾ 0.23 (6)
`9.16 ⫾ 0.18 (9)
`7.18 ⫾ 0.22 (2)
`h␤CGRP
`⬍6 (6)
`7.79 ⫾ 0.14 (5)a
`⬍6 (3)
`(Cys(Et)2,7)h␣CGRP
`⬍6 (6)
`7.46 ⫾ 0.06 (4)a
`⬍6 (3)
`(Cys(ACM)2,7)h␣CGRP
`6.89 ⫾ 0.51 (3)
`6.48 ⫾ 0.28 (4)
`6.73 ⫾ 0.45 (3)
`hAM
`8.07 ⫾ 0.19 (6)b
`6.53 ⫾ 0.09 (6)
`7.12 ⫾ 0.19 (6)
`IMDS
`a Note that these CGRP analogues were weak partial agonists at this receptor, with Emax values of 47.9 ⫾ 5.4 and 22.8 ⫾ 6% for (Cys(Et)2,7)h␣CGRP and
`(Cys(ACM)2,7)h␣CGRP, respectively. These values were generated by comparing the curve maximum asymptotes of the h␣CGRP analogs with that for h␣CGRP itself (set
`at 100%), which was used as the reference full agonist for these experiments.
`b Emax values for IMDS were equivalent to those of h␣CGRP assayed in parallel.
`
`4
`
`

`

`Molecularly and Pharmacologically Distinct Amylin Receptors
`
`1659
`
`(Cys(Et)2,7)-␣CGRP was considerably more efficacious than
`(Cys(Acm)2,7)-␣CGRP with %Emax values of 83.5 ⫾ 7.2 and
`8.1 ⫾ 2.1, respectively (Fig. 4B).
`
`TABLE 2
`Comparison of IMDS and h␣CGRP potency for stimulation of cAMP
`accumulation at human CT, AMY, CGRP, and AM receptors
`Values are presented as mean ⫾ S.E.M.
`
`as seen for the CGRPs (Fig. 3; Table 2). This contrasts with
`the interaction of IMDS at CGRP and AM receptors assayed
`in the same cellular background where IMDS displayed sim-
`ilar high efficacy at all three receptors but differed from the
`activity of h␣CGRP at these receptors, which only had high
`potency at the CGRP1 receptor (Fig. 3; Table 2).
`(Cys(Et)2,7)-␣CGRP and
`The
`linear CGRP analogs
`(Cys(Acm)2,7)-␣CGRP have been used to subclassify CGRP
`receptors into CGRP1 and CGRP2 receptors (Dennis et al.,
`1990, 1991; Poyner et al., 2002). Because AMY receptors can
`also function as high-affinity CGRP receptors, it was of in-
`terest to assess the potency of the linear CGRP analogs at CT
`and AMY receptors. Both analogs had very low potency and
`efficacy at CT(a), AMY2(a), and AMY3(a) receptors, but they
`displayed moderate potency at the AMY1(a) receptor (Table 1;
`Fig. 4A). However, both analogs were only partial agonists at
`the latter receptor exhibiting %Emax responses of 47.9 ⫾ 5.4
`and 22.8 ⫾ 6.0, respectively, for (Cys(Et)2,7)-␣CGRP and
`(Cys(Acm)2,7)-␣CGRP. At the CGRP1 receptor, both analogs
`displayed high potency, pEC50 of 9.4 ⫾ 0.12 (n ⫽ 5) and
`9.08 ⫾ 0.63 (n ⫽ 4) for (Cys(Et)2,7)-␣CGRP and (Cys(Acm)2,7)-
`␣CGRP, respectively, similar to unmodified h␣CGRP [9.51 ⫾
`0.14 (n ⫽ 5)], but they were again partial agonists. However,
`
`Downloaded from
`
`molpharm.aspetjournals.org
`
` at ASPET Journals on September 25, 2016
`
`n
`
`6
`5
`6
`10
`6
`5
`6
`6
`8
`6
`4
`4
`5
`3
`
`Agonist
`
`Receptor
`CT(a)
`
`AMY1(a)
`
`AMY2(a)
`
`pEC50
`6.53 ⫾ 0.09
`IMDS
`6.80 ⫾ 0.04
`h␣CGRP
`8.07 ⫾ 0.19*
`IMDS
`8.70 ⫾ 0.17
`h␣CGRP
`6.25 ⫾ 0.26
`IMDS
`7.24 ⫾ 0.19
`h␣CGRP
`7.12 ⫾ 0.19†
`IMDS
`7.60 ⫾ 0.17
`h␣CGRP
`8.71 ⫾ 0.13
`IMDS
`9.47 ⫾ 0.19
`h␣CGRP
`8.10 ⫾ 0.04
`IMDS
`6.39 ⫾ 0.10
`h␣CGRP
`8.69 ⫾ 0.13
`IMDS
`6.87 ⫾ 0.13
`h␣CGRP
`* P ⬍ 0.05 versus CT(a), AMY2(a), and AMY3(a) receptors.
`† P ⬍ 0.05 versus CT(a), AMY1(a), and AMY2(a) receptors.
`
`AMY3(a)
`
`CGRP1
`
`AM1
`
`AM2
`
`Fig. 3. Induction of cAMP accumulation by IMDS in COS-7 cells tran-
`siently transfected with CT(a)-based receptor phenotypes (A) and CL-
`based receptor phenotypes (B). For CGRP and AM receptors, the response
`across receptors probably represents different levels of receptor expres-
`sion. The Emax for IMDS and h␣CGRP was equivalent for all. The graph
`is of a representative experiment, with triplicate repeats, of at least six
`independent experiments.
`
`Fig. 4. Induction of cAMP accumulation at AMY1(a) (A) or CGRP1 (B)
`receptors by linear CGRP analogs. h␣CGRP (closed squares), (Cys(Et)2,7)-
`␣CGRP (F), and (Cys(Acm)2,7)-␣CGRP (open circles). pEC50 and Emax
`values, respectively, at the CGRP1 receptor were h␣CGRP, 9.51 ⫾ 0.14,
`100% (n ⫽ 5); (Cys(Et)2,7)-␣CGRP, 9.40 ⫾ 0.12, 83.54 ⫾ 7.19% (n ⫽ 5);
`and (Cys(Acm)2,7)-␣CGRP, 9.08 ⫾ 0.63, 8.08 ⫾ 2.09% (n ⫽ 4). The graph
`is of a representative experiment, with triplicate repeats, of at least four
`independent experiments. pEC50 and Emax values for peptides at the
`AMY1(a) receptor are detailed in Table 1.
`
`5
`
`

`

`onist versus when hCT was the agonist (Table 3; Figs. 5, 6, 7,
`H versus D, and 8D), although this was not significant at the
`AMY3(a) receptor. AC413 also seemed to discriminate
`between AMY1(a) versus AMY3(a) receptors when rAMY was
`used as the agonist, being more effective at AMY1(a) (Fig. 8D).
`In competition for 125I-rAMY binding, sCT8-32, AC187, and
`AC413 each displayed high affinity at both AMY1(a) and
`
`TABLE 3
`pKB values for antagonists in antagonizing agonist-induced stimulation
`of cAMP accumulation at human CT and AMY receptor phenotypes
`Values are presented as mean ⫾ S.E.M.
`
`Antagonist & Receptors
`sCT8-32
`CT(a)
`CT(a)
`AMY1(a)
`AMY1(a)
`AMY1(a)
`AMY1(a)
`AMY1(a)
`AMY3(a)
`AMY3(a)
`AC187
`CT(a)
`CT(a)
`AMY1(a)
`AMY1(a)
`AMY1(a)
`AMY1(a)
`AMY1(a)
`AMY3(a)
`AMY3(a)
`AC413
`CT(a)
`CT(a)
`AMY1(a)
`AMY1(a)
`AMY1(a)
`AMY1(a)
`AMY1(a)
`AMY3(a)
`AMY3(a)
`h␣CGRP8-37
`CT(a)
`CT(a)
`AMY1(a)
`AMY1(a)
`AMY1(a)
`AMY1(a)
`AMY1(a)
`AMY3(a)
`AMY3(a)
`rAMY8-37
`CT(a)
`CT(a)
`AMY1(a)
`AMY1(a)
`AMY1(a)
`AMY1(a)
`AMY1(a)
`AMY3(a)
`AMY3(a)
`hAM22-52
`⬍5
`CT(a)
`⬍5
`CT(a)
`⬍5
`AMY1(a)
`⬍5
`AMY1(a)
`⬍5
`AMY1(a)
`⬍5
`AMY1(a)
`AMY1(a)
`N.D.
`⬍5
`AMY3(a)
`⬍5
`AMY3(a)
`N.D., not done; ⬍5, antagonist caused no significant shift of the agonist concen-
`tration effect curve at concentrations of 10⫺5 M.
`
`Downloaded from
`
`molpharm.aspetjournals.org
`
` at ASPET Journals on September 25, 2016
`
`n
`
`7
`7
`7
`11
`11
`12
`4
`6
`6
`
`7
`7
`7
`7
`11
`4
`4
`6
`5
`
`7
`7
`5
`4
`10
`2
`2
`8
`8
`
`5
`4
`7
`11
`9
`14
`6
`8
`7
`
`2
`2
`4
`3
`
`3
`4
`
`1
`1
`3
`3
`1
`1
`
`4
`4
`
`Agonist
`
`pKB
`
`8.17 ⫾ 0.17
`8.22 ⫾ 0.26
`7.95 ⫾ 0.16
`7.78 ⫾ 0.13
`7.80 ⫾ 0.17
`7.68 ⫾ 0.18
`7.61 ⫾ 0.17
`7.87 ⫾ 0.25
`7.92 ⫾ 0.19
`
`7.15 ⫾ 0.23
`6.89 ⫾ 0.25
`7.30 ⫾ 0.11
`8.02 ⫾ 0.18
`7.86 ⫾ 0.20
`7.85 ⫾ 0.26
`7.73 ⫾ 0.27
`7.37 ⫾ 0.33
`7.68 ⫾ 0.22
`
`6.94 ⫾ 0.13
`7.48 ⫾ 0.17
`7.11 ⫾ 0.27
`7.92 ⫾ 0.23
`7.30 ⫾ 0.24
`7.25 ⫾ 0.21
`7.44 ⫾ 0.67
`6.83 ⫾ 0.27
`7.10 ⫾ 0.14
`
`⬍5
`⬍5
`⬍5
`6.62 ⫾ 0.13
`6.79 ⫾ 0.24
`6.78 ⫾ 0.13
`6.56 ⫾ 0.4
`ⱕ5
`6.17 ⫾ 0.26
`
`⬍5
`⬍5
`⬍5
`5.59 ⫾ 0.24
`N.D.
`N.D.
`N.D.
`⬍5
`⬍5
`
`hCT
`rAMY
`hCT
`rAMY
`h␣CGRP
`h␤CGRP
`Tyr0-h␣CGRP
`hCT
`rAMY
`
`hCT
`rAMY
`hCT
`rAMY
`h␣CGRP
`h␤CGRP
`Tyr0-h␣CGRP
`hCT
`rAMY
`
`hCT
`rAMY
`hCT
`rAMY
`h␣CGRP
`h␤CGRP
`Tyr0-h␣CGRP
`hCT
`rAMY
`
`hCT
`rAMY
`hCT
`rAMY
`h␣CGRP
`h␤CGRP
`Tyr0-h␣CGRP
`hCT
`rAMY
`
`hCT
`rAMY
`hCT
`rAMY
`h␣CGRP
`h␤CGRP
`Tyr0-h␣CGRP
`hCT
`rAMY
`
`hCT
`rAMY
`hCT
`rAMY
`h␣CGRP
`h␤CGRP
`Tyr0-h␣CGRP
`hCT
`rAMY
`
`1660
`
`Hay et al.
`
`Antagonist Pharmacology. N-Terminally truncated an-
`alogs of CT and related peptides have traditionally been used
`as “specific” antagonists of the primary receptors at which
`they interact. However, the specificity of interaction across
`the range of CT and AMY receptor phenotypes has not been
`systematically addressed. We have therefore assessed the
`relative effectiveness of these peptide antagonists and a
`number of chimeras of sCT8-32 and rAMY (Fig. 1) as antag-
`onists of CT(a), AMY1(a), and AMY3(a) receptors. Antagonist
`studies were not performed at the AMY2(a) receptor because
`of the weak AMY phenotype we observe in COS-7 cells.
`Of the peptides examined, sCT8-32 was the most effective
`antagonist with a pKB of ⬃8 across all receptors examined. It
`did not display significant selectivity, with a similar pKB
`observed for CT(a), AMY1(a), and AMY3(a) receptors, for each
`of the agonists (Table 3; Figs. 5, A and E, 6, A and E, and 7,
`A and E), although there was a weak trend for lower affinity
`at AMY1(a) receptors with either rAMY or the CGRPs as
`agonists (Fig. 8A).
`In contrast, the CGRP1 receptor antagonist CGRP8-37 was
`selective for AMY receptors over CT receptors (Fig. 8B), with
`no antagonism of agonist responses at CT receptors with
`concentrations of antagonist up to 10⫺5 M (Table 3; Fig. 5, B
`and F). However, CGRP8-37 was only a weak antagonist at
`AMY1(a) and AMY3(a) receptors with pKB values of ⬍7 (Table
`3; Figs. 6, B and F, and 7, B and F). With AMY as agonist,
`CGRP8-37 exhibited weak selectivity for AMY1(a) over
`AMY3(a) receptors, although this did not reach statistical
`significance (t test; p ⫽ 0.11) in the current study. There was
`an apparent agonist-dependent component to antagonism by
`CGRP8-37, with no effect seen at any of the receptors when
`hCT was used as the agonist (Table 3; Figs. 5B, 6B, and 7B).
`In support of the weak effect of AM at these receptors
`(Table 1), AM22-52, an antagonist of AM receptors, had no
`effect at either CT or AMY receptors (Table 3). Confirmation
`of the integrity of AM22-52 was obtained in experiments with
`AM2 receptors, where this peptide is known to be an antag-
`onist (data not shown; Hay et al., 2003). rAMY8-37 was almost
`without activity, exhibiting only very weak antagonist activ-
`ity at AMY1(a) receptors, and only when rAMY was the
`agonist (Table 3).
`The peptide chimeras of rAMY and sCT8-32, AC187 and
`AC413, each had affinity for CT(a), AMY1(a), and AMY3(a)
`receptors but displayed selectivity between receptor pheno-
`types (Table 3; Fig. 8, C and D). AC187 was ⬃10-fold more
`potent an antagonist of AMY1(a) receptors compared with
`CT(a) receptors when rAMY was used as the agonist (Table 3;
`Figs. 5G, 6G, and 8C). Likewise, AC187 was more potent at
`AMY3(a) receptors over CT(a) receptors when rAMY was the
`agonist (Table 3; Figs. 5G, 7G, and 8C), but no significant
`difference was seen between AMY1(a) and AMY3(a) receptors
`(Fig. 8C). As seen with CGRP8-37, there was an apparent
`agonist-dependent effect observed with the antagonist po-
`tency of AC187 when hCT was the agonist, because no sig-
`nificant change in AC187 potency was seen across the three
`receptor types (Table 3; Fig. 8C). Equivalent antagonist be-
`havior was observed for AC413 when hCT was the agonist,
`with no difference in antagonist potency between CT(a),
`AMY1(a), and AMY3(a) receptors (Table 3; Figs. 5D, 6D, 7D,
`and 8D). However, additional receptor-dependent and ago-
`nist-dependent behavior was seen for AC413. For each of the
`receptors, AC413 was more potent when rAMY was the ag-
`
`6
`
`

`

`Molecularly and Pharmacologically Distinct Amylin Receptors
`
`1661
`
`Downloaded from
`
`molpharm.aspetjournals.org
`
` at ASPET Journals on Septem