0031-6997/02/5402-233-246$7.00
`PHARMACO LOGICAL REVIEWS
`Copyright© 2002 by The Am erican Society for Pharmacology and Experimental Therapeutics
`Pharmacol Rev 54:233-246, 2002
`
`Vol. 54, No. 2
`20206/990608
`Printed in U.S.A
`
`International Union of Pharmacology. XXXII. The
`Mammalian Calcitonin Gene-Related Peptides,
`Adrenomedullin, Amylin, and Calcitonin Receptors
`
`DAVID R. POYNER, PATRICK M. SEXTON, IAN MARSHALL, DAVID M. SMITH, REMI QUIRION, WALTER BORN, ROMAN MUFF,
`,JAN A. FISCHER, AND STEVEN M. FOORD
`
`Pharmaceutical Sciences Institute, Aston University, Birmingham, United Kingdom (D.R.P.); Howard Florey Institute of Experimental
`Physiology and Medicine, University of Melbourne, Victoria, Australia (P.M.S.); Department of Pharmacology, University College London,
`London, United Kingdom (l.M.); AstraZeneca Pharmaceuticals, Alderley Edge, Cheshire, United Kingdom (D. M.S.); Douglas Hospital
`Research Centre and Department of Psychiatry, McGill University, Verdun, Quebec, Canada (R.Q.); Research Laboratory for Calcium
`Metabolism, Departments of Orthopedic Surgery and Medicine, University of Zurich, Klinik Balgrist, Zurich, Switzerland (W.A.B., R.M.,
`J.A.F.); and GlaxoSmithKline, Medicines Research Centre, Stevenage, Hertfordshire, United Kingdom (S.M.F.)
`
`This paper is available online at http://pharmrev.aspetjournals.org
`
`Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
`I. Introduction .. ......... .... .... . ............ ... ............ .. . ............. . .. ......... 234
`II. The calcitonin gene-related peptide receptors .................... ..................... .. .. 235
`A. Additional cofactors or receptors ........ ..... .. . .............. .. .. ..... .. . .. .... .. .... 237
`B. Tissue factors ...... . .. . ................... . .. .. ....... ... ..................... .. . . . 237
`C. Contribution of more than one receptor to pA2 values ........ ... .................... . .. 237
`III. The adrenomedullin receptors .......................... .. ...... ............ . .. ... . .... . . 237
`IV. The calcitonin receptors ....... ... .. ..... .... ... .... .... ...... .. ... ... . ... . . ...... ... .. . 239
`V. The amylin receptors ............... ............ ....... . ... .. . ... . .................... . . 240
`VI. Receptor modulation .. ..... . ............ .. . .. . . .............................. .. ........ 241
`A. Expression of mRNA encoding calcitonin-like receptor, calcitonin receptor, and receptor
`activity modifying proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
`VII. Receptor effector mechanisms ........................................................... 242
`VIII. The evolution of receptor activity modifying proteins, the calcitonin peptide family, and its
`receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
`IX. Conclusions and recommendations ................................... . ..... . .......... ... 243
`References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
`
`Abstract--The calcitonin family of peptides com(cid:173)
`prises calcitonin, amylin, two calcitonin gene-re(cid:173)
`lated peptides (CGRPs), and adrenomedullin. The
`first calcitonin receptor was cloned in 199l.lts phar(cid:173)
`macology is complicated by the existence of several
`splice variants. The receptors for the other members
`the family are made up of subunits. The calcitonin(cid:173)
`like receptor (CL receptor) requires a single trans(cid:173)
`membrane domain protein, termed receptor activity
`modifying protein, RAMPl, to function as a CGRP
`receptor. RAMP2 and -3 enable the same CL receptor
`to behave as an adrenomedullin receptor. Although
`the calcitonin receptor does not require RAMP to
`
`bind and respond to calcitonin, it can associate with
`the RAMPs, resulting in a series of receptors that
`typically have high affinity for amylin and varied
`affinity for CGRP. This review aims to reconcile
`what is observed when the receptors are reconsti(cid:173)
`tuted in vitro with the properties they show in na(cid:173)
`tive cells and tissues. Experimental conditions must
`be rigorously controlled because different degrees
`of protein expression may markedly modify pharma(cid:173)
`cology in such a complex situation. Recommenda(cid:173)
`tions, which follow International Union of Pharma(cid:173)
`cology guidelines, are made for the nomenclature of
`these multimeric receptors.
`
`Address correspondence to: Dr. David R. Poyner, Pharmaceutical Sciences Institute, Aston University, Birmingham, UK B4 7ET. E-mail:
`d. r.poyne~aton.ac. uk
`
`233
`
`1
`
`EX2187
`Eli Lilly & Co. v. Teva Pharms. Int'l GMBH
`IPR2018-01423
`
`

`

`234
`
`POYNER ET AL.
`
`I. Introduction
`
`The calcitonin family of peptides comprises five
`known members: calcitonin (CT1
`), amylin (AMY), two
`CT gene-related peptides (aCGRP and f3CGRP), and
`adrenomedullin (AM) (Table 1). Although homology at
`the level of the primary sequence is weak, there are
`stronger relationships between the secondary structures
`of the peptides. They all have a six-amino acid ring
`structure (seven for CT) close to their N termini, formed
`by an intramolecular disulfide bond. This is followed by
`a region of potential amphipathic a-helix, and they all
`are C terminally amidated.
`The peptides are widely distributed in various periph(cid:173)
`eral tissues as well as in the peripheral and central
`nervous system (CNS) and induce multiple biological
`effects including potent vasodilatation (CGRP and AM),
`reduction in nutrient intake (AMY), and decreased bone
`resorption (CT). Due to their structural similarities, the
`peptides share some biological activities, suggesting
`that they interact with similar G protein-coupled recep(cid:173)
`tors (GPCRs). However, there is clear evidence to sup(cid:173)
`port unique biological activities and so distinct recep(cid:173)
`tors. Receptors have been characterized, in vivo and in
`vitro, on the basis of pharmacological responses and
`radioligand binding studies. The eDNA encoding a por(cid:173)
`cine CT receptor was cloned in 1991 (Lin et al., 1991),
`with human (Gorn et al., 1992; Kuestner et al., 1994)
`and rat receptors (Albrandt et al., 1993; Sexton et al.,
`1993) cloned soon after. The CT receptor belongs to the
`"family B" of GPCRs, which typically recognize regula(cid:173)
`tory peptides such as parathyroid hormone, secretin,
`glucagon, and vasoactive intestinal polypeptide (Sexton,
`1999). The rat and human calcitonin receptor-like recep(cid:173)
`tors were identified shortly after (Chang et al., 1993;
`Njuki et al., 1993; Fliihmann et al., 1995). They show 50
`and 54% overall identity with the respective calcitonin
`receptors. Historically, this protein has been abbrevi(cid:173)
`ated to "CRLR"; however, following IUPHAR guidelines
`(Ruffolo et al., 2000), it will be referred to in this article
`as the "CL receptor". (Although this follows the conven(cid:173)
`tion adopted in the field, since it does not bind any
`known ligand by itself, it would be strictly correct to
`refer to it as the "CL protein".)
`Initially, the CL receptor was considered an orphan re(cid:173)
`ceptor. Evidence for CGRP receptor function of the CL
`receptor was first obtained by Aiyar et al. (1996) who
`
`1 Abbreviations: CT, calcitonin; CL, CT-like; CGRP, CT gene(cid:173)
`related peptide; sCT, salmon CT; hC'l', human CT; CTR, C'f receptor;
`AMY, amylin; AM, adrenomedullin; CNS, central nervous system;
`GPCR, G protein-coupled receptor; IUPHAR, International Union of
`Pharmacology; HEK, human embryonic kidney; RAMP, receptor ac(cid:173)
`tivity modifying protein(s); RCP, receptor component protein; CHO,
`Chinese hamster ovary; RAEC, rabbit aortic endothelial cell; UTR,
`untranslated region; BIBN4096BS, 1-piperidinecarboxamide, N-[2-
`[[5-amino-1- [[ 4-( 4-pyridiny lJ-1-piperazinyl] car bony I] pentyl] amino]-
`1-[( 3, 5-di bromo-4-hydroxypheny I Jmethy 1]-2-oxoethyl]-4-( 1,4-dihy(cid:173)
`dro-2-oxo-3(2H)-quinazolinyl).
`
`observed CGRP binding and CGRP-dependent cAMP ac(cid:173)
`cumulation in a single subclone of HEK293 cells stably
`transfected with the hCL receptor encoding eDNA. The
`important breakthrough came when an expression cloning
`approach demonstrated that the CL receptor required a
`single transmembrane domain protein, termed receptor
`activity modifying protein, RAMP1, to function as a CGRP
`receptor (McLatchie et al., 1998) (Fig. 1). The RAMP family
`of proteins comprises three members, RAMP1, -2, and -3.
`RAMP2 and -3 enable the same CL receptor to behave as
`an AM receptor. The RAMPs share a common topological
`organization but less than 30% sequence identity. They are
`intrinsic membrane proteins (predicted sizes: M,. 14,000-
`17,000) with an extracellular N terminus of ~ 100 amino
`acids, a single transmembrane domain, and a short intra(cid:173)
`cellular domain (10 amino acids) (Muff et al., 2001; Sexton
`et al., 2001). The CT receptor does not require RAMP to
`bind and respond to CT, but it can associate with the
`RAMPs. This results in a series of receptors that typically
`have high affinity for AMY, and varied affinity for CGRP
`(Christopoulos et al., 1999; Muff et al., 1999).
`Two related members of"family A" GPCRs, RDC1 and
`L1/G lOD, have been considered as receptors for CGRP
`and AM, respectively (Kapas et al., 1995; Kapas and
`Clark, 1995). This was unexpected because although
`RDC1 and GlOD are homologous to each other, they are
`very different from the CT and CL receptors. In fact,
`several attempts have been made to further characterize
`RDC1- and L1/G10D-induced CGRP and AM responses
`without success (Kennedy et al., 1998; McLatchie et al.,
`1998; Tong et al., 2000). These receptors are now con(cid:173)
`sidered "orphans" once more.
`Detailed studies of the pharmacology displayed by CT
`and CL receptors, expressed with or without all three
`RAMPs, have now been reported by several laboratories
`(Btihlmann et al., 1999; Christopoulos et al., 1999;
`Fraser et al., 1999; Muff et al., 1999; Aldecoa et al., 2000;
`Husmann et al., 2000; Leuthauser et al., 2000; Ti(cid:173)
`lakaratne et al., 2000; Zumpe et al., 2000; Aiyar et al.,
`2001; Oliver et al., 2001). They have revealed receptors
`that bind and respond to CT, CGRP, AM, and AMY. In
`view of the structural similarity between the CT family
`of peptides, it is not surprising that cross-reactivity be(cid:173)
`tween them at their cognate receptors has been demon(cid:173)
`strated. Because two GPCR proteins and three RAMPs
`can reconstitute receptors for the whole CT family of
`peptide ligands, the situation is even more complex. In
`addition, at least some cells appear to express more than
`one RAMP and there is evidence to support the regula(cid:173)
`tion of RAMP expression at the mRNA level.
`This review aims to reconcile what is observed when
`the receptors are reconstituted in vitro with the proper(cid:173)
`ties they show in native cells and tissues. The Nomen(cid:173)
`clature Committee of the IUPHAR subcommittee on cal(cid:173)
`citonin receptors also makes recommendations for the
`nomenclature of these receptors.
`
`2
`
`

`

`haCGRP
`raCGRP
`h!3CGRP
`r!3CGRP
`hAMY
`rAMY
`hAM
`rAM
`hCT
`sCT
`
`CGRP, ADRENOMEDULLIN, AMYLIN, AND CALCITONIN RECEPTORS
`
`235
`
`TABLE 1
`Structures CGRP, amylin, adrenomedullin, and calcitonin
`D T A T C V T H R L A G L L s R S G G v v K N N F V P T N V G s KA
`F
`A C
`s c NT AT c v T H R LAG L L s R S G G V V K D N F V p TN V G s E A
`F
`N TAT c v T H R LAG L L s R S G G M V K S N F V p TN V G S K A
`A C
`F
`s c
`N TAT c v T H R LAG L L s R S G G V V K D N F V p TN V G S K A
`F
`K C NT AT CAT Q R LAN F L V H S S N N F G A I L S s TN V G S N T
`y
`K c
`N TAT CAT Q R LAN F L V R S S N N L G P V L P s TN V G s NT
`y
`G c
`R F G T C T V Q K L A H Q I y Q F T D K D K D NVA P R N K I s p Q G y
`R F G T C T M Q K L A H Q I y Q F T D K D K D G M A p R N K I s p Q G y
`G c
`c G N L S T C M L G T Y T Q D F N K F H T F
`p
`p Q T A I G V G A
`c S N L S T C V L G K L S Q E L H K L Q T y
`p
`p R TNT G S G T
`- ----------
`
`Disulfide bond
`
`a -helix
`
`h, human; r, rat; S 1 salmon.
`hAM is the structure of the 15-521rugment; theN-terminal amino acids are YRQSMNNFQGLRSF. rAM shows the structure of the 13- 50 fragment; the N-lerminal amino
`acids are YRQSMNQGSRST.
`
`II. The Calcitonin Gene-Related Peptide
`Receptors
`
`aCGRP was cloned from the gene encoding CT (Amara
`et al. , 1982). Alternate splicing of the CT gene leads to
`the production, especially in nervous tissues, of aCGRP,
`a 37 -amino acid peptide. A second CGRP homolog·,
`J3CGRP, was subsequently discovered. It differs from
`human aCGRP by three amino acids and in the rat by
`one amino acid. J3CGRP is encoded by its own unique
`gene, with high homology to the CT gene (Steenbergh et
`al., 1985). a- and J3CGRP display similar biological ac(cid:173)
`tivities.
`Historically, CGRP receptors have been divided into
`two classes, CGRP1 and CGRP2 . CGRP 1 receptors are
`more sensitive than CGRP2 receptors to the peptide
`antagonist CGRP8 _37 (taken by many workers to mean a
`pA2 of 7 or greater) (Dennis et al., 1989; Quirion et al.,
`1992). On the other hand, the linear CGRP analogs
`
`[Cys(ACM)2
`7]haCGRP are more potent
`7
`]- and [Cys(Et)2
`•
`•
`agonists at CGRP 2 receptors than at CGRP 1 (Dennis et
`al., 1989; Dumont et al., 1997; Moreno et al., 2002).
`The CGRP1:CGRP2 classification has provided a con(cid:173)
`ceptual framework for the development of CGRP phar(cid:173)
`macology. However, progress in the field has been ham(cid:173)
`pered by lack of suitable selective drugs (Marshall and
`Wisskirchen, 2000). The prototypical tissue expressing
`the CGRP2 receptor is the rat vas deferens (Dennis et
`al., 1989). Here pA2 estimates of CGRP8 _37, measured
`with CGRP as agonist, range from below 5.5 (Giuliani et
`al., 1992) to 6.7 (Longmore et al., 1994). For the proto(cid:173)
`typical CGRP 1 receptor expressing tissue (guinea pig
`left atrium), the range is 6.9 to 7.7 (Dennis et al., 1990;
`Mimeault et al., 1991). In both tissues, there is at least
`a 10-fold spread of values. Although a meta-analysis of
`these data sets confirms that there is a significant dif(cid:173)
`ference, the variation demonstrates that any individual
`
`~
`( ..
`c:)•0:··~~··G)·· -'•.<lXilG) ~G0(i)(!) NH2
`~:fi·<i>•<lXiXOOXi)~(i);o;(00);<0• •0;· .f!. 1· • , ,, " .. e r. Gl0· ··~ ,.,
`
`(i)(!t •• ~ •0 .. .. .• \.\ ~ .. • •J"·~G> ~ llr'' ·•/l.c;Jl;,
`
`NH1Lii.}.i'®0CD(i)~•x.;:.<.~:o..tt~·i •<:X;G "' G:\:;) -8
`
`!'_~; 00•(l)GOO•<iXj)(l)(j)G)0 • 00 · ~
`
`CRLR
`
`"'·"GCi)(ll(i)•' Ci) o'G)G • ' " ·<i·Gl·
`
`, Ci). ;0
`
`FIG 1. Schematic diagram of human CL receptor (CRLR) and RAMP 1. Stars show glycosylation consensus sites on CL receptor. Residues unique
`to CL receptor over the human C'l' receptor, or unique to RAMPl over RAMP2 or RAMP3 are indicated in bold circles. Arrows show predicted signal
`peptide cleavage sites.
`
`3
`
`

`

`236
`
`POYNER ET AL.
`
`pA2 value reported for the antagonist must be treated
`with caution. Values in the range of 6.5 to 7.5 are likely
`to be particularly hard to interpret under the CGRP 1 :
`CGRP2 classification. A wide range of pA2 values have
`been reported for CGRP8_37 in different preparations; in
`the rat for example: 8.0 in kidney, 7.9 in cerebral artery,
`7.6 in mesenteric artery, 6.9 in pulmonary and coronary
`arteries, 6.6 in caudal artery, 6.5 in uterus, 6.1 in vas
`deferens, 5.9 in internal anal sphincter, <6 in colon, and
`< 5 in thoracic aorta (Poyner and Marshall, 2001). Al(cid:173)
`though it is difficult to conceive that a single receptor
`subtype can account for such a wide range of pA2 values,
`there do not appear to be any natural breaks in the data.
`Recently, several nonpeptide antagonists have been
`introduced; the best characterized is BIBN4096BS (al(cid:173)
`though, strictly, this is a low molecular weight antago(cid:173)
`nist because BIBN is a highly modified peptide). It
`shows a marked selectivity for human over rat receptors
`(Doods et al., 2000), and it is more potent than CGRP8 _3 7
`in both species (Doods et al., 2000; Wu et al., 2000).
`Recent work has revealed that the relative affinity of
`BIBN4096BS is related to the species of origin for the
`RAMPl, rather than the receptor protein. Subsequent
`chimeric protein and point mutation analysis have iden(cid:173)
`tified tryptophan 74 of RAMPl as a key residue for the
`higher affinity of human CGRP receptors (Mallee et al.,
`2002).
`The status of the CGRP2-selective agonists is unclear.
`Agonists are not the tools of choice to define receptor
`subtypes. Waugh et
`al.
`(1999)
`reported
`that
`[Cys(ACM)2
`7)haCGRP is a partial agonist in the porcine
`•
`coronary artery; this could explain its apparent selectiv(cid:173)
`ity in other systems. Wu et al. (2000) have shown that
`[Cys(ACM) 2
`7)haCGRP and [Cys(Et)2
`7)haCGRP have
`•
`•
`similar potencies in both the rat atria and vas deferens
`and apparently act through an adrenomedullin-acti(cid:173)
`vated receptor, even though CGRP8_37 and BIBN4096BS
`demonstrate heterogeneity in the receptors responding
`to CGRP in these preparations.
`In summary, there is good evidence for heterogeneity
`among receptors that respond to CGRP in tissues, de(cid:173)
`rived from data with antagonists. Although CGRP 8 _37 is
`not easy to use, the limited data available from nonpep(cid:173)
`tide antagonists broadly support the conclusions that
`were based on this compound. The "selective" nonpep(cid:173)
`tide antagonists show promise, although as yet they
`have been little used. Questions remain about the extent
`of the heterogeneity of CGRP receptors, particularly
`whether the CGRP2 receptors are a homogeneous group,
`what the boundaries of this group are in terms of antag(cid:173)
`onist affinity, and whether CGRP is the endogenous
`ligand for all the receptors that have been described as
`belonging to this category.
`There is good evidence for the presence of CL receptor/
`RAMPl complexes on the surfaces of cells. Coimmuno(cid:173)
`precipitation experiments carried out in total cell ex(cid:173)
`tracts revealed stable interactions between RAMPl and
`
`both the immature (58 kDa) and mature (66 kDa) gly(cid:173)
`cosylated forms of CL receptor. Whole cell coimmuno(cid:173)
`precipitation and confocal microscopy indicated that the
`interaction was maintained once the receptor reached
`the cell surface (Leuthauser et al., 2000; Hilairet et al.,
`2001a,b). This interaction is also maintained during ag(cid:173)
`onist-mediated internalization of the receptor (Ku(cid:173)
`wasako et al., 2000). The covalent cross-linking of an
`-85-kDa complex [composed of one CL receptor (66 kDa)
`and one RAMPl molecule (17 kDa)], using the mem(cid:173)
`brane
`impermeable bis(sulfosuccinimidyl) suberate
`(BS3
`), further demonstrated that the interaction has a
`1:1 stoichiometry. Cross-linking experiments with 125I(cid:173)
`CGRP also showed that both the CL receptor/RAMP!
`complex and RAMPl were radiolabeled, indicating that:
`1) RAMPl and CL receptor remain associated upon
`CGRP binding; and 2) residues within RAMPllie close
`to, or may be part of, the CGRP binding pocket. These
`data demonstrate the existence of a meta-stable complex
`between the RAMPl and CL receptor, which is main(cid:173)
`tained upon activation of the receptor by CGRP (Hilairet
`et al., 200la,b).
`The receptor formed by the heterodimerization of CL
`receptor and RAMPl in recombinant systems is similar
`enough to that observed for the CGRP1 receptor in na(cid:173)
`tive tissues and cell lines to suggest that the combina(cid:173)
`tion occurs naturally and has physiological significance.
`The pA2 for CGRP 8 _37 for human CL receptor expressed
`with human RAMPl in HEK293 cells has been reported
`as 7.6 (Aiyar et al., 1996). This value correlates well with
`those obtained for the CGRP receptor in cell lines such
`as SK-N-MC (7.5-8.7) (Muff et al., 1992; Longmore et
`al., 1994; Entzeroth et al., 1995; Zimmermann et al.,
`1995; Poyner et al., 1998) and in human cerebral artery
`(7.7) (Edvinsson et al., 2001). Data are not yet available
`for the antagonist affinities of the rat CL receptor/rat
`RAMPl complex, but the rat CL receptor/human
`RAMPl complex has a pA2 of 7.5 (calculated from the
`data of Leuthauser et al., 2000). Thus, in the rat and
`human, there are good grounds for assuming that the
`majority of receptors with a pA2 for CGRP8_37 in excess
`of 7 correspond io CL reC~_l.liur/RAMPl. There are areas
`where CL receptor encoding mRNA is poorly expressed
`or apparently absent, e.g., in the cerebellum, but which
`show high levels of CGRP binding (Dotti-Sigrist et al.,
`1988; Fliihmann et al., 1997). Although this may simply
`reflect problems of trying to equate protein with mRNA
`expression, if workers find a CGRP1 phenotype in these
`tissues, they should exercise due caution in assuming
`that it is necessarily CL receptor/RAMP!. In species
`where no data on the CL receptor are available, such as
`the guinea pig, receptors with a pA2 for CGRP 8 _37 in
`excess of 7 are usually considered to be CGRP 1; prag(cid:173)
`matically, it seems reasonable for this practice to con(cid:173)
`tinue.
`Nevertheless, caution should be exercised in recombi(cid:173)
`nant systems because different deg-rees of protein ex-
`
`4
`
`

`

`CGRP, ADRENOMEDULLIN, AMYLIN, AND CALCITONIN RECEPTORS
`
`237
`
`pression may markedly modify pharmacology in such a
`complex situation. This particularly applies to experi(cid:173)
`ments where RAMPs and receptors from different spe(cid:173)
`cies are used.
`There is evidence that the CGRP receptor complex
`might interact with another protein. A cytosolic protein,
`RCP (receptor component protein), was identified on the
`basis of its ability to potentiate the CGRP-mediated
`responses in Xenopus laeuis oocytes in much the same
`way as was RAMPl (although the two proteins show no
`similarity) (Luebke et al., 1996). RCP appears to be
`required for NIH-3T3 cells to show CGRP responsive(cid:173)
`ness. If mRNA encoding RCP is complexed with anti(cid:173)
`sense oligonucleotides, there is a reduction in NIH-3T3
`cell CGRP responsiveness (with appropriate controls be(cid:173)
`ing unaltered). Some immunoprecipitation data imply a
`direct interaction between CL receptor and RCP (Evans
`et al., 2000). CGRP receptors can be reconstituted
`through the expression of CL receptor/RAMP! in Xeno(cid:173)
`pus oocytes or insect cells without exogenous RCP
`(McLatchie et al., 1998; Aldecoa et al., 2000). However,
`this is not to say that RCP has no role; it may be that
`these systems express endogenous RCP. Recent work
`has shown the reconstitution ofCGRP and AM receptors
`in the yeast Saccharomyces cereuisiae through the coex(cid:173)
`pression of RAMPs with CL receptor (Miret et al., 2001).
`Yeast may also share an RCP homolog, but it is more
`distantly related again. It will take more work to define
`the role of RCP.
`Although many receptors responding to CGRP are
`likely to correspond to CL receptor/RAMP!, these are
`unlikely to explain the behavior of those found, for ex(cid:173)
`ample, in the rat vas deferens or aorta or cell lines such
`as the human-derived Col 29 cells (Cox and Tough,
`1994). What might explain these data? Several alterna(cid:173)
`tives include additional receptors or RAMPs, additional
`tissue factors, or the contribution of more than one re(cid:173)
`ceptor to the pA2 values for CGRP8 _37 that have been
`determined.
`
`A. Additional Cofactors or Receptors
`As the human genome sequencing project refines its
`data, it will soon become apparent whether another
`GPCR will be discovered to account for the observations
`suggesting the existence of CGRP2 receptors. It may be
`that some very labile accessory protein is responsible for
`promoting a putative CGRP2 receptor form, perhaps by
`interacting with the CL receptor. The mRNA for the CL
`receptor is present in rat vas deferens, the prototypical
`CGRP2 receptor-expressing tissue (Moreno et al., 2002),
`as well as other tissues showing pharmacology similar to
`the putative CGRP2 type, e.g., porcine coronary artery
`(Rorabaugh et al., 2001). A possible contributor may be
`G proteins, as recent work with CT and AMY receptors
`has indicated that modulation of the level and type of G
`protein a subunit can profoundly influence phenotype
`(Tilakaratne et al., 2000; Smyth et al., 2001). Another
`
`potential cofactor is the RCP (see above, Moreno et al.,
`2002).
`
`B. Tissue Factors
`A number of workers have considered that tissue(cid:173)
`specific factors such as proteolysis (of ligand, CL recep(cid:173)
`tor, or RAMPl) may play a role in the apparent CGRP
`receptor heterogeneity (e.g., Longmore et al., 1994;
`Rorabaugh et al., 2001). However, others have found
`that peptidase inhibitors make little difference to CGRP
`pharmacology
`(Tomlinson
`and
`Poyner,
`1996;
`Wisskirchen et al., 1998).
`
`C. Contribution of More than One Receptor to pA2
`Values
`The complexes of CL receptor with RAMP2 and -3 as
`well as the various CT receptor/RAMP complexes all
`generate receptors that interact with CGRP (see above).
`Little work has been done on the pharmacology of the
`majority of these complexes but there are some poten(cid:173)
`tially relevant data. Human aCGRP is a potent agonist
`at the human CT(aJ receptor/human RAMPl complex
`(EC50 of 0.8 nM) (see below for discussion of CT receptor
`nomenclature). This response is antagonized by salmon
`calcitonin (8-32) (sCT8 _ 32) but not 100 nM CGRP8 _37
`(Leuthii.user et al., 2000). This complex is probably bet(cid:173)
`ter considered an AMY receptor than a CGRP receptor,
`but without use of an appropriate antagonist such as
`sCT8 _ 32, this would not be apparent (see below). Aiyar et
`al. (2001) have reported that porcine CL receptor ex(cid:173)
`pressed with either human RAMP2 or RAMP3 shows
`high-affinity CGRP binding but the functional pharma(cid:173)
`cology is closer to that of adrenomedullin receptors.
`Given that CGRP 2 receptor-expressing tissues may
`sometimes respond to amylin and AM (e.g., Poyner et al.,
`1999; Wisskirchen et al., 1999), these other complexes
`may mediate some ofthe effects ofCGRP. Use ofsCT8 _ 32
`(which would antagonize complexes containing the CT
`receptor but not the CL receptor) might be useful in
`further defining a CGRP2 phenotype.
`Currently, there is no molecular correlate for a CGRP2
`receptor. As noted above, it is possible that the class
`represents more than one pharmacologically distinct en(cid:173)
`tity. Indeed, some workers have noted "atypical" CGRP
`receptors (Wisskirchen et al., 1998; Esfandyari et al.,
`2000), although frequently these are all considered as
`being CGRP2 receptors; a tendency that might obscure
`real differences. Furthermore, CGRP may not be the
`endogenous ligand for all of these receptors. Given these
`problems, it is recommended that authors use the term
`"CGRP2 receptor" with care.
`
`III. The Adrenomedullin Receptors
`
`AM is a peptide that was isolated in 1993 from human
`pheochromocytomas (Kitamura et al., 1993). Human
`AM is 52 amino acids long. The N-terminal 12 amino
`
`5
`
`

`

`238
`
`POYNER E1' AL.
`
`acids can be removed with little change in potency (Lin
`et al., 1994). The general pharmacology of AM has been
`reviewed elsewhere (Hinson et al., 2000).
`CL receptor and RAMP2 or -3 reconstitute AM recep(cid:173)
`tors that show the pharmacological characteristics of the
`native receptors (Table 2). RAMP2 and RAMP3 enable
`CL receptor to generate AM receptors that are pharma(cid:173)
`colog·ically similar. The biochemistry of the CL receptor/
`RAMP2 or the CL receptor/RAMP3 complexes suggests
`they form in the endoplasmic reticulum and remain
`tightly associated as they are delivered to the cell sur(cid:173)
`face (Hilairet et al., 200lb), and this is supported by
`confocal microscopic data (Kuwasako et al., 2000). Only
`the maturely glycosylated forms of the CL receptor/
`RAMP2 or CL receptor/RAMP3 complexes at the cell
`surface appear to bind 1251-AM (Hilairet et al., 2001a).
`Some biological activities of AM are mediated by
`CGRP receptors. At these receptors AM behaves as a
`potent competitor for 1251-aCGRP binding sites, and the
`production of cAMP induced by both CGRP and AM can
`be inhibited by CGRP8_37. Because it is common for the
`CL receptor to be coexpressed with combinations of
`RAMPs, it is likely that CGRP and AM receptors are
`coexpressed in the same cells.
`However, there are activities of AM that are clearly
`mediated by a unique AM receptor because they cannot
`be antagonized by CGRP8 _37 . Examples are AM-induced
`vasodilatation in the guinea pig pulmonary artery,
`tachycardia, hypotensive effects in Long-Evans rats,
`and the control of aldosterone production by the rat
`adrenal (Rebuffat et al., 2002). A cellular model of a
`unique AM receptor is provided by a rabbit aortic endo(cid:173)
`thelial cell (RAEC) line and the neuroblastoma/glioma
`NG 108-15 hybrid cell line (Zimmermann et al., 1996;
`
`Muff et al., 1998). Upon transfection of RAECs with
`RAMPl encoding eDNA, expression of a CGRP 1 recep(cid:173)
`tor, sensitive to CGRP8 _37, is also observed. These data
`imply expression of an endogenous CL receptor/RAMP2
`AM receptor in RAECs and its conversion into a CGRP 1
`receptor by competitive interaction of CL receptor with
`coexpressed exogenous RAMPl.
`1251-CGRP and 1251-AM binding studies define tissues
`that contain CGRP, AM, or mixtures of both receptors.
`Binding sites labeled by 1251-AM bind CGRP and AMY
`with over 200-fold lower affinities compared with AM
`(Poyner et al., 1999). Furthermore, AM binding sites in
`the CNS have a unique distribution compared with
`those for CGRP or AMY. Thus, the binding data strongly
`suggests that CGRP and AM interact with distinct sites
`(Owji et al., 1995; Coppock et al., 1996; Zimmermann et
`al., 1996; Upton et al., 1997; Juaneda et al., 2000). AM
`pharmacology would be greatly facilitated by the avail(cid:173)
`ability of selective antagonists. AM22_52 behaves as a
`competitive antagonist and blocks a variety of AM activ(cid:173)
`ities. It has been reported to selectively antagonize AM
`in cell lines, e.g., NG108-15 neuroblastoma cells (Zim(cid:173)
`mermann et al., 1996). However, in vivo it seems much
`less effective (Santiago et al., 1995; Champion et al.,
`1997). In the vas deferens, it was active by itself in
`inhibiting electrically stimulated contractions, and
`whereas it could additionally antagonize the response to
`AM, it had similar effects on the responses to CGRP and
`to some extent, AMY. Any lack of a specific effect of
`AM22_52 in contrast to some other reports might argue
`for heterogeneity among AM receptors. Recent studies of
`the reconstituted CGRP and AM receptors in yeast sug(cid:173)
`gest AM22_52 and CGRP8 •37 are selective for the CL re(cid:173)
`ceptor/RAMP! and CL receptor/RAMP2 combinations in
`
`TABLE 2
`Pharmacological profile for CL receptor/RAMP complexes
`
`-----------------------
`
`Cell
`
`References
`
`-------------------------------------
`
`Receptor
`
`rtCGRP
`
`{JCGRP
`
`hCL receptor/hRAMP1
`
`rCL receptor/rRAMPl
`rCL receptorlhllAMP1
`
`rCL receptor/mRAMP1
`pCL receptor/hRAMP1
`
`hCL receptor/hRAMP2
`
`r CL receptor/rRAMP2
`rCL receptor/hRAMP2
`
`rCL receptor/mRAMP2
`pCL receptot·/hRAMP2*
`hCL receptor/hRAMP3
`
`11
`
`9.8
`9.3
`8.4
`8.3
`8.3
`8.2
`9.8
`10.2
`6.5
`6.8
`7.7
`> 7
`
`10
`10.3
`9.6
`8.9
`0.3
`7.9
`8.2
`8.4
`10
`9.5
`5.9
`6.3
`
`> 7
`>6
`>6
`6.7
`
`AM
`
`AMzz-.'i2
`6.4
`
`> 6
`-;..6
`> 6
`
`AMY
`
`NS
`
`7.8
`:>6
`> 6
`
`NS
`NS
`
`>6
`>6
`>6
`
`CGRP8~37
`McLatchie et a!., 1998
`Swiss31'3
`8.1
`9.5
`Aiyar et a!., 1996
`7.8
`9
`HEK293
`9.1
`HEK293
`6.8
`Aiyar et al., 2001
`8.2
`8.8
`Oliver eta!., 2001
`UMR106
`-;..7
`Buhlmann eta!., 1999
`COS-7
`8.9
`8.8
`6.9
`Buhlmann et al., 1999
`UMR106
`Aldccoa et al. , 2000
`Schneider
`6.9
`8.8
`COS-7
`6.8
`8.8
`Husmann eta!. , 2000
`7.7
`9.18
`Elshourbagy et a!., 1998
`HEK293
`Aiyar et a!., 2001
`HEK293
`8.2
`9.1
`7.8
`9.2
`7.1
`McLatchie et a!., 1998
`Swiss3T3
`9.1
`7.2
`Fraser et a!., 1999
`HEK293
`7.7
`7.0
`8Ji
`>5
`Aiyar et a!., 2001
`HEK293
`>6
`7
`9.3
`Oliver et a!., 2001
`UMR106
`8.5
`7.1
`8.8
`Buhlmann et a!., 1999
`COS-7
`7.0
`8.6
`9.5
`7.4
`Buhlmann et a!., 1999
`UMR106
`6,7
`7.9
`9.1
`7.0
`Aldecoa et al., 2000
`Schneider
`8.5
`8.7
`7.0
`6.8
`Husmann et al., 2000
`COS-7
`7.14
`9.0/8.6
`7.7/6.2
`8.8/7.2
`8.617.5
`Aiyar et a!., 2001
`HEK293
`7.1
`7.1
`8 .