WHAT MAKES A CGRP 2 RECEPTOR?

Transcription

1 Clinical and Experimental Pharmacology and Physiology (2007) 34, doi: /j x Blackwell Publishing Asia BRIEF REVIEW CGRP Brief Review 2 receptors WHAT MAKES A CGRP 2 RECEPTOR? DL Hay Proteomics and Biomedicine Research Group, School of Biological Sciences, University of Auckland, Auckland, New Zealand SUMMARY 1. Heterogeneity in the receptors for the neuropeptide calcitonin gene-related peptide (CGRP) has been apparent for nearly 20 years. This is most clearly manifested in the observation of CGRP sensitive and -insensitive populations of CGRP-activated receptors. The pa 2 values for CGRP 8 37 in excess of 7 are widely considered to be the result of antagonism of CGRP 1 receptors, whereas those below 7 are believed to be the consequence of antagonism of a second population of receptors, namely CGRP 2 receptors. 2. However, a multitude of pa 2 values exist for CGRP 8 37, spanning several log units, and as such no obvious clusters of values are apparent. Understanding the molecular nature of the receptors that underlie this phenomenon is likely to aid the development of selective pharmacological tools to progress our understanding of the physiology of CGRP and related peptides. Because there is active development of CGRP agonists and antagonists as therapeutics, such information would also further this pursuit. 3. The CGRP 1 receptor is pharmacologically and molecularly well defined as a heterodimer of the calcitonin receptor-like receptor (CL) and receptor activity modifying protein (RAMP) 1. The CL/RAMP1 complex is highly sensitive to CGRP Conversely, the constituents of the CGRP 2 receptor have not been identified. In fact, there is little evidence for a distinct molecular entity that represents the CGRP 2 receptor. 4. Recent pharmacological characterization of receptors related to CGRP 1 has revealed that some of these receptors may explain CGRP 2 receptor pharmacology. Specifically, AMY 1(a) (calcitonin receptor/ramp1) and AM 2 (CL/RAMP3) receptors can be activated by CGRP but are relatively insensitive to CGRP Correspondence: Debbie L Hay, Proteomics and Biomedicine Research Group, School of Biological Sciences, University of Auckland, 3 Symonds Street, Private Bag 92019, New Zealand. dl.hay@auckland.ac.nz Presented at the Denis Wade Johnson and Johnson New Investigator Symposium, Annual Scientific Meeting of the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists, Melbourne, December Received 2 July 2006; revision 15 November 2006; accepted 4 January This, along with other supporting data, suggests that the CGRP 2 receptor that has been extensively reported in the literature may, in fact, be an amalgamation of contributions from a variety of CGRP-activated receptors. The use of appropriate combinations of agonists and antagonists, along with receptor expression studies, could allow such receptors to be separated. Key words: adrenomedullin, amylin, calcitonin gene-related peptide, calcitonin, CGRP 1 receptor, CGRP 2 receptor, receptor activity modifying protein (RAMP). A BRIEF INTRODUCTION TO CALCITONIN GENE-RELATED PEPTIDE AND ITS RECEPTORS The vasodilatory neuropeptide calcitonin gene-related peptide (CGRP) exists in two forms, denoted a and b or I and II. The acgrp isoform is encoded by the calcitonin gene (CALCA) and tissuespecific mrna processing determines the primary product in any given region. 1,2 For example, in the central nervous system, acgrp predominates over calcitonin. The bcgrp isoform is coded for by a separate gene (CALCB) on chromosome 11 and is thought to have arisen by gene duplication. 3,4 These peptides belong to a wider family that also includes adrenomedullin (AM) 1 and 2, calcitonin and amylin. All these peptides have two conserved cysteine residues near their N-termini that form a disulphide bridge. The amidated C- terminal residue in all peptides is a prerequisite for biological activity. This family, which will be referred to as the CGRP family in the present review, contains several physiologically and clinically relevant peptides. Adrenomedullin and CGRP are extremely potent vasodilators 5,6 and anti-cgrp agents are putative migraine targets. 7 9 Adrenomedullin is a potent angiogenic factor and AM gene-deficient mice are non-viable, likely due to impaired blood vessel formation. 10,11 Amylin is important for maintaining glycaemic control and an analogue of this peptide is now marketed for treating Type 1 and Type 2 diabetes. 12 Calcitonin is used in the treatment of osteoporosis. 13 Non-peptide therapeutics that selectively target individual receptors for these peptides would be useful in many conditions. To help develop these, it is important to understand the molecular pharmacology of the receptors for this peptide family. Many peptides, including the CGRP family, mediate their actions via G-protein-coupled receptors (GPCR). The GPCR form the largest family of cell-surface proteins that are capable of interacting with an extracellular stimulus and transducing that stimulus to produce a reaction inside a cell. 14 The GPCR are the target of approximately 50% of all drugs and respond to stimuli as diverse

2 964 DL Hay List of abbreviations: AM Adrenomedullin CTR Calcitonin receptor AMY Amylin receptor GPCR G-Protein-coupled receptor CGRP Calcitonin gene-related peptide RAMP Receptor activity modifying protein CGRP 1 Heterodimer of CL and RAMP1 RCP Receptor component protein CL Calcitonin receptor-like receptor Table 1 Agonist (pec 50 ) and antagonist (pa 2 /pk B ) potencies at cloned human calcitonin gene-related peptide, amylin, calcitonin and adrenomedullin receptors expressed in Cos-7 cells Receptor CGRP 1 54 AM 1 64 AM 2 CT (a) 51 AMY 1(a) 51 AMY 3(a) 51 Agonists (pec 50 ) hacgrp hbcgrp ham (Cys(Acm), 2,7 )hacgrp NS NS 7.46 NS (Cys(Et), 2,7 )hacgrp 9.09 < < 6 ramy Antagonists (pa 2 /pk B ) BIBN4096BS < 5 < 5 64 < CGRP < Weak partial agonist. In most studies, calcitonin gene-related peptide (CGRP) binds with approximately 100-fold lower affinity than adrenomedullin. 83 Values in bold are key comparisons between receptors of relevance to this review and Fig. 1. CT (a) denotes the insert negative form of the calcitonin receptor. References from which data are taken are noted next to the receptor name; values from other studies are indicated as appropriate. hacgrp, hbcgrp, human a and b calcitonin gene-related peptide, respectively; ham, human adrenomedullin; ramy, rat amylin; NS, no significant effect. as light, odours, small chemicals, peptides and large proteins. 14 Their molecular properties make them highly accessible to drugs and selective for specific stimuli, just two reasons why they are proven drug targets. Although all GPCR have seven transmembranespanning domains, an extracellular N-terminus and an intracellular C-terminus, they can be divided into several subclasses based on signature amino acid sequences. The Class 1 (rhodopsin) subfamily is the largest and generally binds small molecules and short peptides. Receptors in this class have been studied extensively, culminating recently in the elucidation of the crystal structure of rhodopsin. 15,16 The Class 2 (secretin) family receptors bind larger peptides in the range of 27 to approximately 50 amino acids and are much less understood. 17 This class of receptor mediates the actions of CGRP and related peptides. 18 Other examples of Class 2 receptors are the glucagon receptor, glucagon-like peptide 1 receptor and parathyroid hormone receptors. 19 Class 3 receptors include the metabotropic glutamate and GABA B receptors. The characterized GPCR for the CGRP family are the Class 2 receptors, the calcitonin receptor (CTR) and the calcitonin receptorlike receptor (CL). 18 There are several splice variants of CTR. 18 The most well-characterized splice variant of human CTR is positive (CT (b) ) or negative (CT (a) ) for a 16 amino acid insert in the first intracellular loop. The presence of the insert modifies G-protein coupling. There are no known splice variants of CL. Both CL and CTR are unusual receptors because peptide specificity is governed by receptor activity modifying proteins (RAMP) that modify the pharmacology and function of these GPCR. 18,20 The discovery of RAMP undermined the historical notion that the GPCR itself is sufficient for hormone/drug recognition and activity. Receptor activity modifying proteins form a unique family of three proteins, each with a single transmembrane-spanning domain, short intracellular C-terminal tail (approximately nine amino acids) and long extracellular N-terminus (approximately 100 amino acids). 20 They are quite different to GPCR. In addition to interacting with CL and CTR, there is now evidence that RAMP interact with a further four Class 2 GPCR, 21 as well as with members of Class 1 (N Tilakaratne et al., unpubl. data, 2002) and Class Several comprehensive reviews can be consulted for further information on RAMP As a result of CL and CTR interactions with RAMP, the International Union of Pharmacology (IUPHAR) nomenclature recognizes that CGRP interacts with CL/ RAMP1 (CGRP 1 ) receptors, whereas AM interacts with CL/ RAMP2 (AM 1 ) or CL/ RAMP3 (AM 2 ) receptors. The CTR without RAMP is sufficient for calcitonin binding, but CTR with RAMP1, 2 or 3 are amylin AMY 1, AMY 2 and AMY 3 receptors, respectively. 18 However, this is somewhat simplified and the receptors also interact with related peptides, in addition to their cognate ligands (Table 1). The purpose of the present review is to consider how the recent pharmacological delineation of these molecular entities may correspond to and potentially explain the pharmacological CGRP receptor subtypes that have been apparent for many years.

3 CGRP 2 receptors 965 HISTORICAL DEVELOPMENT OF CGRP RECEPTOR SUBTYPES Some of the earliest functions attributed to CGRP included stimulation of noradrenergic sympathetic outflow, 26 inhibition of gastric acid secretion 28,28 and the signature activity, vasodilation. 5 It was soon accepted that CGRP produced its effects by acting through specific receptors and radioligand binding studies have identified the presence of such receptors in the brain and many peripheral tissues. 29 The identification of the 8 37 fragment of CGRP (CGRP 8 37 ) as an inhibitor of human CGRP-induced activation of adenylate cyclase activity in rat liver plasma membranes signalled the arrival of the first useful tool for pharmacologically characterizing CGRP receptors. 30 It was this antagonist (plus some other C-terminal fragments of CGRP) and the linear agonist (Cys(Acm), 2,7 )hacgrp that first pointed towards the existence of CGRP receptor subtypes. 31,32 In these studies, some CGRP receptors (typified by those on the rat atria) were antagonized more effectively by the CGRP fragments than those on tissues such as the guinea-pig vas deferens. In contrast, (Cys(Acm), 2,7 )hacgrp was a more potent agonist in the vas deferens than the atria. These pivotal studies led to the subclassification of CGRP receptors into antagonist-sensitive receptors (designated CGRP 1 ) and antagonist-insensitive receptors (the CGRP 2 subtype). With greater activity in the vas deferens than atria, it was logical then to consider (Cys(Acm), 2,7 )hacgrp as a CGRP 2 receptor-selective agonist. This classification scheme has been fundamental to the study of CGRP receptors and has been reviewed extensively; these reviews should be consulted for more information on particular topics of interest. 18,33,34 The description of CGRP 1 and CGRP 2 receptor phenotypes has served those who study CGRP receptors well; however, it is questionable whether the CGRP 2 receptor is a single receptor type or whether it is, in fact, explained by multiple molecular entities. Studies over the years have shown that the CGRP antagonist CGRP 8 37 actually displays a marked range of affinity values (pa 2 /pk B ) in functional studies. 33,35 For example, recent examination of CGRP 8 37 pa 2 estimates from rat tissues revealed four groupings of values: (i) perfused blood vessels; (ii) isolated blood vessels/atrium; (iii) vas deferens/internal anal sphincter; and (iv) thoracic aorta/colon. 35 Whether these reflect real molecular differences in receptors has yet to be revealed; molecular candidates for CGRP receptors are discussed further below. One of the key issues in characterizing CGRP receptors is that although these subtypes appear to be obvious in functional studies, they are not revealed in competition binding assays. Affinity estimates for CGRP 8 37 in binding studies are similar in CGRP 1 - and CGRP 2 - expressing tissues For example, binding affinity values for CGRP 8 37 in membranes prepared from large porcine coronary arteries (historically considered to express CGRP 2 receptors) were almost identical to those in membranes prepared from HEK293 cells transfected with porcine CL (a CGRP 1 receptor component). 37 Various CGRP family and CGRP-derived peptides have been tested in binding studies at CGRP receptors, but the affinity profiles obtained do not appear to correlate with functionally identified CGRP receptor subtypes. Other peptide probes have been developed to try to distinguish CGRP receptor subtypes. In addition to CGRP 8 37 and (Cys(Acm), 2,7 )hacgrp, another popular tool used to differentiate CGRP receptors is (Cys(Et), 2,7 )acgrp. Replacement of the acetaminomethyl moiety in (Cys(Acm), 2,7 )hacgrp by an ethylamide group resulted in the compound (Cys(Et), 2,7 )acgrp, which has greater potency at inhibiting the rat vas deferens twitch response than (Cys(Acm), 2,7 ) hacgrp. 39 It was shown that (Cys(Et), 2,7 )acgrp produced only weak effects in the guinea-pig atria, consistent with earlier data for the putative CGRP 2 -selective agonist (Cys(Acm), 2,7 )hacgrp and, therefore, suggesting that (Cys(Et), 2,7 )acgrp may also be a selective CGRP 2 receptor agonist. 39 However, the robustness of both ligands as CGRP 2 agonists is debatable and this will be expanded upon through the course of the present article. By far the most selective pharmacological tool for studying CGRP-activated receptors is BIBN4096BS, a compound developed by Boehringer Ingelheim as a migraine therapy. 40 It is a very effective CGRP 1 receptor antagonist with selectivity for human over rodent receptors and for CGRP over AM and amylin receptors. 41,42 It is interesting to note that heterogeneity is also evident with this antagonist; BIBN4096BS was approximately 10-fold more effective at inhibiting CGRP responses in rat atria than vas deferens and there is also a small degree of separation in pk B values in different CGRPresponsive cell lines. 43,44 However, owing to the limited availability of this compound, the apparent heterogeneity has not been investigated extensively. NON-MOLECULAR EXPLANATIONS FOR CGRP RECEPTOR HETEROGENEITY There is certainly extensive evidence of heterogeneous CGRP pharmacology, but there is no clear explanation for it. Several hypotheses have been put forward. Species differences (rat atria vs guinea-pig vas deferens) were proposed as an explanation for initial observations of heterogeneity; however, a spread of pa 2 values for CGRP 8 37 has since been observed in tissues from the same species and in discrete cell lines. 33,43,45,46 A range of other possibilities for the heterogeneity has also been suggested, including experimental artefacts, differential metabolism of antagonists, the use of nonequilibrium conditions, problems of tissue accessibility and proteolysis. Such explanations probably do contribute; in one study in the rat vas deferens, the antagonistic effect of acgrp 8 37 was potentiated in the presence of the neutral endopeptidase inhibitor thiorphan (pa 2 increased from < 6 6.6). 47 In the case of the four clusters of CGRP 8 37 pa 2 values described above for rat tissues, it is likely that some of this can be explained by methodological differences. For example, receptors in perfused blood vessels may have better access to CGRP 8 37 than those on tissues in organ baths that rely on diffusion. Nevertheless, these possibilities certainly do not explain all instances of heterogeneity 35 and, in some studies, potential experimental artefacts, such as temperature and the effect of protease inhibitors, have been formally tested and ruled out The very nature of the commonly used pharmacological tools for studying CGRP receptors introduces several practical difficulties. For example, in studies where CGRP 8 37 is a weak antagonist, it is very difficult to achieve high enough concentrations of the peptide to perform a full Schild analysis. Single concentration information is useful, but it cannot be as precise as functional affinity estimates based on several antagonist concentrations. In addition, CGRP 8 37 seems to be rather difficult to work with; even in cells transfected with CGRP 1 receptors, it is not uncommon for pk B values to vary more than 10-fold across experiments (DL Hay, unpubl. obs., 2005). The linear agonists are the most problematic; (Cys(Acm), 2,7 ) hacgrp is

4 966 DL Hay a partial agonist, making any result obtained highly dependent on the coupling efficiency and receptor reserve of the tissue used at a given time, a well-recognized difficulty with trying to use agonists to characterize receptors. 51,52 This property also means that (Cys(Acm), 2,7 )hacgrp can be an effective antagonist under the right circumstances, although this has not been formally tested on many occasions. 53,54 Interestingly, (Cys(Et), 2,7 )hacgrp can behave as a potent agonist of molecularly defined CGRP 1 receptors (Table 1), which questions its previous status as a CGRP 2 -selective agonist. These factors, and the apparent inconsistencies that result, make data interpretation with respect to the activities of these ligands across studies difficult. Differential signalling offers another interesting perspective on CGRP receptor heterogeneity. In a study of the antagonistic effects of CGRP 8 37 on different CGRP-induced signalling pathways in MG63 cells, the actions of the antagonist were subtly different depending on the pathway stimulated by CGRP. 55 Thus, the pa 2 for antagonism of extracellular signal-regulated kinase (ERK) dephosphorylation at 8.1 was statistically lower than the value of 8.7, 8.8 and 8.8 for antagonism of CGRP-stimulated camp production, camp response element-binding protein (CREB) phosphorylation and p38 mitogen-activated protein kinase (MAPK) phosphorylation, respectively. 55 It will be interesting to see whether other studies report similar differences in the future. Although only a single report, articles of this nature epitomise how the intricacy of cellular signalling/ environment can influence receptor pharmacology. This has recently been reviewed comprehensively by Nelson and Challis. 56 Their description of other families of receptors that display distinct pharmacological phenotypes with respect to antagonists but for which molecular correlates have been difficult to define is enlightening. For example, the phenotypically characterized a 1L -adrenoceptor displays anomalously low functional affinity for the antagonist prazosin, reminiscent of the CGRP 2 phenomenon. Because no cdna corresponding to the a 1L -phenotype has been identified, other ways in which it may be generated have been proposed. For example, there is evidence that this receptor may be an alternative conformation of the a 1A -adrenoceptor, in common with the acceptance of the b 4 - adrenoceptor phenotype as an alternative state of the b 1 -adrenoceptor. Tissue- and cell-dependent pharmacology for a given receptor is relatively common and can result from subtle differences in the cellular milieu that we increasingly recognize as having an influence on antagonist (inverse agonist) as well as agonist pharmacology. 56 The interaction of distinct proteins with a given receptor has the potential to alter the confirmation of that receptor and, thus, the way in which it interacts with its ligands. Such possible explanations should not be neglected for understanding the nature of CGRP 2 receptors. MOLECULAR NATURE OF CGRP 1 RECEPTORS As described earlier, IUPHAR has defined the CGRP 1 receptor as a heterodimeric composite of CL and RAMP1 (Fig. 1). 18 Pharmacological characterization of these proteins, heterologously expressed in cell lines, certainly seems to match historic data for CGRP receptors expressed endogenously in cell lines such as rat L6 myocyte and human SK-N-MC neuroblastoma, gold standard cell lines for studying CGRP receptors. 20,54,57 These cell lines have been shown to express CL and RAMP1 in conjunction with CGRP 1 -like pharmacology. 58 Reported pa 2 values for CGRP 8 37 at RAMP1/CL complexes range from 7.5 to 9.3 and can thus be considered CGRP 8 37 sensitive (Table 1). 20,54,57 Supporting studies have been reviewed elsewhere. 18 Although generally supporting RAMP1/CL as CGRP 1 receptors, the story from tissues that express CL and RAMP1 mrna is not so straightforward; CGRP 8 37 is not always as effective as may be expected. For example, in human arteries that were shown to express both components, estimated antagonistic potencies (pk i ) of CGRP 8 37 ranged between 6.9 and 8, 59 straddling the arbitrary characterization boundary between CGRP 2 (< 7) and CGRP 1 (> 7). 33 That study 60 highlights the real difficultly in interpreting tissue studies in the face of the expression of multiple receptors that can interact with CGRP and its fragments (see below); the artery segments also expressed Fig. 1 Diagram showing the molecular composition of CGRP 1 receptors and receptors that may contribute to CGRP 2 pharmacology. CT (a) denotes the insert negative form of the calcitonin receptor. EC, extracellular; IC, intracellular; RAMP, receptor activity modifying protein; CL, calcitonin receptor-like receptor.

5 CGRP 2 receptors 967 RAMP2 and RAMP3 in varying amounts, potentially complicating the observed phenotype. Northern blot analysis of rat tissues confirmed that CGRP-responsive tissues contain CL and RAMP1, but frequently also RAMP2 and RAMP3, 60 reflecting the fact that tissues contain complex collections of cells that likely express more than one type of receptor that may interact with CGRP. These candidate receptors will be expanded upon further below. In fact, both SK-N-MC and L6 cells also express RAMP2 and, therefore, have the potential to express functional AM receptors in addition to their well-characterized CGRP 1 receptors. 58 There are actually very few examples where a single RAMP mrna has been reported in isolation. This is likely to be because RAMP also interact with other receptors and have a broader role. This serves to illustrate that there is likely to be complex regulation of the receptor subunits in tissues or cells that natively express them. In the absence of well-characterized tools for detecting protein expression of these subunits, it will remain unclear which of the detected mrna species in any given experiment mirror functional receptor complexes at the cell surface that generate an observed pharmacology. In 1996, a protein that conferred CGRP responsiveness in Xenopus oocytes was described. 61 This 148 amino acid protein was cloned from guinea-pig organ of Corti and does not share the classical seven transmembrane domains of the other receptors described for the CGRP family of peptides. 61 It has since been discovered that this protein, termed receptor component protein (RCP), does not form a binding site for CGRP but is a component of the CGRP receptor signalling system It was shown that RCP coimmunoprecipitated with CL, suggesting a direct interaction between the two proteins. In fact, RCP is now considered to be an integral component of the CGRP 1 receptor. 18 POTENTIAL MOLECULAR CORRELATES FOR CGRP 2 RECEPTORS Although CGRP 1 receptors now have a molecular correlate, the situation for CGRP 2 receptors is rather more ambiguous: CGRP 2 - like pharmacology has not been reproduced in pharmacological studies with CL and RAMP1. Initially, it was hoped that the colonic epithelial cell line Col-29 would serve as a means of identifying CGRP 2 receptors because this is the phenotype these cells had initially displayed. 45 However, later pharmacological analysis, paired with molecular studies, revealed CGRP 1 pharmacology along with expression of CL and RAMP1. 58 Relevant to the present review is evidence that combinations of CTR, CL and RAMP, in addition to CL/RAMP1, also have significant affinity for CGRP and its fragments. 18 This has given hope that these molecular entities may yet explain the CGRP 2 receptor. Adrenomedullin receptors In general, the AM 1 receptor has little affinity for CGRP and, so, is not considered to be a likely candidate for a CGRP 2 receptor; pa 2 values for CGRP 8 37 at this receptor are approximately Conversely, the AM 2 receptor, in rodents, shows reasonable CGRP binding, 65 a moderate pec 50 for acgrp (7.37) and has a pa 2 for CGRP 8 37 of below 7 (6.2; values from Cos-7 cells). 64 These are characteristics of a CGRP 2 receptor. 33,34 At human AM 2 receptors, there seems to be a greater separation between AM and CGRP 65,66 and CGRP 8 37 has a pa 2 of close to 7 (at least in Cos-7 cells). 64 These data suggest that AM 2 receptors could account for some of the observations of low CGRP 8 37 pa 2 values. Amylin receptors Recent studies using (Cys(Acm), 2,7 )acgrp and (Cys(Et), 2,7 )acgrp also pointed towards AMY receptors as potential contributors to CGRP 2 pharmacology; the AMY 1(a) receptor was sensitive to the stimulatory effects of these two linear analogues. 67 Further detailed pharmacological analysis of these receptors identified a spectrum of agonist and antagonist behaviour at AMY receptors that supports this hypothesis. 52 In particular, the AMY 1(a) receptor was potently activated by acgrp (pec ) but antagonized only weakly by CGRP 8 37 (pk B 6.6; Table 1). 52 In support of this, receptors responsive to amylin but weakly inhibited by CGRP 8 37 have been reported in the guinea-pig and rat vas deferens. 48,49 Although the activity of the linear agonists at AMY 1(a) receptors was used initially as supporting evidence that this receptor may contribute to observations of CGRP 2 receptor pharmacology, the selectivity of (Cys(Et), 2,7 )hacgrp as a specific CGRP 2 receptor agonist is questionable owing to potent activity at CGRP 1 receptors (e.g. Table 1). Therefore, this alone is not sufficient evidence to support the hypothesis. Conversely, as a competitive antagonist, CGRP 8 37 is less susceptible to the influence of the cellular environment and, therefore, data generated with this ligand provide more robust evidence that these receptors may explain CGRP 2 receptor pharmacology. The AMY 2 receptor has not yet been characterized extensively in functional studies, but in binding studies, the CGRP affinity for this receptor seems to be dependent on the calcitonin receptor splice variant expressed. 68 Indeed, these splice variants add a further layer of complexity; there are several of these 18 and the pharmacological consequences of their interactions with RAMP are little studied. More CTR splice variants, in conjunction with RAMP, could be CGRP responsive and contribute to observations of apparent CGRP 2 pharmacology. The AMY 3(a) receptor may also be modestly activated by acgrp (pec ) and weakly antagonized by CGRP 8 37 (pk B 6.2; Table 1). 52 There was no significant receptor stimulation by (Cys(Acm), 2,7 )hacgrp and (Cys(Et), 2,7 )hacgrp (as measured by camp) at AMY 3(a) receptors. 52 Conclusions On the basis of the ability to be activated by CGRP, coupled with weak antagonism by CGRP 8 37, AM 2, AMY 1(a) and AMY 3(a) receptors could all be considered as potential candidates for CGRP 2 receptors. The studies demonstrate how expressed receptors can show CGRP 2 - like pharmacology. Nevertheless, it is always difficult to correlate studies of this type with real situations in tissues. To support the cell culture studies, it is necessary to try to compare pharmacological profiles with receptor expression in prototypical CGRP 2 receptorexpressing tissues, such as the rat vas deferens. However, this is not that straightforward; commercially available RAMP antibodies, which are necessary to identify RAMP protein expression, do not behave consistently (DL Hay, unpubl. obs., 2005). It is worth noting that the rat vas deferens contains mrna for CL and all three RAMP proteins and so could functionally express both CGRP 1 and AM 2 receptors. 60 Interestingly, BIBN4096BS shows a range of affinities at each of the cloned CGRP-activated receptors and, due to

6 968 DL Hay its specificity for RAMP1-based receptors, is a very useful pharmacological tool (Table 1). 42 ARE THERE OTHER CGRP RECEPTOR SUBTYPES? There are two main issues for discussion here. The first is aspects of pharmacology that do not clearly fit within any current receptor classification scheme and the other is alternative molecular entities that have been reported as CGRP receptors. Pharmacology In terms of pharmacology, there is a handful of studies in which atypical activity of CGRP and related peptides has been observed, leading to speculation that alternative CGRP receptors exist. For example, in the rat colon, 69 only CGRP effectively stimulated ion transport; (Cys(Acm), 2,7 )hacgrp, (Cys(Et), 2,7 )hacgrp, amylin and AM were weak, suggesting that CGRP 1 (AM is a potent agonist at CGRP 1 receptors), AM 2 or AMY 1(a) receptors were not involved. Furthermore, the actions of CGRP were not inhibited by 1 mmol/l CGRP 8 37, but tetrodotoxin was able to block the weak stimulation produced by (Cys(Et), 2,7 )hacgrp (although not CGRP). 69 In the rat vas deferens, pk B values for BIBN4096BS against (Cys(Et), 2,7 )hacgrp and human AM were unexpectedly higher than against human or rat acgrp or human bcgrp. 43 In many cases, acgrp and bcgrp appear to act similarly, as they do at all of the cloned receptors that have been discussed in the present article (although, overall, bcgrp probably has slightly higher affinity). There are, however, circumstances where their effects can be differentiated to a greater extent. 70,71 For example, in bone marrow white cells, acgrp stimulated osteogenesis whereas bcgrp reportedly had no effect. 72 In a study using human extracranial arteries, CGRP 8 37 was more effective at inhibiting acgrp than bcgrp, although the difference was less than 10-fold. 73 In osteoblastic MG63 cells, the effects of (Cys(Acm), 2,7 )hacgrp were signalling pathway dependent, with (Cys(Acm), 2,7 )hacgrp being an agonist for ERK dephosphorylation but an antagonist of CGRP-stimulated camp production or CREB/p38 MAPK phosphorylation. 55 Although these observations are interesting, they currently stand as isolated reports. They need to be confirmed in independent studies so that their significance can be ascertained. Other molecular entities The first reported candidate CGRP receptor was a canine orphan seven transmembrane receptor known as RDC-1. When transfected into Cos-7 cells, RDC-1 yielded CGRP-specific camp responses that were effectively blocked by CGRP Adrenomedullin could also stimulate camp levels in cells transfected with RDC-1 with potency that may be expected for a CGRP receptor. 74 However, attempts to further characterize this protein as a CGRP receptor were unsuccessful. 20,75 Expression of RDC-1 in Xenopus oocytes and HEK293 cells did not change the cellular response to CGRP. 20 A description of correlations between CGRP receptor binding and proposed CGRP receptor molecular counterparts found no correlation between CGRP binding and RDC-1 mrna in rat tissues. 59 According to IUPHAR guidelines generated by the appropriate nomenclature subcommittee, the receptor is no longer considered to be a candidate CGRP receptor. 18 Interestingly, it was recently reported that the chemokine SDF-1/CXCL12 could bind to and signal through RDC In some studies, CL mrna was undetectable in cerebellum, leading to speculation that another molecular entity could be responsible for the high levels of CGRP binding present in this brain region. 77,78 In accordance with this, there have been reports of a CGRP-B receptor, based on the generation of a monoclonal antibody to cerebellar CGRP receptors. The antibody has been reported to detect a 66 kda protein that is different from CL. 78 This receptor has not been cloned or characterized pharmacologically and it is not known how it may relate to the CGRP 2 receptor, although the antibody did detect receptor expression in rat vas deferens as well as other rat tissues. 78 However, CL has been detected in rat cerebellum in another study and so still could account for CGRP receptors here. 59 There is also an indication in the patent literature that further sequences that encode CGRP receptors may exist. 79 Although these studies are interesting, these proteins are not yet sufficiently well characterized to be established as bona fide CGRP receptors. A further consideration is that other factors may interact with known receptor components to generate CGRP 2 receptors. As mentioned previously, the contribution of calcitonin receptor splice variants has not been explored. There may be an unknown cofactor or known receptors may simply act in some subtle way with RCP and/or G-proteins to modulate receptor phenotype (see above). 57 Receptor dimerization could also be a contributing factor. Further expansion on a recent report demonstrating phenotypic differences in amylin receptors coupled to different G-proteins is awaited with interest (M Morfis and PM Sexton, unpubl. data, 2005). RECOMMENDATIONS FOR DIFFERENTIATING CGRP RECEPTORS Because there are few selective tools for CGRP family receptors, they can best be differentiated using selected combinations of the agonists and antagonists that are available. When dealing with clonal cell lines and, therefore, a situation where multiple agonists and antagonists can be tested at a variety of concentrations, this is relatively straightforward. Blockade of CGRP activity by low concentrations of BIBN4096BS (Table 1), the only highly selective ligand, in such cells would essentially rule out AM and AMY receptors that contain RAMP2 or RAMP3 because BIBN4096BS sensitivity is largely governed by RAMP1. 41,42 Weak agonism by amylin and weak antagonism by salmon calcitonin 8 32 would then point towards the involvement of CGRP 1 over AMY 1 receptors. 52,54 The AM 2 receptors could be resolved from CGRP 1 receptors by weak antagonism by CGRP 8 37 coupled with equal potency of AM and AM and modest antagonism by AM ,64 However, in tissues, differentiating CGRP receptors is much more difficult given the likely multiplicity of receptors present and other limitations. Nonetheless, the careful use of combinations of the most selective agonists and antagonists (particularly BIBN4096BS) would be beneficial. Calcitonin gene-related peptide (8 37) is still considered a useful tool, in combination with other agents. Conversely, it is recommended that the linear CGRP agonists be used with great caution. The apparent CGRP 2 selectivity of (Cys(Et), 2,7 )hacgrp has been strongly

7 CGRP 2 receptors 969 disputed because it is an effective agonist of CGRP 1 receptors 55,58,80 and, as a partial agonist, the properties of (Cys(Acm), 2,7 )hacgrp are likely to be too system dependent to be useful. It is also advised that potency data for other agonists be compared cautiously across studies because the receptors appear to behave differently in different expression systems. This is particularly apparent for the amylin receptors that contain the insert positive form of CTR (CT (b) ). 68 With further study, the newly identified peptide AM 2 (intermedin) may be a useful addition in pharmacological studies. 81,82 As mentioned earlier, receptor component expression studies should be performed alongside pharmacology studies wherever possible to aid data interpretation. CONCLUSIONS There is no doubt that there are distinct populations of receptors that are responsive to CGRP. This is clearly seen in the wide range of reported pa 2 values for the antagonist CGRP Although differences across species, differential metabolism of antagonists, problems of tissue accessibility and proteolysis are all likely to contribute to this phenomenon, these do not account for all observations. Many cloned receptors that are responsive to CGRP have now been characterized pharmacologically and a range of CGRP 8 37 sensitivities are apparent. In particular, the AM 2 and AMY 1(a) receptors show CGRP 2 -like pharmacology. However, it is important to point out that most of the functional pharmacological characterization of cloned receptors with antagonists has been performed using human receptors. In contrast, most studies reporting CGRP receptor heterogeneity have been in non-human tissues. Therefore, receptor profiles generated for human receptors that support the hypothesis that CGRP 2 receptors may be accounted for by receptors such as AM 2 or AMY 1 need to be confirmed for rodent receptors. Interestingly, the CGRP 2 phenomenon is strikingly similar to that of a phenotypically characterized a 1L -adrenoceptor, which displays anomalously low functional affinity for the antagonist prazosin. 56 This and other examples of atypical pharmacology have been reviewed recently, 56 offering insight into many of the issues described for CGRP 2 receptors in the present article. Those studying CGRP receptors can learn from these examples and evaluate whether the approaches used to understand anomalous pharmacology in other receptor families could usefully be applied to CGRP 2 receptors. Researchers should be cautious with overinterpretation of small differences in pa 2 values, use multiple tools wherever possible and combine this with molecular analysis to try to grasp what CGRP 2 receptors really are. At this stage, it is prudent to question when it is appropriate to use the term CGRP 2 receptor. Because CGRP 2 receptor molecular structure cannot be defined, this phenotype is quite possibly explained by an amalgamation of contributions by AM 2, AMY 1 and other receptors. Therefore, the term should be only be used after careful consideration. ACKNOWLEDGEMENTS The author s work reported herein was supported by the New Zealand Lottery Health Commission and the Maurice and Phyllis Paykel Trust. The author thanks Dr David R Poyner (School of Life and Health Sciences, Aston University, Birmingham, UK) for his critique of this manuscript. REFERENCES 1. Amara SG, Jonas V, Rosenfeld MG, Ong ES, Evans RM. 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