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Regulation of signal transduction by calcitonin gene-related peptide receptors
Review
Regulation of signal transduction by calcitonin gene-related peptide receptors Christopher S Walker1, Alex C Conner2, David R Poyner3 and Debbie L Hay1 1
School of Biological Sciences, and Centre for Brain Research, University of Auckland, Auckland, New Zealand Warwick Medical School, University of Warwick, Coventry, United Kingdom 3 School of Life and Health Sciences, Aston University, Birmingham, United Kingdom 2
Calcitonin gene-related peptide (CGRP) plays a pivotal role in migraine, activating its cognate receptor to initiate intracellular signalling. This atypical receptor comprises a distinct assembly, made up of a G protein-coupled receptor (GPCR), a single transmembrane protein, and an additional protein that is required for Gas coupling. By altering the expression of individual receptor components, it might be possible to adjust cellular sensitivity to CGRP. In recognition of the increasing clinical significance of CGRP receptors, it is timely to review the signalling pathways that might be controlled by this receptor, how the activity of the receptor itself is regulated, and our current understanding of the molecular mechanisms involved in these processes. Like many GPCRs, the CGRP receptor appears to be promiscuous, potentially coupling to several G proteins and intracellular pathways. Their precise composition is likely to be cell type–dependent, and much work is needed to ascertain their physiological significance. Calcitonin gene-related peptide (CGRP) is a 37 amino acid vasodilatory neuropeptide. A little over a decade ago, the molecular identity of the CGRP receptor was confirmed. It was discovered that receptor activity-modifying protein (RAMP)1 was required for the G-protein-coupled receptor (GPCR) that is known as the calcitonin receptor-like receptor (CLR) to form a functional CGRP receptor. This receptor is a potential drug target for several diseases, including migraine [1] and cardiovascular disease [2]. Speculatively, drugs targeting the CGRP receptor system might also be beneficial for conditions such as flushing, pain, obesity and sepsis [1–4]. A detailed knowledge of CGRP receptor signalling and its regulation is essential to understanding the biological roles of CGRP and how they might be exploited for therapeutic benefit. Given the increasing volume of clinical data for CGRP receptor antagonists in migraine, we review the potential consequences of blocking the activation of this receptor. We examine recent advances describing the signalling, desensitisation and internalisation of the CGRP receptor, along with the molecular mechanisms that underlie these processes.
What is the CGRP receptor? Historically, two distinct subtypes of the CGRP receptor (CGRP1 and CGRP2) were described. However, the molecular and pharmacological characterisation of receptors for peptides closely related to CGRP [adrenomedullin (AM) and amylin] have rendered this classification obsolete [5]. The term ‘CGRP receptor’ is now used only for the combination of CLR and RAMP1 (Figure 1). For functionality, an additional protein called receptor component protein (RCP) is required. This atypical receptor configuration illustrates how the CGRP receptor adds to our growing appreciation of the ingenious mechanisms used by GPCRs to regulate their activities. CLR co-expression with RAMP2 or RAMP3 corresponds to AM1 or AM2 receptors, respectively, which both display some affinity for CGRP. The pharmacology of the CGRP receptor has been comprehensively defined in several species, displaying the highest affinity for CGRP, with significantly lower affinity for AM [5,6]. It should be noted that CGRP has high affinity for the human amylin subtype 1 (AMY1) receptor in heterologous expression systems [7], although this requires confirmation in other species. In principle, changes in Glossary of terms AM: adrenomedullin AMY1: amylin 1 receptor subtype CGRP: calcitonin gene-related peptide CLR: calcitonin receptor-like receptor CREB: cAMP response element binding protein CTR: calcitonin receptor ECE-1: endothelin-converting enzyme-1 GPCR: G protein-coupled receptor GRK: GPCR kinase ICL: intracellular loop JNK: c-Jun N-terminal kinase MAPK: mitogen-activated protein kinase NO: nitric oxide NOS: NO synthase Olcegepant: BIBN4096, 1-[N2-[3,5-dibromo-N-[[4-(3,4-dihydro-2(1H)-oxoquinazolin-3-yl)-1-piperidinyl]carbonyl]-D-tyrosyl]-l-lysyl]-4-(4-pyridinyl)piperazine PI3K: phosphoinositide 3-kinase PKA: protein kinase A PKB: protein kinase B PKC: protein kinase C PLC: phospholipase C PTX: pertussis toxin RCP: receptor component protein Telcagepant: MK-0974, [N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide]
Corresponding author: Hay, D.L. ([email protected]).
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0165-6147/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2010.06.006 Trends in Pharmacological Sciences 31 (2010) 476–483
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Figure 1. CGRP receptor components and important residues for receptor signalling and internalisation. The CGRP receptor is formed by CLR (blue), RAMP1 (yellow) and RCP (orange). Functionally important residues are shown as single letter abbreviations. Amino acid residues are numbered from the start of the predicted N-terminal signal peptide (Swiss-Prot Q16602). Several amino acids within the CLR C-terminus (N400-C436) and I312 at the ICL3/TM5 junction are required for effective CGRP-mediated internalisation. Important features of the CGRP receptor, including the TM6 ‘kink’ (P343) and the putative eighth helix (G388-W399) in CLR are illustrated. Abbreviations; C’, C-terminal; ECL, extracellular loop; ICL, intracellular loop; N’, N-terminal; TM, transmembrane.
the expression of CGRP-responsive receptors in disease, along with that of CGRP, could be physiologically relevant, even though CGRP potency might be lower at these receptors. Furthermore, the CGRP concentrations used in many studies are typically sufficient to activate multiple CGRPresponsive receptors, complicating interpretation. Therefore, it might not always be possible to definitively assign a biological function or signalling event to the CGRP receptor, even if the agonist is CGRP. CGRP receptor antagonists Recently, there has been considerable interest in the role of CGRP in migraine and the development of specific CGRP receptor antagonists for treating this disease. The first CGRP receptor antagonist was developed shortly after the identification of CGRP, when it was discovered that truncation of the first seven amino acids resulted in a peptide that acted as a competitive inhibitor of CGRP. Subsequently, CGRP8–37 has been used extensively to probe and elucidate the actions of CGRP. However, similar to its parent, this fragment displays affinity for related receptors. Several non-peptide CGRP receptor specific antagonists have now been developed, which appear to target the interface between CLR and RAMP1, making them more selective for the CGRP receptor than for the AM or AMY receptors [8–12]. These antagonists display significant species selectivity, being preferentialy selective for human rather than rodent receptors [13–15]. The best recognised of these antagonists, which display efficacy for treating migraine, are telcagepant (MK-0974; [N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5b]pyridin-1-yl)piperidine-1-carboxamide]) and olcegepant (BIBN4096; 1-[N2-[3,5-dibromo-N-[[4-(3,4-dihydro-2(1H) -oxoquinazolin-3-yl)-1-piperidinyl]carbonyl]-D-tyrosyl]-llysyl]-4-(4-pyridinyl)piperazine) [15,16]. Several mechan-
isms might contribute to their efficacy, including reductions in CGRP-mediated vasodilation in the cerebral vasculature, neurotransmission and release of inflammatory mediators [1]. These antagonists represent valuable tools for defining the signalling pathways activated specifically by the CGRP receptor. It is important to characterise these so that the clinical consequences of blocking CGRP receptor activity can be better understood. Molecular mechanisms of receptor activation and intracellular signalling Similar to other GPCRs, the binding of CGRP to its receptor is thought to facilitate conformational changes in the receptor that lead to downstream signalling. As other reviews have comprehensively described the literature surrounding the structure–function relationships of the CGRP receptor with respect to ligand binding [17], this section will concentrate on receptor regions of functional importance. We summarise the important structural motifs and amino acid residues in both CLR and RAMP1 that are reportedly responsible for G protein coupling, trafficking and internalisation. CLR CLR, which belongs to the B family of GPCRs, comprises the main functional unit of the CGRP receptor (Figure 1). Aside from its apparent obligate requirement for RAMP to allow CLR to bind peptide and signal, its activation is known to involve several crucial elements, in common with other GPCRs (Figure 1). These include the presence of a proline ‘kink’ in transmembrane helix (TM)6 similar to the rhodopsin-like family A GPCRs [18] and a potential ‘DRY’ motif equivalent around residues R173, H177 and E233 [19,20]. There is also evidence suggesting stabilisation of the CLR interaction with Gas by RCP [21]. Within the three intracellular loops (ICL) of CLR, there are several 477
Review proposed areas of importance for G protein interactions (Figure 1). These include basic arginine and lysine residues (R173 at the ICL1/TM2 interface and K249 of ICL2) in addition to lesser, possibly structural roles for several adjacent, hydrophobic residues including L169, V245, W246 and W254 [19,22]. It appears that both R173 and K249 have the potential for direct interactions with the G protein. A third basic residue (R336) in ICL3, which also appears to be important for receptor activation, might be involved in stabilising the active conformation; R336 and K333 are, putatively, part of a basic–xx–basic motif that is conserved across family A and B GPCRs. There is also an interesting potential Gas coupling motif in the proximal region of ICL3 comprising three hydrophobic residues (I312, L316 and L320), possibly forming an epitope as an intracellular extension of TM5 [22]. The C-terminus of CLR has been examined in two complementary studies as part of both the CGRP receptor [23] and the AM1 receptor [24]. Residues G388–W399 of CLR form a potential eighth helix (Figure 1), as observed in family A GPCRs [25], which is needed for cell-surface expression. Although the RAMP type (RAMP1 versus RAMP2), agonist (CGRP versus AM) type, and cell line used in these studies differed, there is broad agreement as to the regions involved in cell surface expression and internalisation (see below). Receptor modifications that affect internalisation might play a role in the regulation of signalling simply by altering the length of time that the receptor is at the cell surface. By examining a series of nested deletion mutants in the C-terminal of CLR, Kuwasako and coworkers revealed that a region corresponding to the putative eighth helix was important for coupling to Gas [24]. Pertussis toxin (PTX) sensitivity was used in an indirect assay to indicate potential regions of coupling to Gai. This showed a complicated pattern of activity; the authors identified residues E414–R417 as being particularly important, although some effects were seen with residues 388–404 [24]. Although our understanding of activation of the CGRP receptor in molecular detail is still limited, it is possible to suggest a general pattern of activation based around the ‘flowering’ model from the GPCR super-family as a whole. Receptor activation results in movement of the TM regions, changing the intracellular surface of the receptor and its contacts with G proteins. Further work is required to describe those specific contacts. RAMP1 RAMP1 is required for the translocation of CLR to the cell surface, and participates in ligand binding; it is therefore essential for CGRP receptor signalling [17,26]. It is known that overexpression of RAMP1 sensitises both vascular smooth muscle cells and trigeminal ganglia neurons to CGRP, and might be limiting for CGRP signalling [27,28]. Although the N-terminus of RAMP1 is important for both ligand binding and receptor trafficking, no clear roles for the short (approximately 10 amino acids) C-terminus of RAMP1 in CGRP receptor signalling have been identified. This region contains an endoplasmic reticulum retention signal [29], but C-terminal truncation of RAMP1 had no apparent effect on CGRP receptor function [30,31]. 478
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Nevertheless, RAMPs in other receptor complexes have been shown to modulate signalling, internalisation and trafficking [11]. RCP RCP is an intracellular peripheral membrane protein that associates with the CGRP receptor (Figure 1). RCP expression typically correlates with CGRP responsiveness in many tissues [32]. Knockdown of RCP expression reduced CGRP-mediated cAMP production, without affecting binding to the CGRP receptor or cAMP accumulation via adenosine or b2-adrenergic receptors [21]. Thus, RCP is likely to be essential for effective Gas coupling (see below) and might play a unique role in signal transduction via CLR. Whether RCP aids CGRP receptor coupling to Gas alone requires investigation. Regardless of this, RCP adds sophistication to the control of CGRP activity, and its relative expression might have important implications in diseases in which CGRP is involved. CGRP receptor-mediated signal transduction Activation of the CGRP receptor is generally accepted to result in Gas-mediated activation of adenylate cyclase, with a subsequent increase in cAMP and activation of protein kinase A (PKA) [33] (Figure 2a). However, like many GPCRs, it is expected that the CGRP receptor can couple to additional G proteins and other proteins, such as arrestins, to confer signalling. For the CGRP receptor, this includes Gaq/11-mediated phospholipase C (PLC)b1 activation, mitogen-activated protein kinase (MAPK) activation, and nitric oxide (NO) production, as well as a host of transcriptional events [2,34,35]. Cellular background is likely to be an important determinant of specific intracellular coupling and receptor phenotype for the CGRP receptor. For example, the complement of signalling proteins varies between cell types, and differential intracellular phosphorylation of several GPCRs is known to modulate tissue-specific signalling events [36]. Amylin receptors, which are closely related to the CGRP receptor, display differential receptor phenotypes when expressed in different cell lines [37], and G protein expression levels can influence amylin binding [38]. Gas coupled signal transduction The CGRP receptor-mediated cAMP response, downstream of Gas, is the best understood signal transduction pathway for CGRP. As a consequence, CGRP receptor function has commonly been characterised by measuring cAMP as a marker for receptor activation [6,8,9,21]. This has been particularly useful in cell lines that express the CGRP receptor (e.g. SK-N-MC) [33]. This pathway also seems important in more complex physiological systems, but confidently ascribing a CGRP-mediated signal transduction event specifically to the CGRP receptor has been more difficult. This is where the selective non-peptide CGRP receptor antagonists, as described above, are particularly valuable. Physiological functions and signalling events can now be unambiguously assigned to the CGRP receptor. This is evident from recent studies on cultured neurons, in which olcegepant blocked CGRP-stimulated cAMP accumulation [28,39] and PKA-dependent pain responses [40].
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Figure 2. CGRP receptor-mediated intracellular signalling. (a) Gas signalling increases AC (green) activity, elevating intracellular cAMP, activating PKA and subsequently many potential downstream effectors. (b) The CGRP receptor might also couple to Gai/o, reducing AC (red) activity, decreasing intracellular cAMP and reducing PKA activity. (c) CGRP signalling via Gaq activates PLC-b, which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into IP3 and diacylglycerol (DAG), resulting in elevated intracellular Ca2+ and PKCe activation. (d) The CGRP receptor might also utilise Ga-independent signalling, and Gbg- or b-arrestin-mediated signalling pathways. Arrows represent reported pathways; broken arrows represent potential or inferred pathways. AC, adenylate cyclase; ER, endoplasmic reticulum.
PKA has been implicated in numerous biological effects of CGRP, including vasodilation [2] and neural function [41], confirming the importance of Gas activation by CGRP receptors. PKA inhibitors such as H-89 have been extensively used to inhibit the actions of CGRP, including vascular and lymphatic dilation [42,43]. Studies have also confirmed the involvement of PKA, downstream of CGRP receptor activation, in regulating the activity of various downstream signalling components, including K+ channels [44], L-type Ca2+ channels [45] and cAMP response element binding protein (CREB) [46]. CREB expression can also be upregulated by CGRP [47]. Interestingly, PKA can also directly activate endothelial nitric oxide synthase (NOS), increasing nitric oxide (NO) production, which is essential for vasodilation [2]. Gai/o coupled signal transduction There is some evidence for Gai/o signalling by the CGRP receptor, which is traditionally identified by sensitivity to PTX (Figure 2b). The CGRP-mediated stimulation of Ca2+ transients in rat nodose neurons and the activation of cJun N-terminal kinase (JNK) in SK-N-MC cells (which express endogenous CGRP receptors) both displayed PTX sensitivity [48,49]. Interestingly, both these processes involve neural derived cells, in which Gai/o is abundantly expressed. Rat cardiac cells also displayed PTX sensitivity in CGRP-mediated activation of K+ channels [50]. Other studies using PTX in non-neural cell lines transfected with CGRP receptors have yielded mixed results. In HEK293
cells, CGRP stimulated PTX-insensitive Ca2+ transients and mitogen-activated protein kinase (MAPK) activation [extracellular signal regulated kinase (ERK)2 and p38] [51,52]. However, in Swiss 3T3 cells, and more recently in HEK293 cells, overexpressing CLR and RAMP2, PTX treatment resulted in potentiation of CGRP or AM-stimulated cAMP accumulation, respectively [24,53]. This could be interpreted as reduced CGRP receptor-mediated Gai activity, but it is also possible that the usual cellular balance of stimulatory and inhibitory processes is changed with PTX treatment, thus favouring stimulatory mechanisms via the CGRP receptor. Gaq/11 coupled signal transduction CGRP has been reported to activate several signalling components downstream of Gaq/11, including PLCb and protein kinase C (PKC) [34,54] (Figure 2c). Thus, Gaq/11coupling might explain some of the functions of CGRP observed in absence of cAMP accumulation. For instance, in osteoblastic OHS-4 cells, which express CLR, CGRP stimulated the PLCb1-dependent Ca2+ release from the endoplasmic reticulum, in the absence of cAMP accumulation [34,35]. However, calcitonin also induced Ca2+ transients comparably to CGRP, indicating the presence of a functional calcitonin receptor (CTR) that might also be activated by CGRP when RAMP1 is present; that is the AMY1 receptor. [7,35]. This receptor, expressed alone or with RAMPs, couples to Gaq/11, which can activate Ca2+ transients via PLC [38]. Therefore, it is unclear whether 479
Review CGRP-stimulated Gaq/11 signalling is driven by CLR or CTR in OHS-4 cells. The difficulty in firmly assigning a particular G protein to a given cellular response is not insignificant, given the interconnectivity of cellular signalling pathways. Thus, the Gaq/11 pathway is frequently studied in cellular backgrounds in which significant cAMP accumulation would also be expected in response to CGRP. For example, in HEK293 cells, CGRP stimulates cAMP accumulation, Ca2+ transients and inositol 1,4,5-triphosphate (IP3) accumulation, suggesting both Gas and Gaq/11 involvement [51]. Xenopus oocytes expressing CLR and RAMP1 apparently tested negative for Gaq/11 coupling [53], although this might reflect an inability of human CLR to couple Xenopus Gaq/11 effectively. Both Ca2+ transients and IP3 accumulation can be activated independently of Gaq/11, such as by Gas or Gbg, and therefore the extent to which CGRP receptors can couple to Gaq is still unclear [55]. Specific PKC inhibitors have shown that PKC activation, in conjunction with PKA, contributes to the effects of CGRP in controlling neuron function. CGRP has been shown to regulate mechanical hyperalgesia [56], P2X3 expression [57,58] and tetrodotoxin receptor Na+ channel activity [59] via both PKC and PKA. Compelling evidence for the involvement of PKC in CGRP signalling comes from alveolar epithelial cells, in which CGRP treatment protected epithelial cells from hypoxia-induced cell death. This was associated with the translocation of PKC epsilon (PKCe) to the cell membrane, and cell death could be prevented by the PKC inhibitor, calphostin C and the PKCe-specific inhibitor, PKCV1–2 [60]. Irrespective of whether activation of PKC occurs directly through Gaq/11 or through more convoluted mechanisms involving Gas or other pathways, this kinase appears to play important roles in CGRPmediated signal transduction in particular cell types. Ga-independent signalling pathways? Although GPCRs are best known for mediating their actions via the Ga subunits of G proteins, Gbg- and barrestin-coupled signal transduction is increasingly found [55,61] (Figure 2d). Fine-tuning of signalling can occur via a multitude of mechanisms, and therefore, in common with other GPCRs, CGRP receptor activation might elicit interactions that modulate many signals and receptor responses [55]. Gbg signalling Bioluminescence resonance energy transfer studies have shown that Gg subunits can associate with the CGRP receptor [62]. Beyond this, however, Gbg signalling via the CGRP receptor has not specifically been investigated. Gbg signalling is known to activate downstream effectors including ion channels and PLCb [55]. b-arrestin signalling Although no specific evidence for CGRP receptor-mediated signalling through b-arrestin has been described, b-arrestin associates with CGRP receptors and aids in CGRP receptor desensitisation and internalisation [62–64]. It is therefore plausible that barrestin could be involved in the activation of ERK1/2, p38, JNK and phosphoinositide 3-kinase (PI3K) by CGRP [61]. 480
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Protein kinase B/Akt Protein kinase B (PKB)/Akt might also be involved in CGRP-mediated signal transduction and biological functions, although the mechanisms regulating its activation are unclear. In endothelial cells, both CGRP and AM activate Akt via phosphorylation [65], and AM activates Akt in cultured adipocytes, potentially through CGRP receptors [66]. Similarly, in the dorsal horn, both AM and capsaicin (which releases CGRP and other neuropeptides) stimulated Akt activation, an effect that was blocked by the PI3K inhibitors wortmannin and LY294002 [67]. In a separate study, wortmannin blocked CGRP stimulated ERK2 activation, also implicating PI3K and Akt in CGRP signalling [52]. MAPK cascades The MAPK cascades represent major downstream effectors of CGRP; activation of ERK1/2, JNK and p38 have been observed in several studies, with outcomes ranging from cell proliferation [68,69] to regulation of neural function [70]. These might be activated by Ga-dependent or by alternative mechanisms. Interestingly, in CGRP-treated osteoblastic MG63 cells, the PKA inhibitor H-89 blocked CREB activation, partially blocked ERK activation, and had no effect on JNK activation, suggesting that both PKA dependent and independent pathways are involved [46]. CGRP-stimulated cellular proliferation in HaCat keratinocytes is associated with p38, ERK1/2 and JNK activation [68], and in lung alveolar epithelial cells, the ERK inhibitor U0126 effectively blocked CGRP-induced proliferation and ERK activation [69]. In cultured trigeminal ganglia glial cells, CGRP induced ERK1/2, JNK and p38 activation and subsequently increased inducible NOS expression and NO production [71]. Furthermore, MAPK expression can also be upregulated by CGRP in glial cells [47]. Chronic morphine tolerance and associated increases in p38 and ERK1/2 activation in the spinal cord are effectively blocked by olcegepant, providing good evidence for CGRP receptor involvement [70]. However, in a separate study, capsaicin-stimulated ERK activation in trigeminal ganglia neurons in vivo was not affected by olcegepant [72]. Summary of CGRP-receptor mediated signal transduction The potential intracellular consequences of CGRP receptor activation are numerous. Understanding these and their interactions in both health and disease might permit the development of novel therapeutics that target specific pathways [73]. To take full advantage of these processes, it is essential to determine how the CGRP receptor is itself regulated. Receptor regulation: desensitisation and internalisation To ensure continued GPCR responsiveness, receptor activation and intracellular signalling are carefully regulated. For GPCRs, although the processes vary in terms of mechanism and time scale, the connected processes of desensitisation, internalisation and recycling/degradation of receptors are typically involved [74] (Figure 3). For CLR, there has been some controversy; desensitisation of CLRmediated CGRP or AM responsiveness has been observed
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Figure 3. Agonist-mediated regulation of CGRP receptor function. The CGRP receptor, located at the plasma membrane (1), binds CGRP and is activated, facilitating intracellular signalling (2). The activated CGRP receptor is a target for GRKs, which phosphorylate serine/threonine residues in CLR, resulting in receptor desensitisation (other kinases e.g. PKA and PKC might also be involved) (3). b-Arrestin interacts with the phosphorylated CGRP receptor (4). The CGRP receptor/b-Arrestin complex and ECE-1 (red) might undergo clathrin/AP2/dynamin-dependent endocytosis (5), before the CGRP receptor is internalised (6) and trafficked to degradation (7) or recycling pathways (8). The CGRP receptor is trafficked to either the lysosomal pathway, where CLR and RAMP1 are degraded (7) or recycled via an endosome, where acidification facilitates ECE-1-mediated degradation of CGRP, promoting dissociation of b-arrestin and dephosphorylation (8). The CGRP receptor is resensitised and recycled back to the plasma membrane (9). AP2, adaptor protein 2; P, phosphate.
in some, but not all studies [33]. More recent work, including several mutagenesis studies in transfected cell lines, has begun to clarify the biology. Desensitisation Homologous desensitisation of CGRP signalling has been shown for several pathways, including cAMP in various cell types [33], Akt in endothelial cells [65], and Ca2+ transients in transfected HEK293 cells [75]. Protein kinases are known to play an important part in the desensitisation of many GPCRs, including the CGRP receptor, although it is not always clear which of these is involved. For example, it is still controversial as to whether PKA is involved in CGRP receptor desensitisation in SK-N-MC cells [33,71]. CLR appears to possess PKC phosphorylation sites [33], and there is some evidence of PKC involvement in CGRP receptor desensitisation using PKC inhibitors [76,77]. GPCR kinases (GRKs) initiate desensitisation by phosphorylating serine/threonine residues in activated receptors. This facilitates b-arrestin binding, reducing G protein-mediated signalling but potentially recruiting other signalling molecules [55,61]. In the case of CGRP receptors, the knockdown of GRK-6, but not GRK-2 or -5 was found to be important for the CGRP-mediated desensitisation of porcine CLR [77]. Interestingly, the overexpression of GRK-2, -3 or -4, but not GRK-5 or -6, in HEK293 cells expressing human CLR and RAMP2, increased internalisation efficiency [24]. As data are very limited and some discrepancies exist between studies, it remains to be established which GRKs are involved in CGRP receptor desensitisation. Internalisation and trafficking Following activation by CGRP, the CGRP receptor forms a complex with b-arrestin, which is essential for its dynamin/ clathrin dependent internalisation [62,63,75]. Several
regions of CLR are important for CGRP receptor internalisation. These include a conserved isoleucine residue (I312) at the ICL3/TM5 junction [19], the putative eighth helix (G388–W399), and a dispersed area including several serine/threonine residues (N400–L436) [23,24]. It follows that at least some of these regions are involved in receptor deactivation by GRKs and binding to barrestin. Recent studies in SK-N-MC cells have suggested that the duration of CGRP receptor stimulation has profound effects upon its fate. Following acute stimulation (1 hour), CGRP receptors were preferentially targeted to recycling pathways, whereas with chronic stimulation (8 hours), CGRP receptors were targeted for degradation via endosomes and lysosomes [78]. These results are in contrast to studies performed in HEK293 cells, in which overexpressed CLR was noted be inefficiently recycled, with the majority of CLR targeted to the lysosomes for degradation [75]. In both studies, acute stimulation was performed with 100 nM CGRP, but cell line-dependent factors, receptor detection methods and receptor expression levels could contribute to these apparent differences between studies. Further studies revealed the importance of endothelin-converting enzyme (ECE)-1. ECE-1 might internalise with CGRP-bound CGRP receptors, which in acidified endosomes degrade CGRP, resulting in destabilisation of the CGRP receptor/b-arrestin complex and resensitisation [64]. This process might also terminate b-arrestin-mediated ERK signalling [79]. Conclusion The increasing clinical significance of CGRP coupled with the unique molecular composition of the CGRP receptor makes understanding its signalling and regulation particularly important, but also a distinct challenge to comprehend. There is evidence for promiscuous CGRP 481
Review receptor signalling, through Gas, Gai/o, Gaq/11 and other pathways, although the evidence supporting Gas-independent coupling is currently limited. Controversy surrounding CGRP receptor regulation still exists. It is likely that differences in cellular background both explain and confound the data. Nevertheless, a picture of CGRP receptormediated signalling and regulation is beginning to emerge. With the advent of specific receptor antagonists, this picture can be refined, allowing the precise determination of the complex intracellular signalling networks activated by CGRP receptors. Defining these and the fate of activated CGRP receptors is essential for our understanding of CGRP biology, and its role in diseases such as migraine and hypertension. Once the signalling pathways important for CGRP action in specific disease states are known, molecules can be screened against these pathways, facilitating the development of pathway-selective drugs [73]. These could be more efficacious, with fewer off-target effects. It is important to focus on understanding CGRP receptor signalling and regulation in primary cell cultures and in vivo models at physiological expression levels of receptor. Of particular interest will be how CGRP receptor signalling and regulation change in disease states. Disclosure No author declares any conflict of interest. References 1 Doods, H. et al. (2007) CGRP antagonists: unravelling the role of CGRP in migraine. Trends Pharmacol. Sci. 28, 580–587 2 Brain, S.D. and Grant, A.D. (2004) Vascular actions of calcitonin generelated peptide and adrenomedullin. Physiol. Rev. 84, 903–934 3 Hay, D.L. and Poyner, D.R. (2009) Calcitonin gene-related peptide, adrenomedullin and flushing. Maturitas 64, 104–108 4 Danaher, R.N. et al. (2008) Evidence that alpha-calcitonin gene-related peptide is a neurohormone that controls systemic lipid availability and utilization. Endocrinology 149, 154–160 5 Hay, D.L. et al. (2008) International Union of Pharmacology. LXIX. Status of the calcitonin gene-related peptide subtype 2 receptor. Pharmacol. Rev. 60, 143–145 6 Bailey, R.J. and Hay, D.L. (2006) Pharmacology of the human CGRP1 receptor in Cos 7 cells. Peptides 27, 1367–1375 7 Hay, D.L. et al. (2005) Pharmacological discrimination of calcitonin receptor: receptor activity-modifying protein complexes. Mol. Pharmacol. 67, 1655–1665 8 Hay, D.L. et al. (2003) CL/RAMP2 and CL/RAMP3 produce pharmacologically distinct adrenomedullin receptors: a comparison of effects of adrenomedullin22-52, CGRP8-37 and BIBN4096BS. Br. J. Pharmacol. 140, 477–486 9 Hay, D.L. et al. (2006) Determinants of 1-piperidinecarboxamide, N-[2[[5-amino-l-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1[(3,5-dibromo-4 hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2oxo-3(2H)-quinazolinyl) (BIBN4096BS) affinity for calcitonin generelated peptide and amylin receptors—the role of receptor activity modifying protein 1. Mol. Pharmacol. 70, 1984–1991 10 Salvatore, C.A. et al. (2006) Identification and pharmacological characterization of domains involved in binding of CGRP receptor antagonists to the calcitonin-like receptor. Biochemistry 45, 1881–1887 11 Sexton, P.M. et al. (2009) Modulating receptor function through RAMPs: can they represent drug targets in themselves? Drug Discov. Today 14, 413–419 12 Miller, P.S. et al. (2010) Non-peptidic antagonists of the CGRP receptor, BIBN4096BS and MK-0974, interact with the calcitonin receptor-like receptor via methionine-42 and RAMP1 via tryptophan-74. Biochem. Biophys. Res. Commun. 391, 437–442 13 Mallee, J.J. et al. (2002) Receptor activity-modifying protein 1 determines the species selectivity of non-peptide CGRP receptor antagonists. J. Biol. Chem. 277, 14294–14298
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formation in CGRP receptor-positive osteosarcoma cells (OHS-4). Cytokine 11, 200–207 Tobin, A.B. et al. (2008) Location, location, location: site-specific GPCR phosphorylation offers a mechanism for cell-type-specific signalling. Trends Pharmacol. Sci. 29, 413–420 Tilakaratne, N. et al. (2000) Amylin receptor phenotypes derived from human calcitonin receptor/RAMP coexpression exhibit pharmacological differences dependent on receptor isoform and host cell environment. J. Pharmacol. Exp. Ther. 294, 61–72 Morfis, M. et al. (2008) Receptor activity-modifying proteins differentially modulate the G protein-coupling efficiency of amylin receptors. Endocrinology 149, 5423–5431 Takhshid, M.A. et al. (2006) Characterization and effects on cAMP accumulation of adrenomedullin and calcitonin gene-related peptide (CGRP) receptors in dissociated rat spinal cord cell culture. Br. J. Pharmacol. 148, 459–468 Han, J.S. et al. (2005) Critical role of calcitonin gene-related peptide 1 receptors in the amygdala in synaptic plasticity and pain behavior. J. Neurosci. 25, 10717–10728 Han, J.S. et al. (2010) Facilitation of synaptic transmission and pain responses by CGRP in the amygdala of normal rats. Mol. Pain 6, 10 Champion, H.C. et al. (2003) Responses to human CGRP, ADM, and PAMP in human thymic arteries. Am. J. Physiol. Regul. Integr. Comp. Physiol. 284, R531–537 Hosaka, K. et al. (2006) Calcitonin gene-related peptide activates different signaling pathways in mesenteric lymphatics of guinea pigs. Am. J. Physiol. Heart Circ. Physiol. 290, H813–822 Wellman, G.C. et al. (1998) ATP-sensitive K+ channel activation by calcitonin gene-related peptide and protein kinase A in pig coronary arterial smooth muscle. J. Physiol. 507 (Pt 1), 117–129 Hosokawa, S. et al. (2010) Calcitonin gene-related peptide- and adrenomedullin-induced facilitation of calcium current by different signal pathways in nucleus tractus solitarius. Brain Res. 1327, 47–55 Kawase, T. et al. (2005) Immature osteoblastic MG63 cells possess two calcitonin gene-related peptide receptor subtypes that respond differently to [Cys(Acm)(2,7)] calcitonin gene-related peptide and CGRP(8-37). Am. J. Physiol. Cell Physiol. 289, C811–818 Vause, C.V. and Durham, P.L. (2010) Calcitonin gene-related peptide differentially regulates gene and protein expression in trigeminal glia cells: findings from array analysis. Neurosci. Lett. 473, 163–167 Wiley, J.W. et al. (1992) The peptide CGRP increases a high-threshold Ca2+ current in rat nodose neurones via a pertussis toxin-sensitive pathway. J. Physiol. 455, 367–381 Disa, J. et al. (2000) Involvement of cAMP-dependent protein kinase and pertussis toxin-sensitive G-proteins in CGRP mediated JNK activation in human neuroblastoma cell line. Neuropeptides 34, 229– 233 Kim, D. (1991) Calcitonin-gene-related peptide activates the muscarinic-gated K+ current in atrial cells. Pflugers Arch. 418, 338– 345 Aiyar, N. et al. (1999) Calcitonin gene-related peptide receptor independently stimulates 3’,5’-cyclic adenosine monophosphate and Ca2+ signaling pathways. Mol. Cell Biochem. 197, 179–185 Parameswaran, N. et al. (2000) Activation of multiple mitogenactivated protein kinases by recombinant calcitonin gene-related peptide receptor. Eur. J. Pharmacol. 389, 125–130 Main, M.J. et al. (1998) The CGRP receptor can couple via pertussis toxin sensitive and insensitive G proteins. FEBS Lett. 441, 6–10 Morara, S. et al. (2008) Calcitonin gene-related peptide (CGRP) triggers Ca2+ responses in cultured astrocytes and in Bergmann glial cells from cerebellar slices. Eur. J. Neurosci. 28, 2213–2220 Ritter, S.L. and Hall, R.A. (2009) Fine-tuning of GPCR activity by receptor-interacting proteins. Nat. Rev. Mol. Cell Biol. 10, 819–830 Sun, R.Q. et al. (2004) Calcitonin gene-related peptide receptor activation produces PKA- and PKC-dependent mechanical hyperalgesia and central sensitization. J. Neurophysiol. 92, 2859–2866
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57 Fabbretti, E. et al. (2006) Delayed upregulation of ATP P2X3 receptors of trigeminal sensory neurons by calcitonin gene-related peptide. J. Neurosci. 26, 6163–6171 58 Simonetti, M. et al. (2008) Mechanisms mediating the enhanced gene transcription of P2X3 receptor by calcitonin gene-related peptide in trigeminal sensory neurons. J. Biol. Chem. 283, 18743–18752 59 Natura, G. et al. (2005) Calcitonin gene-related peptide enhances TTXresistant sodium currents in cultured dorsal root ganglion neurons from adult rats. Pain 116, 194–204 60 Wang, W. et al. (2005) Endogenous calcitonin gene-related peptide protects human alveolar epithelial cells through protein kinase C epsilon and heat shock protein. J. Biol. Chem. 280, 20325–20330 61 De Fea, K. (2008) Beta-arrestins and heterotrimeric G-proteins: collaborators and competitors in signal transduction. Br. J. Pharmacol. 153 (Suppl 1), S298–309 62 Heroux, M. et al. (2007) Assembly and signaling of CRLR and RAMP1 complexes assessed by BRET. Biochemistry 46, 7022–7033 63 Hilairet, S. et al. (2001) Agonist-promoted internalization of a ternary complex between calcitonin receptor-like receptor, receptor activitymodifying protein 1 (RAMP1), and beta-arrestin. J. Biol. Chem. 276, 42182–42190 64 Padilla, B.E. et al. (2007) Endothelin-converting enzyme-1 regulates endosomal sorting of calcitonin receptor-like receptor and betaarrestins. J. Cell Biol. 179, 981–997 65 Nikitenko, L.L. et al. (2006) Adrenomedullin and CGRP interact with endogenous calcitonin-receptor-like receptor in endothelial cells and induce its desensitisation by different mechanisms. J. Cell Sci. 119, 910–922 66 Iemura-Inaba, C. et al. (2008) Role of adrenomedullin system in lipid metabolism and its signaling mechanism in cultured adipocytes. Am. J. Physiol. Regul. Integr. Comp. Physiol. 295, R1376–1384 67 Ma, W. et al. (2006) A role for adrenomedullin as a pain-related peptide in the rat. Proc. Natl. Acad. Sci. U. S. A. 103, 16027–16032 68 Yu, X.J. et al. (2009) Calcitonin gene-related peptide increases proliferation of human HaCaT keratinocytes by activation of MAP kinases. Cell Biol. Int. 33, 1144–1148 69 Kawanami, Y. et al. (2009) Calcitonin gene-related peptide stimulates proliferation of alveolar epithelial cells. Respir. Res. 10, 8 70 Wang, Z. et al. (2009) Cell-type specific activation of p38 and ERK mediates calcitonin gene-related peptide involvement in tolerance to morphine-induced analgesia. FASEB. J. 23, 2576–2586 71 Vause, C.V. and Durham, P.L. (2009) CGRP stimulation of iNOS and NO release from trigeminal ganglion glial cells involves mitogenactivated protein kinase pathways. J. Neurochem. 110, 811–821 72 Sixt, M.L. et al. (2009) Calcitonin gene-related peptide receptor antagonist olcegepant acts in the spinal trigeminal nucleus. Brain 132, 3134–3141 73 Kenakin, T.P. (2009) Cellular assays as portals to seventransmembrane receptor-based drug discovery. Nat. Rev. Drug Discov. 8, 617–626 74 Kelly, E. et al. (2008) Agonist-selective mechanisms of GPCR desensitization. Br. J. Pharmacol. 153 (Suppl 1), S379–388 75 Kuwasako, K. et al. (2000) Visualization of the calcitonin receptor-like receptor and its receptor activity-modifying proteins during internalization and recycling. J. Biol. Chem. 275, 29602–29609 76 Pin, S.S. and Bahr, B.A. (2008) Protein kinase C is a common component of CGRP receptor desensitization induced by distinct agonists. Eur. J. Pharmacol. 587, 8–15 77 Aiyar, N. et al. (2000) Involvement of G protein-coupled receptor kinase-6 in desensitization of CGRP receptors. Eur. J. Pharmacol. 403, 1–7 78 Cottrell, G.S. et al. (2007) Post-endocytic sorting of calcitonin receptorlike receptor and receptor activity-modifying protein 1. J. Biol. Chem. 282, 12260–12271 79 Cottrell, G.S. et al. (2009) Endosomal endothelin-converting enzyme-1: a regulator of beta-arrestin-dependent ERK signaling. J. Biol. Chem. 284, 22411–22425
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Regulation of signal transduction by calcitonin gene-related peptide receptors Christopher S Walker1, Alex C Conner2, David R Poyner3 and Debbie L Hay1 1
School of Biological Sciences, and Centre for Brain Research, University of Auckland, Auckland, New Zealand Warwick Medical School, University of Warwick, Coventry, United Kingdom 3 School of Life and Health Sciences, Aston University, Birmingham, United Kingdom 2
Calcitonin gene-related peptide (CGRP) plays a pivotal role in migraine, activating its cognate receptor to initiate intracellular signalling. This atypical receptor comprises a distinct assembly, made up of a G protein-coupled receptor (GPCR), a single transmembrane protein, and an additional protein that is required for Gas coupling. By altering the expression of individual receptor components, it might be possible to adjust cellular sensitivity to CGRP. In recognition of the increasing clinical significance of CGRP receptors, it is timely to review the signalling pathways that might be controlled by this receptor, how the activity of the receptor itself is regulated, and our current understanding of the molecular mechanisms involved in these processes. Like many GPCRs, the CGRP receptor appears to be promiscuous, potentially coupling to several G proteins and intracellular pathways. Their precise composition is likely to be cell type–dependent, and much work is needed to ascertain their physiological significance. Calcitonin gene-related peptide (CGRP) is a 37 amino acid vasodilatory neuropeptide. A little over a decade ago, the molecular identity of the CGRP receptor was confirmed. It was discovered that receptor activity-modifying protein (RAMP)1 was required for the G-protein-coupled receptor (GPCR) that is known as the calcitonin receptor-like receptor (CLR) to form a functional CGRP receptor. This receptor is a potential drug target for several diseases, including migraine [1] and cardiovascular disease [2]. Speculatively, drugs targeting the CGRP receptor system might also be beneficial for conditions such as flushing, pain, obesity and sepsis [1–4]. A detailed knowledge of CGRP receptor signalling and its regulation is essential to understanding the biological roles of CGRP and how they might be exploited for therapeutic benefit. Given the increasing volume of clinical data for CGRP receptor antagonists in migraine, we review the potential consequences of blocking the activation of this receptor. We examine recent advances describing the signalling, desensitisation and internalisation of the CGRP receptor, along with the molecular mechanisms that underlie these processes.
What is the CGRP receptor? Historically, two distinct subtypes of the CGRP receptor (CGRP1 and CGRP2) were described. However, the molecular and pharmacological characterisation of receptors for peptides closely related to CGRP [adrenomedullin (AM) and amylin] have rendered this classification obsolete [5]. The term ‘CGRP receptor’ is now used only for the combination of CLR and RAMP1 (Figure 1). For functionality, an additional protein called receptor component protein (RCP) is required. This atypical receptor configuration illustrates how the CGRP receptor adds to our growing appreciation of the ingenious mechanisms used by GPCRs to regulate their activities. CLR co-expression with RAMP2 or RAMP3 corresponds to AM1 or AM2 receptors, respectively, which both display some affinity for CGRP. The pharmacology of the CGRP receptor has been comprehensively defined in several species, displaying the highest affinity for CGRP, with significantly lower affinity for AM [5,6]. It should be noted that CGRP has high affinity for the human amylin subtype 1 (AMY1) receptor in heterologous expression systems [7], although this requires confirmation in other species. In principle, changes in Glossary of terms AM: adrenomedullin AMY1: amylin 1 receptor subtype CGRP: calcitonin gene-related peptide CLR: calcitonin receptor-like receptor CREB: cAMP response element binding protein CTR: calcitonin receptor ECE-1: endothelin-converting enzyme-1 GPCR: G protein-coupled receptor GRK: GPCR kinase ICL: intracellular loop JNK: c-Jun N-terminal kinase MAPK: mitogen-activated protein kinase NO: nitric oxide NOS: NO synthase Olcegepant: BIBN4096, 1-[N2-[3,5-dibromo-N-[[4-(3,4-dihydro-2(1H)-oxoquinazolin-3-yl)-1-piperidinyl]carbonyl]-D-tyrosyl]-l-lysyl]-4-(4-pyridinyl)piperazine PI3K: phosphoinositide 3-kinase PKA: protein kinase A PKB: protein kinase B PKC: protein kinase C PLC: phospholipase C PTX: pertussis toxin RCP: receptor component protein Telcagepant: MK-0974, [N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide]
Corresponding author: Hay, D.L. ([email protected]).
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0165-6147/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2010.06.006 Trends in Pharmacological Sciences 31 (2010) 476–483
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Figure 1. CGRP receptor components and important residues for receptor signalling and internalisation. The CGRP receptor is formed by CLR (blue), RAMP1 (yellow) and RCP (orange). Functionally important residues are shown as single letter abbreviations. Amino acid residues are numbered from the start of the predicted N-terminal signal peptide (Swiss-Prot Q16602). Several amino acids within the CLR C-terminus (N400-C436) and I312 at the ICL3/TM5 junction are required for effective CGRP-mediated internalisation. Important features of the CGRP receptor, including the TM6 ‘kink’ (P343) and the putative eighth helix (G388-W399) in CLR are illustrated. Abbreviations; C’, C-terminal; ECL, extracellular loop; ICL, intracellular loop; N’, N-terminal; TM, transmembrane.
the expression of CGRP-responsive receptors in disease, along with that of CGRP, could be physiologically relevant, even though CGRP potency might be lower at these receptors. Furthermore, the CGRP concentrations used in many studies are typically sufficient to activate multiple CGRPresponsive receptors, complicating interpretation. Therefore, it might not always be possible to definitively assign a biological function or signalling event to the CGRP receptor, even if the agonist is CGRP. CGRP receptor antagonists Recently, there has been considerable interest in the role of CGRP in migraine and the development of specific CGRP receptor antagonists for treating this disease. The first CGRP receptor antagonist was developed shortly after the identification of CGRP, when it was discovered that truncation of the first seven amino acids resulted in a peptide that acted as a competitive inhibitor of CGRP. Subsequently, CGRP8–37 has been used extensively to probe and elucidate the actions of CGRP. However, similar to its parent, this fragment displays affinity for related receptors. Several non-peptide CGRP receptor specific antagonists have now been developed, which appear to target the interface between CLR and RAMP1, making them more selective for the CGRP receptor than for the AM or AMY receptors [8–12]. These antagonists display significant species selectivity, being preferentialy selective for human rather than rodent receptors [13–15]. The best recognised of these antagonists, which display efficacy for treating migraine, are telcagepant (MK-0974; [N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5b]pyridin-1-yl)piperidine-1-carboxamide]) and olcegepant (BIBN4096; 1-[N2-[3,5-dibromo-N-[[4-(3,4-dihydro-2(1H) -oxoquinazolin-3-yl)-1-piperidinyl]carbonyl]-D-tyrosyl]-llysyl]-4-(4-pyridinyl)piperazine) [15,16]. Several mechan-
isms might contribute to their efficacy, including reductions in CGRP-mediated vasodilation in the cerebral vasculature, neurotransmission and release of inflammatory mediators [1]. These antagonists represent valuable tools for defining the signalling pathways activated specifically by the CGRP receptor. It is important to characterise these so that the clinical consequences of blocking CGRP receptor activity can be better understood. Molecular mechanisms of receptor activation and intracellular signalling Similar to other GPCRs, the binding of CGRP to its receptor is thought to facilitate conformational changes in the receptor that lead to downstream signalling. As other reviews have comprehensively described the literature surrounding the structure–function relationships of the CGRP receptor with respect to ligand binding [17], this section will concentrate on receptor regions of functional importance. We summarise the important structural motifs and amino acid residues in both CLR and RAMP1 that are reportedly responsible for G protein coupling, trafficking and internalisation. CLR CLR, which belongs to the B family of GPCRs, comprises the main functional unit of the CGRP receptor (Figure 1). Aside from its apparent obligate requirement for RAMP to allow CLR to bind peptide and signal, its activation is known to involve several crucial elements, in common with other GPCRs (Figure 1). These include the presence of a proline ‘kink’ in transmembrane helix (TM)6 similar to the rhodopsin-like family A GPCRs [18] and a potential ‘DRY’ motif equivalent around residues R173, H177 and E233 [19,20]. There is also evidence suggesting stabilisation of the CLR interaction with Gas by RCP [21]. Within the three intracellular loops (ICL) of CLR, there are several 477
Review proposed areas of importance for G protein interactions (Figure 1). These include basic arginine and lysine residues (R173 at the ICL1/TM2 interface and K249 of ICL2) in addition to lesser, possibly structural roles for several adjacent, hydrophobic residues including L169, V245, W246 and W254 [19,22]. It appears that both R173 and K249 have the potential for direct interactions with the G protein. A third basic residue (R336) in ICL3, which also appears to be important for receptor activation, might be involved in stabilising the active conformation; R336 and K333 are, putatively, part of a basic–xx–basic motif that is conserved across family A and B GPCRs. There is also an interesting potential Gas coupling motif in the proximal region of ICL3 comprising three hydrophobic residues (I312, L316 and L320), possibly forming an epitope as an intracellular extension of TM5 [22]. The C-terminus of CLR has been examined in two complementary studies as part of both the CGRP receptor [23] and the AM1 receptor [24]. Residues G388–W399 of CLR form a potential eighth helix (Figure 1), as observed in family A GPCRs [25], which is needed for cell-surface expression. Although the RAMP type (RAMP1 versus RAMP2), agonist (CGRP versus AM) type, and cell line used in these studies differed, there is broad agreement as to the regions involved in cell surface expression and internalisation (see below). Receptor modifications that affect internalisation might play a role in the regulation of signalling simply by altering the length of time that the receptor is at the cell surface. By examining a series of nested deletion mutants in the C-terminal of CLR, Kuwasako and coworkers revealed that a region corresponding to the putative eighth helix was important for coupling to Gas [24]. Pertussis toxin (PTX) sensitivity was used in an indirect assay to indicate potential regions of coupling to Gai. This showed a complicated pattern of activity; the authors identified residues E414–R417 as being particularly important, although some effects were seen with residues 388–404 [24]. Although our understanding of activation of the CGRP receptor in molecular detail is still limited, it is possible to suggest a general pattern of activation based around the ‘flowering’ model from the GPCR super-family as a whole. Receptor activation results in movement of the TM regions, changing the intracellular surface of the receptor and its contacts with G proteins. Further work is required to describe those specific contacts. RAMP1 RAMP1 is required for the translocation of CLR to the cell surface, and participates in ligand binding; it is therefore essential for CGRP receptor signalling [17,26]. It is known that overexpression of RAMP1 sensitises both vascular smooth muscle cells and trigeminal ganglia neurons to CGRP, and might be limiting for CGRP signalling [27,28]. Although the N-terminus of RAMP1 is important for both ligand binding and receptor trafficking, no clear roles for the short (approximately 10 amino acids) C-terminus of RAMP1 in CGRP receptor signalling have been identified. This region contains an endoplasmic reticulum retention signal [29], but C-terminal truncation of RAMP1 had no apparent effect on CGRP receptor function [30,31]. 478
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Nevertheless, RAMPs in other receptor complexes have been shown to modulate signalling, internalisation and trafficking [11]. RCP RCP is an intracellular peripheral membrane protein that associates with the CGRP receptor (Figure 1). RCP expression typically correlates with CGRP responsiveness in many tissues [32]. Knockdown of RCP expression reduced CGRP-mediated cAMP production, without affecting binding to the CGRP receptor or cAMP accumulation via adenosine or b2-adrenergic receptors [21]. Thus, RCP is likely to be essential for effective Gas coupling (see below) and might play a unique role in signal transduction via CLR. Whether RCP aids CGRP receptor coupling to Gas alone requires investigation. Regardless of this, RCP adds sophistication to the control of CGRP activity, and its relative expression might have important implications in diseases in which CGRP is involved. CGRP receptor-mediated signal transduction Activation of the CGRP receptor is generally accepted to result in Gas-mediated activation of adenylate cyclase, with a subsequent increase in cAMP and activation of protein kinase A (PKA) [33] (Figure 2a). However, like many GPCRs, it is expected that the CGRP receptor can couple to additional G proteins and other proteins, such as arrestins, to confer signalling. For the CGRP receptor, this includes Gaq/11-mediated phospholipase C (PLC)b1 activation, mitogen-activated protein kinase (MAPK) activation, and nitric oxide (NO) production, as well as a host of transcriptional events [2,34,35]. Cellular background is likely to be an important determinant of specific intracellular coupling and receptor phenotype for the CGRP receptor. For example, the complement of signalling proteins varies between cell types, and differential intracellular phosphorylation of several GPCRs is known to modulate tissue-specific signalling events [36]. Amylin receptors, which are closely related to the CGRP receptor, display differential receptor phenotypes when expressed in different cell lines [37], and G protein expression levels can influence amylin binding [38]. Gas coupled signal transduction The CGRP receptor-mediated cAMP response, downstream of Gas, is the best understood signal transduction pathway for CGRP. As a consequence, CGRP receptor function has commonly been characterised by measuring cAMP as a marker for receptor activation [6,8,9,21]. This has been particularly useful in cell lines that express the CGRP receptor (e.g. SK-N-MC) [33]. This pathway also seems important in more complex physiological systems, but confidently ascribing a CGRP-mediated signal transduction event specifically to the CGRP receptor has been more difficult. This is where the selective non-peptide CGRP receptor antagonists, as described above, are particularly valuable. Physiological functions and signalling events can now be unambiguously assigned to the CGRP receptor. This is evident from recent studies on cultured neurons, in which olcegepant blocked CGRP-stimulated cAMP accumulation [28,39] and PKA-dependent pain responses [40].
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Figure 2. CGRP receptor-mediated intracellular signalling. (a) Gas signalling increases AC (green) activity, elevating intracellular cAMP, activating PKA and subsequently many potential downstream effectors. (b) The CGRP receptor might also couple to Gai/o, reducing AC (red) activity, decreasing intracellular cAMP and reducing PKA activity. (c) CGRP signalling via Gaq activates PLC-b, which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into IP3 and diacylglycerol (DAG), resulting in elevated intracellular Ca2+ and PKCe activation. (d) The CGRP receptor might also utilise Ga-independent signalling, and Gbg- or b-arrestin-mediated signalling pathways. Arrows represent reported pathways; broken arrows represent potential or inferred pathways. AC, adenylate cyclase; ER, endoplasmic reticulum.
PKA has been implicated in numerous biological effects of CGRP, including vasodilation [2] and neural function [41], confirming the importance of Gas activation by CGRP receptors. PKA inhibitors such as H-89 have been extensively used to inhibit the actions of CGRP, including vascular and lymphatic dilation [42,43]. Studies have also confirmed the involvement of PKA, downstream of CGRP receptor activation, in regulating the activity of various downstream signalling components, including K+ channels [44], L-type Ca2+ channels [45] and cAMP response element binding protein (CREB) [46]. CREB expression can also be upregulated by CGRP [47]. Interestingly, PKA can also directly activate endothelial nitric oxide synthase (NOS), increasing nitric oxide (NO) production, which is essential for vasodilation [2]. Gai/o coupled signal transduction There is some evidence for Gai/o signalling by the CGRP receptor, which is traditionally identified by sensitivity to PTX (Figure 2b). The CGRP-mediated stimulation of Ca2+ transients in rat nodose neurons and the activation of cJun N-terminal kinase (JNK) in SK-N-MC cells (which express endogenous CGRP receptors) both displayed PTX sensitivity [48,49]. Interestingly, both these processes involve neural derived cells, in which Gai/o is abundantly expressed. Rat cardiac cells also displayed PTX sensitivity in CGRP-mediated activation of K+ channels [50]. Other studies using PTX in non-neural cell lines transfected with CGRP receptors have yielded mixed results. In HEK293
cells, CGRP stimulated PTX-insensitive Ca2+ transients and mitogen-activated protein kinase (MAPK) activation [extracellular signal regulated kinase (ERK)2 and p38] [51,52]. However, in Swiss 3T3 cells, and more recently in HEK293 cells, overexpressing CLR and RAMP2, PTX treatment resulted in potentiation of CGRP or AM-stimulated cAMP accumulation, respectively [24,53]. This could be interpreted as reduced CGRP receptor-mediated Gai activity, but it is also possible that the usual cellular balance of stimulatory and inhibitory processes is changed with PTX treatment, thus favouring stimulatory mechanisms via the CGRP receptor. Gaq/11 coupled signal transduction CGRP has been reported to activate several signalling components downstream of Gaq/11, including PLCb and protein kinase C (PKC) [34,54] (Figure 2c). Thus, Gaq/11coupling might explain some of the functions of CGRP observed in absence of cAMP accumulation. For instance, in osteoblastic OHS-4 cells, which express CLR, CGRP stimulated the PLCb1-dependent Ca2+ release from the endoplasmic reticulum, in the absence of cAMP accumulation [34,35]. However, calcitonin also induced Ca2+ transients comparably to CGRP, indicating the presence of a functional calcitonin receptor (CTR) that might also be activated by CGRP when RAMP1 is present; that is the AMY1 receptor. [7,35]. This receptor, expressed alone or with RAMPs, couples to Gaq/11, which can activate Ca2+ transients via PLC [38]. Therefore, it is unclear whether 479
Review CGRP-stimulated Gaq/11 signalling is driven by CLR or CTR in OHS-4 cells. The difficulty in firmly assigning a particular G protein to a given cellular response is not insignificant, given the interconnectivity of cellular signalling pathways. Thus, the Gaq/11 pathway is frequently studied in cellular backgrounds in which significant cAMP accumulation would also be expected in response to CGRP. For example, in HEK293 cells, CGRP stimulates cAMP accumulation, Ca2+ transients and inositol 1,4,5-triphosphate (IP3) accumulation, suggesting both Gas and Gaq/11 involvement [51]. Xenopus oocytes expressing CLR and RAMP1 apparently tested negative for Gaq/11 coupling [53], although this might reflect an inability of human CLR to couple Xenopus Gaq/11 effectively. Both Ca2+ transients and IP3 accumulation can be activated independently of Gaq/11, such as by Gas or Gbg, and therefore the extent to which CGRP receptors can couple to Gaq is still unclear [55]. Specific PKC inhibitors have shown that PKC activation, in conjunction with PKA, contributes to the effects of CGRP in controlling neuron function. CGRP has been shown to regulate mechanical hyperalgesia [56], P2X3 expression [57,58] and tetrodotoxin receptor Na+ channel activity [59] via both PKC and PKA. Compelling evidence for the involvement of PKC in CGRP signalling comes from alveolar epithelial cells, in which CGRP treatment protected epithelial cells from hypoxia-induced cell death. This was associated with the translocation of PKC epsilon (PKCe) to the cell membrane, and cell death could be prevented by the PKC inhibitor, calphostin C and the PKCe-specific inhibitor, PKCV1–2 [60]. Irrespective of whether activation of PKC occurs directly through Gaq/11 or through more convoluted mechanisms involving Gas or other pathways, this kinase appears to play important roles in CGRPmediated signal transduction in particular cell types. Ga-independent signalling pathways? Although GPCRs are best known for mediating their actions via the Ga subunits of G proteins, Gbg- and barrestin-coupled signal transduction is increasingly found [55,61] (Figure 2d). Fine-tuning of signalling can occur via a multitude of mechanisms, and therefore, in common with other GPCRs, CGRP receptor activation might elicit interactions that modulate many signals and receptor responses [55]. Gbg signalling Bioluminescence resonance energy transfer studies have shown that Gg subunits can associate with the CGRP receptor [62]. Beyond this, however, Gbg signalling via the CGRP receptor has not specifically been investigated. Gbg signalling is known to activate downstream effectors including ion channels and PLCb [55]. b-arrestin signalling Although no specific evidence for CGRP receptor-mediated signalling through b-arrestin has been described, b-arrestin associates with CGRP receptors and aids in CGRP receptor desensitisation and internalisation [62–64]. It is therefore plausible that barrestin could be involved in the activation of ERK1/2, p38, JNK and phosphoinositide 3-kinase (PI3K) by CGRP [61]. 480
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Protein kinase B/Akt Protein kinase B (PKB)/Akt might also be involved in CGRP-mediated signal transduction and biological functions, although the mechanisms regulating its activation are unclear. In endothelial cells, both CGRP and AM activate Akt via phosphorylation [65], and AM activates Akt in cultured adipocytes, potentially through CGRP receptors [66]. Similarly, in the dorsal horn, both AM and capsaicin (which releases CGRP and other neuropeptides) stimulated Akt activation, an effect that was blocked by the PI3K inhibitors wortmannin and LY294002 [67]. In a separate study, wortmannin blocked CGRP stimulated ERK2 activation, also implicating PI3K and Akt in CGRP signalling [52]. MAPK cascades The MAPK cascades represent major downstream effectors of CGRP; activation of ERK1/2, JNK and p38 have been observed in several studies, with outcomes ranging from cell proliferation [68,69] to regulation of neural function [70]. These might be activated by Ga-dependent or by alternative mechanisms. Interestingly, in CGRP-treated osteoblastic MG63 cells, the PKA inhibitor H-89 blocked CREB activation, partially blocked ERK activation, and had no effect on JNK activation, suggesting that both PKA dependent and independent pathways are involved [46]. CGRP-stimulated cellular proliferation in HaCat keratinocytes is associated with p38, ERK1/2 and JNK activation [68], and in lung alveolar epithelial cells, the ERK inhibitor U0126 effectively blocked CGRP-induced proliferation and ERK activation [69]. In cultured trigeminal ganglia glial cells, CGRP induced ERK1/2, JNK and p38 activation and subsequently increased inducible NOS expression and NO production [71]. Furthermore, MAPK expression can also be upregulated by CGRP in glial cells [47]. Chronic morphine tolerance and associated increases in p38 and ERK1/2 activation in the spinal cord are effectively blocked by olcegepant, providing good evidence for CGRP receptor involvement [70]. However, in a separate study, capsaicin-stimulated ERK activation in trigeminal ganglia neurons in vivo was not affected by olcegepant [72]. Summary of CGRP-receptor mediated signal transduction The potential intracellular consequences of CGRP receptor activation are numerous. Understanding these and their interactions in both health and disease might permit the development of novel therapeutics that target specific pathways [73]. To take full advantage of these processes, it is essential to determine how the CGRP receptor is itself regulated. Receptor regulation: desensitisation and internalisation To ensure continued GPCR responsiveness, receptor activation and intracellular signalling are carefully regulated. For GPCRs, although the processes vary in terms of mechanism and time scale, the connected processes of desensitisation, internalisation and recycling/degradation of receptors are typically involved [74] (Figure 3). For CLR, there has been some controversy; desensitisation of CLRmediated CGRP or AM responsiveness has been observed
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Figure 3. Agonist-mediated regulation of CGRP receptor function. The CGRP receptor, located at the plasma membrane (1), binds CGRP and is activated, facilitating intracellular signalling (2). The activated CGRP receptor is a target for GRKs, which phosphorylate serine/threonine residues in CLR, resulting in receptor desensitisation (other kinases e.g. PKA and PKC might also be involved) (3). b-Arrestin interacts with the phosphorylated CGRP receptor (4). The CGRP receptor/b-Arrestin complex and ECE-1 (red) might undergo clathrin/AP2/dynamin-dependent endocytosis (5), before the CGRP receptor is internalised (6) and trafficked to degradation (7) or recycling pathways (8). The CGRP receptor is trafficked to either the lysosomal pathway, where CLR and RAMP1 are degraded (7) or recycled via an endosome, where acidification facilitates ECE-1-mediated degradation of CGRP, promoting dissociation of b-arrestin and dephosphorylation (8). The CGRP receptor is resensitised and recycled back to the plasma membrane (9). AP2, adaptor protein 2; P, phosphate.
in some, but not all studies [33]. More recent work, including several mutagenesis studies in transfected cell lines, has begun to clarify the biology. Desensitisation Homologous desensitisation of CGRP signalling has been shown for several pathways, including cAMP in various cell types [33], Akt in endothelial cells [65], and Ca2+ transients in transfected HEK293 cells [75]. Protein kinases are known to play an important part in the desensitisation of many GPCRs, including the CGRP receptor, although it is not always clear which of these is involved. For example, it is still controversial as to whether PKA is involved in CGRP receptor desensitisation in SK-N-MC cells [33,71]. CLR appears to possess PKC phosphorylation sites [33], and there is some evidence of PKC involvement in CGRP receptor desensitisation using PKC inhibitors [76,77]. GPCR kinases (GRKs) initiate desensitisation by phosphorylating serine/threonine residues in activated receptors. This facilitates b-arrestin binding, reducing G protein-mediated signalling but potentially recruiting other signalling molecules [55,61]. In the case of CGRP receptors, the knockdown of GRK-6, but not GRK-2 or -5 was found to be important for the CGRP-mediated desensitisation of porcine CLR [77]. Interestingly, the overexpression of GRK-2, -3 or -4, but not GRK-5 or -6, in HEK293 cells expressing human CLR and RAMP2, increased internalisation efficiency [24]. As data are very limited and some discrepancies exist between studies, it remains to be established which GRKs are involved in CGRP receptor desensitisation. Internalisation and trafficking Following activation by CGRP, the CGRP receptor forms a complex with b-arrestin, which is essential for its dynamin/ clathrin dependent internalisation [62,63,75]. Several
regions of CLR are important for CGRP receptor internalisation. These include a conserved isoleucine residue (I312) at the ICL3/TM5 junction [19], the putative eighth helix (G388–W399), and a dispersed area including several serine/threonine residues (N400–L436) [23,24]. It follows that at least some of these regions are involved in receptor deactivation by GRKs and binding to barrestin. Recent studies in SK-N-MC cells have suggested that the duration of CGRP receptor stimulation has profound effects upon its fate. Following acute stimulation (1 hour), CGRP receptors were preferentially targeted to recycling pathways, whereas with chronic stimulation (8 hours), CGRP receptors were targeted for degradation via endosomes and lysosomes [78]. These results are in contrast to studies performed in HEK293 cells, in which overexpressed CLR was noted be inefficiently recycled, with the majority of CLR targeted to the lysosomes for degradation [75]. In both studies, acute stimulation was performed with 100 nM CGRP, but cell line-dependent factors, receptor detection methods and receptor expression levels could contribute to these apparent differences between studies. Further studies revealed the importance of endothelin-converting enzyme (ECE)-1. ECE-1 might internalise with CGRP-bound CGRP receptors, which in acidified endosomes degrade CGRP, resulting in destabilisation of the CGRP receptor/b-arrestin complex and resensitisation [64]. This process might also terminate b-arrestin-mediated ERK signalling [79]. Conclusion The increasing clinical significance of CGRP coupled with the unique molecular composition of the CGRP receptor makes understanding its signalling and regulation particularly important, but also a distinct challenge to comprehend. There is evidence for promiscuous CGRP 481
Review receptor signalling, through Gas, Gai/o, Gaq/11 and other pathways, although the evidence supporting Gas-independent coupling is currently limited. Controversy surrounding CGRP receptor regulation still exists. It is likely that differences in cellular background both explain and confound the data. Nevertheless, a picture of CGRP receptormediated signalling and regulation is beginning to emerge. With the advent of specific receptor antagonists, this picture can be refined, allowing the precise determination of the complex intracellular signalling networks activated by CGRP receptors. Defining these and the fate of activated CGRP receptors is essential for our understanding of CGRP biology, and its role in diseases such as migraine and hypertension. Once the signalling pathways important for CGRP action in specific disease states are known, molecules can be screened against these pathways, facilitating the development of pathway-selective drugs [73]. These could be more efficacious, with fewer off-target effects. It is important to focus on understanding CGRP receptor signalling and regulation in primary cell cultures and in vivo models at physiological expression levels of receptor. Of particular interest will be how CGRP receptor signalling and regulation change in disease states. Disclosure No author declares any conflict of interest. References 1 Doods, H. et al. (2007) CGRP antagonists: unravelling the role of CGRP in migraine. Trends Pharmacol. Sci. 28, 580–587 2 Brain, S.D. and Grant, A.D. (2004) Vascular actions of calcitonin generelated peptide and adrenomedullin. Physiol. Rev. 84, 903–934 3 Hay, D.L. and Poyner, D.R. (2009) Calcitonin gene-related peptide, adrenomedullin and flushing. Maturitas 64, 104–108 4 Danaher, R.N. et al. (2008) Evidence that alpha-calcitonin gene-related peptide is a neurohormone that controls systemic lipid availability and utilization. Endocrinology 149, 154–160 5 Hay, D.L. et al. (2008) International Union of Pharmacology. LXIX. Status of the calcitonin gene-related peptide subtype 2 receptor. Pharmacol. Rev. 60, 143–145 6 Bailey, R.J. and Hay, D.L. (2006) Pharmacology of the human CGRP1 receptor in Cos 7 cells. Peptides 27, 1367–1375 7 Hay, D.L. et al. (2005) Pharmacological discrimination of calcitonin receptor: receptor activity-modifying protein complexes. Mol. Pharmacol. 67, 1655–1665 8 Hay, D.L. et al. (2003) CL/RAMP2 and CL/RAMP3 produce pharmacologically distinct adrenomedullin receptors: a comparison of effects of adrenomedullin22-52, CGRP8-37 and BIBN4096BS. Br. J. Pharmacol. 140, 477–486 9 Hay, D.L. et al. (2006) Determinants of 1-piperidinecarboxamide, N-[2[[5-amino-l-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1[(3,5-dibromo-4 hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2oxo-3(2H)-quinazolinyl) (BIBN4096BS) affinity for calcitonin generelated peptide and amylin receptors—the role of receptor activity modifying protein 1. Mol. Pharmacol. 70, 1984–1991 10 Salvatore, C.A. et al. (2006) Identification and pharmacological characterization of domains involved in binding of CGRP receptor antagonists to the calcitonin-like receptor. Biochemistry 45, 1881–1887 11 Sexton, P.M. et al. (2009) Modulating receptor function through RAMPs: can they represent drug targets in themselves? Drug Discov. Today 14, 413–419 12 Miller, P.S. et al. (2010) Non-peptidic antagonists of the CGRP receptor, BIBN4096BS and MK-0974, interact with the calcitonin receptor-like receptor via methionine-42 and RAMP1 via tryptophan-74. Biochem. Biophys. Res. Commun. 391, 437–442 13 Mallee, J.J. et al. (2002) Receptor activity-modifying protein 1 determines the species selectivity of non-peptide CGRP receptor antagonists. J. Biol. Chem. 277, 14294–14298
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