New Insights into the Regulation of CGRP-Family Receptors

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Review

New Insights into the Regulation of CGRP-Family Receptors Joseph J. Gingell,1,2 Erica R. Hendrikse,1,2 and Debbie L. Hay1,2,* The calcitonin gene-related peptide (CGRP) receptor system has emerged as an important drug target for migraine. This is highlighted by the recent regulatory approval of the first drug targeting the CGRP signalling pathway, the CGRP receptor antibody erenumab. The cellular compartments in which receptors are found affects drug access and whether they can exert their effects. G proteincoupled receptors (GPCRs) were thought to signal only at the cell surface, but it is now recognised that some GPCRs, including the CGRP receptor, undergo sustained signalling from endosomes, once internalised in response to ligand. What does this mean for drugs like erenumab? This review covers recent insights into the regulation of CGRP family receptors and examines what implications this may have on drug activity.

Highlights The CGRP system is an important target for treating migraine with monoclonal antibodies against a CGRP receptor or the CGRP peptide itself, having recently received regulatory approval. The CLR/RAMP1 CGRP receptor can signal from within endosomes, creating distinct pools of receptors within cells. The activity of this receptor is blocked by the antagonist antibody erenumab. However, whether erenumab can access CGRP receptors in endosomes is unknown.

Overview of Receptor Regulation Like other G protein-coupled receptors (GPCR) (see Glossary), calcitonin gene-related peptide (CGRP) receptors can trigger diverse signalling pathways and undergo regulatory control [1]. These regulatory processes are essential for ensuring appropriate levels of cellular responsiveness to endogenous ligands, but these processes can also act to influence the effects or effectiveness of drugs. Canonically, GPCR activation is followed by association with GPCR kinases (GRK), which phosphorylate serine and threonine residues in the GPCR C terminus and intracellular loops. Receptor phosphorylation is followed by the recruitment of b-arrestin, which mediates desensitisation, internalisation, and intracellular trafficking. GPCRs can also be phosphorylated by second messenger-dependent kinases, protein kinase A (PKA) and protein kinase C (PKC), which do not appear to result in arrestin recruitment, but can mediate desensitisation [2]. Arrestins assist in receptor internalisation by interacting with components of the clathrin endocytotic pathway. Receptor internalisation was thought to lead only to the termination of signalling, however this view has been challenged, with recent reports showing sustained GPCR signalling from within endosomes [3–5]. GPCR targeted drugs are usually thought to act on receptors at the cell surface. Therefore, internalised receptors which are still capable of signalling, potentially represent distinct pools of receptors that could be exploited by drugs. Alternatively, the receptors within cellular compartments could be inaccessible to current drugs, limiting their ability to influence receptor function. Therefore, this review will focus on the internalisation and recently discovered endosomal signalling of the receptors in the CGRP family (Figure 1, Key Figure). It will also consider the possible implications of these processes on the behaviour and development of drugs that target the CGRP family of receptors, focussing on CGRP.

Therapeutic Relevance of CGRP and Related Peptides CGRP is a 37 amino acid neuropeptide that is expressed throughout the nervous system, especially the sensory subcomponent of the peripheral and central nervous systems. CGRP belongs to a small peptide family that includes calcitonin (CT), amylin, adrenomedullin (AM) and

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CGRP receptors are regulated via several processes, but whether these change in migraine, or in response to CGRP targeted drugs, such as erenumab, is not known.

1 School of Biological Sciences, University of Auckland, Auckland, New Zealand 2 Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand

*Correspondence: [email protected] (D.L. Hay).

https://doi.org/10.1016/j.tips.2018.11.005 © 2018 Elsevier Ltd. All rights reserved.

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Key Figure

Glossary

Internalisation and Fate of CGRP Receptors

AMY1: refers to a subtype of receptors for the peptide hormone amylin. This is dual receptor for CGRP and amylin, formed by CTR and RAMP1. RAMP2 and RAMP3 form the AMY2 and AMY3 receptors with CTR. CGRP: a 37 amino acid neuropeptide that is highly expressed in sensory nerves. Two variants of CGRP exist, a and b, which are derived from distinct genes. Clathrin endocytotic pathway: the clathrin endocytotic pathway transports membrane or extracellular cargo from the cell membrane into the cell. This involves a clathrin coat assembling around the membrane to form a clathrin coated pit in a process mediated by adaptor proteins. The clathrin coated pit is excised from the membrane in a dynamin dependent process to form a clathrin coated vesicle. The clathrin endocytotic pathway can be blocked by hypertonic medium, which inhibits the formation of clathrin coated pits. CLR: the calcitonin-like receptor which is a member of the class B/ secretin-like GPCR family. CT: the 32 amino acid peptide ligand calcitonin that is active in controlling bone homeostasis. CTR: the calcitonin receptor that is the most closely related receptor to CLR, and is also a class B/secretinfamily GPCR. DiscoveRx b-arrestin assay: this assay measures b-arrestin recruitment to GPCRs, using enzyme fragment complementation. b-arrestin is conjugated to a b-galactosidase deletion mutant, while the GPCR is C terminally tagged with the deleted fragment of b-galactosidase. b-arrestin interaction with the receptor forces complementation of the enzyme fragments. The resulting enzyme activity can be measured to determine b-arrestin recruitment. Dynamin: dynamin is GTPase involved in the budding and scission of vesicles from membranes. ECE-1: endothelin converting enzyme 1 is best known for its role in the proteolytic processing of endothelin precursors, but has also been reported to degrade CGRP. ERK: extracellular signal-regulated kinase, a widely expressed

CGRP CTR

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Figure 1. The calcitonin-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) (1) cointernalise with calcitonin gene-related peptide (CGRP) into endosomes in an arrestin-dependent manner (2). These endosomes have several fates. They can enter a degradation pathway and be sorted to lysosomes (3), can participate in sustained signalling though protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) (4), or can enter a recycling pathway to return to the cell surface (5). It is not yet clear if these fates are mutually exclusive. The recycling can be controlled by endothelin converting enzyme 1 (ECE-1), which breaks down CGRP in the recycling endosome, leading to disengagement of arrestin from the receptor complex. Although the calcitonin receptor (CTR) CTR/RAMP1 complex is also a high affinity CGRP receptor, nothing is known of its regulatory processes (6).

adrenomedullin 2/intermedin [6,7]. This peptide family has considerable therapeutic relevance. For example, pramlintide is an amylin receptor agonist that is approved clinically for the treatment of type 1 and type 2 diabetes, and has antiobesity effects [8]. Salmon CT has previously been used to treat osteoporosis, and an AM antibody is being developed for sepsis [9,10]. Recent attention has been focussed on CGRP, which has emerged as an important drug target for migraine. Some of the key findings linking CGRP to migraine include work showing that infusion of CGRP can trigger migraine-like headaches, and that CGRP concentrations are elevated in migraine sufferers [11,12]. Furthermore, an early small molecule CGRP receptor antagonist (olcegepant) showed clinical efficacy, followed by others, such as telcagepant to provide vital target validation [12]. Such studies have now led to regulatory approval for three drugs: a monoclonal 2

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antibody, erenumab, that targets a CGRP receptor, and two monoclonal antibodies that target the CGRP peptide itself (galcanezumab and fremanezumab) [13]. A fourth antibody, eptinezumab is also in the pipeline, together with several small molecule CGRP receptor antagonists, including rimegepant, ubrogepant, and atogepant [12]. Interestingly, erenumab is the first Food and Drug Administration (FDA)-approved monoclonal antibody drug that targets a GPCR [13], and as such is incredibly important for pharmacology. Despite the clear clinical relevance of CGRP [14], the relationship between CGRP receptor regulation and disease pathophysiology is not well understood. Furthermore, the potential influence of the new drugs on CGRP receptor regulation, or of CGRP receptor regulation on the effectiveness of the new drugs has not been considered. Hence, it is pivotal to understand these processes. For example, in migraine sufferers it is unclear what effect, if any, elevated CGRP levels have on CGRP receptor signalling, trafficking, and expression. Similarly, where there is chronic blockade of CGRP signalling in the presence of a CGRP blocking drug, how do CGRP receptors respond to this?

Introduction to CGRP Receptors The CGRP receptor system is complicated, with receptors formed from two GPCRs, the calcitonin-like receptor (CLR) and the calcitonin receptor (CTR), interacting with receptor activity-modifying protein 1 (RAMP1) to form heterodimers (Figure 1). These are named the CGRP and AMY1 receptors, respectively [7]. Further receptors for related peptides are formed by CLR interacting with RAMP2 and RAMP3 to form high affinity receptors for the AM peptides (AM1 and AM2 receptors, respectively). When expressed alone, the CTR is a receptor for CT, but forms high affinity receptors for the peptide amylin when expressed with RAMPs. The CTR/ RAMP1 heteromer (AMY1) acts in vitro as a dual receptor for amylin and CGRP, although its physiological relevance is not well understood [15]. An additional protein, receptor component protein (RCP), has been suggested to be essential for CLR signalling [16]. However, the precise mechanism is not understood and the picture is further complicated by RCP also being identified as a component of RNA polymerase III, which suggests that it may have broader roles in cell function [17].

Internalisation of the CLR-Based Receptors A key part of receptor regulation is removal of receptors from the cell surface – internalisation. Multiple studies have provided evidence that CLR-based receptors are internalised into endosomes following agonist stimulation (Figure 1). Green fluorescent protein (GFP)-labelled CLR and fluorescent antibodies against tagged RAMPs were first used to monitor this [18]. The CLR was internalised in response to CGRP when coexpressed with RAMP1 (CGRP receptor), and in response to AM when coexpressed with either RAMP2 or RAMP3 (AM1 and AM2 receptors) [19,20]. The labelled receptor components (CLR and RAMP) colocalised together, suggesting that both receptor components cointernalise [18]. Overall, the data support a role for a clathrindependent pathway in CLR internalisation [1,18]. CGRP receptor internalisation has been further investigated with a variety of approaches [3,21–23]. Both CLR and RAMP1 have been colocalised with early endosomal markers and fluorescently labelled CGRP, showing that the peptide-bound receptor complex is internalised [22]. Collectively, there is robust evidence that the tagged CLR/RAMP1 CGRP receptor is internalised into endosomes in response to CGRP in transfected cell models (mostly HEK293 cells).

intracellular signalling molecule that forms part of the mitogen-activated protein kinase pathway. GPCR: G protein-coupled receptor, a family of approximately 800 members that are characterised by seven transmembrane domains, an extracellular N terminus, three extracellular loops, three intracellular loops, and an intracellular C terminal region that often forms an eighth helix running parallel to the membrane. GRK: G protein-coupled receptor kinases are a family of proteins that phosphorylate GPCRs on their intracellular domains. PAC1: pituitary adenylate cyclaseactivating polypeptide 1 receptor. PDZ domain: this refers to protein interaction modules of a specific composition that function in the assembly of protein–protein complexes. The name comes from three proteins: P, 95 kDa protein involved in signalling at the postsynaptic density; D, Drosophila melanogaster Discs large protein; and Z, zonula occludens 1 protein. PDZ motif: is a short linear amino acid sequence present in the intracellular C terminus of many membrane proteins that can interact with PDZ domains present in other proteins. PKA: protein kinase A is a second messenger-dependent protein kinase that is activated by cAMP, and can phosphorylate GPCRs on their intracellular domains. PKC: protein kinase C is a second messenger-dependent protein kinase that is activated by calcium and diacylglycerol, and can phosphorylate GPCRs on their intracellular domains. Radioligand binding: radioligand binding is used to quantify interactions between a receptor and radiolabelled drug. RCP: receptor component protein, a member of a small family of related proteins that is important for CGRP receptor signalling.

CGRP receptor internalisation has also been observed in spinal neurons and human neuroblastoma cells (SK-N-MC) that endogenously express CGRP receptors [3,21]. Furthermore, in microvascular endothelial cells (MVECs), stimulation with AM, but not CGRP, induced CLR

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internalisation [24]. These studies provide evidence that the CLR is internalised in physiologically relevant systems, although the data are still sparse.

Internalisation of CTR-Based Receptors Numerous studies have provided evidence of agonist-induced CTR internalisation [25–31]. Most studies have used radiolabelled forms of CT to monitor this. For example, treatment of a human breast cancer cell line (T47D) with salmon CT at 37  C, followed by washing, resulted in a time-dependent reduction in subsequent radiolabelled iodinated (125I)-salmon CT binding [26]. Recovery of binding occurred in the absence of peptide, but was prevented by inhibitors of protein synthesis [26]. This observation was replicated in lung cancer and osteogenic sarcoma cell lines [25]. The degree of internalisation may depend on CTR splice variant [32], with impaired internalisation of the CT(b) receptor, compared to CT(a) (Box 1) [31]. More recent studies directly measuring CTR internalisation have suggested that this process may not be agonist dependent. GFP-labelled CTR appeared to constitutively internalise and recycle back to the cell surface [33]. This is supported by a study using fluorescently labelled full length salmon CT and the antagonist salmon CT8-32, which were both internalised along with an antibody against the myc-tagged CTR [34]. These data suggest that CTR internalisation does not always depend on activation by an agonist and might be constitutive. This is in contrast to the data that exist for CLR, suggesting that these two closely related receptors may have distinct regulatory mechanisms. The internalisation of the CTR/RAMP complexes has not been investigated to date. Given the high affinity of the CTR/RAMP1 (AMY1) receptor complex for CGRP, and the existence of an amylin analogue drug (pramlintide), this is both a surprising and significant knowledge gap [7,8]. It is not known whether CTR/RAMP complexes behave differently from CTR alone. Given that amylin responsiveness is at least partially retained following prolonged infusion in animal models and that patients remain sensitive to pramlintide, it is tempting to speculate that CTR/RAMP complexes are also regulated differently to CLR/RAMP complexes [8].

Endosomal Signalling of CGRP Receptors Endosomal GPCR signalling is an area of much interest recently, and multiple GPCRs have now been reported as capable of signalling from endosomes, including the b2 adrenergic receptor (AR), the thyrotropin receptor, the V2 vasopressin receptor, the PAC1 receptor, and the parathyroid hormone 1 (PTH1) receptor [4,35–39]. There are now several examples where receptor signalling from endosomes appears to have distinct consequences from receptor signalling at the cell surface, or for mediating a particular effect. Importantly, in some cases, endosomal signalling may have specific physiological consequences. A recent study has reported that the CGRP receptor can signal from endosomes (Figure 1) [3]. A modified form of a CGRP receptor peptide antagonist CGRP8–37, conjugated to the

Box 1. GPCR and CTR Splice Variants Splice variants are generated through alternate mRNA splicing, whereby inclusion/exclusion of exons leads to different mRNA and thus protein sequences. Approximately 50% of GPCRs contain at least two exons and can potentially generate splice variants. CTR can exist as several splice variants, which differ between species [32]. For human receptors, the most common form of CTR lacks a 16 amino acid insert in the first intracellular loop. This is termed CT(a). When an extra exon in intracellular loop 1 is included to encode those 16 amino acids, the receptor is named CT(b). Another variant of CT(a) exists, which instead has a truncation of the first 47 amino acids in its N terminus, forming D(1– 47)hCT(a). The overall importance of GPCR splice variants is not well understood and is an area that could be very fruitful for future research.

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membrane lipid cholestanol was developed (Chol-CGRP8–37), and was used to demonstrate that sustained CGRP receptor endosomal signalling potentially has physiological consequences. In animals treated with CGRP8–37 and Chol-CGRP8–37, a transient antinociceptive effect was observed with CGRP8–37, but with Chol-CGRP8–37 a sustained antinociceptive effect was observed [3]. Although this is compelling data, future studies will be needed to confirm that the in vivo effects observed are due to Chol-CGRP8–37 predominantly blocking endosomal signalling, as opposed to other mechanisms being at work. For example, attachment of lipids can improve the metabolic stability of peptides, and it is possible that this could contribute to the mechanism of action of Chol-CGRP8–37 [40]. This potentially exciting study will hopefully lead to further investigation of the mechanisms that drive and terminate this endosomal signalling. So far, only the CLR/RAMP1 receptor has been considered for endosomal signalling and it will be interesting to determine how RAMP2 and RAMP3 affect these processes in CLR complexes and whether this occurs for CTR or CTR/RAMP complexes, such as the AMY1 receptor. Characterisation of the endosomal signalling profiles of the two AM receptor subtypes or of the two high affinity CGRP receptors, for example, could reveal elegant RAMP-dependent regulatory mechanisms that govern receptor responsiveness or sustained signalling.

Mechanisms of Receptor Regulation Post-Translational Modifications In addition to endosomal signalling, receptor internalisation can lead to receptor degradation by trafficking to the lysosomal pathway or recycling back to the cell surface. Post-translational modifications, such as phosphorylation and ubiquitination on intracellular receptor domains, play an important role in the regulation of GPCRs [41]. The CLR C terminus contains numerous predicted or potential phosphorylation sites (Figure 2A). This is supported by experimental evidence that CLR can be phosphorylated after CGRP stimulation, although no individual phosphorylated residues have been pinpointed [21,23]. In the CGRP receptor, C terminal truncation resulted in a significant reduction in receptor internalisation, while retaining cell surface expression [42]. A similar pattern was observed in the AM1 receptor where a CLR mutant truncated at residue 448, removing a

(A)

(B)

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ICL3 ICL3 ICL2

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Figure 2. Post-Translational Modification Sites and Motifs within the CLR, CTR, and RAMP C Termini. Potential serine/threonine phosphorylation sites within the predicted intracellular domains of the (A) calcitonin-like receptor (CLR), (B) calcitonin receptor (CTR), and (C) receptor activity-modifying proteins (RAMPs) were predicted using the NetPhos3.1 server [78]. Other intracellular serine/threonine and tyrosine residues are also highlighted. Potential intracellular ubiquitination sites were determined by the Ubpred server [79]; none were predicted in CTR or RAMPs. Two distinct clusters, cluster 1 and cluster 2, are present within the CLR C terminus that form arrestin codes (Px(x)PxxP, Px(x)PxxD/E motifs) (Box 3). Note that the PDZ motifs in (B) and (C) include potential phosphorylation sites. Snake plots are adapted from the GPCRdb [80]. Abbreviations: ECL, extracellular loop; ICL, intracellular loop.

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serine and threonine rich segment, had significantly reduced internalisation in response to AM without any loss of cell surface expression, or reduction in signalling [43]. While truncation studies are helpful in confirming that the C terminus is important, these will remove more than just phosphorylation sites, and such studies should ideally be followed up with more detailed analysis of specific sites. Transient stimulation of the CGRP receptor leads to its recycling back to the cell surface, whereas prolonged stimulation (>2 hours) leads to trafficking to lysosomes and degradation (Figure 1) [22]. GPCR trafficking from endosomes to the lysosomal degradation pathway typically involves ubiquitination and subsequent recognition by ubiquitin-binding endosomal sorting protein complexes (Box 2) [41,44]. However, the CGRP receptor appears to be an exception with no CLR ubiquitination detected after CGRP stimulation [22]. By contrast, the CLR is ubiquitinated following stimulation of the AM1 receptor with AM [45]. In both AM1 receptor transfected cells (HEK293) and cell lines with endogenous AM-responsive receptors [human microvascular endothelial cells (HMEC-1) and primary MVECs], the CLR is targeted to lysosomes for degradation following stimulation with AM [24]. Unlike the CGRP receptor, recycling of the AM1 receptor has not been observed, with both transient and prolonged AM stimulation resulting in lysosomal degradation. Interestingly, elimination of potential ubiquitination sites on the CLR did not affect either internalisation or postendocytic trafficking of the AM1 receptor, indicating that the observed ubiquitination was not a prerequisite for CLR degradation. Differences in ubiquitination between the CGRP and AM1 receptors shows that RAMPs can alter the post-translational modification status of CLR, though the mechanisms through which this occurs are uncertain. The mechanisms and details of CTR internalisation are sparse. Radiolabelling with 32P revealed that the CTR C terminus is phosphorylated in response to agonist stimulation, in a process not dependent on PKA or PKC [46], but there appear to be fewer potential phosphorylation sites than in the CLR, indicating it would belong to the Class A GPCR trafficking subtype (Figure 2B) (Box 3). In porcine CTR, progressive truncation of the C terminal tail increased receptor affinity for salmon CT, but resulted in reduced internalisation [47]. There are currently no studies of CTR ubiquitination.

Box 2. Mechanisms of GPCR Trafficking Several mechanisms have been identified through which GPCRs are sorted from early endosomes to lysosomes, the best characterised being the endosomal sorting complexes required for transport (ESCRT) machinery. This conserved machinery recognises ubiquitinated protein cargo and directs it towards the lysosomal degradation pathway rather than recycling or alternative trafficking pathways. The process involves multiple protein complexes working cooperatively to identify and sort the cargo, forming intraluminal vesicles that are transported to lysosomes. One component of the earlystage ESCRT machinery is hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), which has ubiquitinbinding domains [81]. In cells with reduced HRS expression, CLR was retained in HRS vesicles in response to sustained CGRP activation and was not trafficked to lysosomes [82]. Therefore, despite lacking ubiquitination in response to CGRP stimulation, CLR in this context may interact with HRS and the ESCRT complex. In the AM1 receptor, CLR or RAMP2 degradation were not affected by HRS knockdown, suggesting that other endosomal sorting proteins may be involved [45]. Overexpression of HRS in this cellular context appears to disrupt the normal endocytic system, resulting in hyperubiquitination of the CLR under basal conditions, as well as impeding degradation of the ubiquitinated CLR. A second sorting machinery is the actin-sorting nexin 27 (SNX27)-retromer tubule (ASRT) complex. The ASRT complex is a multiprotein complex that is involved in the recycling of internalised GPCRs to the plasma membrane [83]. A PDZ domain in SNX27, a member of the ASRT machinery, recognises PDZ motifs present in the C termini of GPCRs such as PTH1R and the b2AR (and other membrane proteins including RAMP3 [66]). Trafficking from endosomes is driven via tubules, leading to either recycling to the plasma membrane, or retrograde transport to the Golgi apparatus [84].

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Box 3. Arrestin Codes GPCRs are broadly divided into classes based on b-arrestin interactions. In this nomenclature, those classified as Class A undergo shorter interactions with b-arrestin and are recycled rapidly to the cell surface, while those in Class B undergo prolonged interactions with b-arrestin. This classification is distinct from that which otherwise defines receptors into families (i.e., class A/family A/rhodopsin, etc.). Phosphorylation-dependent arrestin recruitment to GPCRs is a widely observed phenomenon. The crystal structures of rhodopsin bound to visual arrestin [85], and the vasopressin 2 receptor C terminal tail bound to b-arrestin 1 [86], have given insights into the molecular mechanism of how arrestins sense receptor phosphorylation. Distinct patterns of phosphorylated and negatively charged resides in the receptor C terminal tail that form electrostatic interactions with positively charged pockets in arrestin were observed [85]. Based on these and other observations, motifs consisting of a phosphorylated serine or threonine and negatively charged aspartic acid and glutamic acid residues arranged in distinct patterns ([P]x(x)[P]xx[P], [P]x(x)[P]xx[D/E]) were identified and are termed arrestin codes. Bioinformatic analysis of the C terminal tails of GPCRs revealed a correlation with the number of arrestin codes present and Class A and B nomenclature regarding arrestin interactions [85], with multiple codes present in Class B GPCRs, but one or no codes present in the Class A GPCRs. These motifs or arrestin codes provide a common mechanism for how GPCRs can form high affinity interactions with arrestin despite the lack of sequence conservation in the C termini. Applying this analysis to the CGRP receptor family suggests that the CLR would belong to Class B, and CTR to Class A (see Figure 2 in main text), which is broadly consistent with the available experimental data.

RAMPs have short intracellular C termini (10 amino acids), but potential sites for post-translational modification are present (Figure 2C). However, there is no evidence that RAMPs themselves are phosphorylated or ubiquitinated. Whilst this has not been studied across every receptor subtype, nor in response to different ligands, no RAMP1 phosphorylation has been observed at the CGRP receptor in response to agonist stimulation [23]. Further, no RAMP ubiquitination has been found at the CGRP receptor [22] or the AM1 receptor [45]. It is, however, possible that RAMPs could influence post-translational modification within CLR or CTR through an allosteric mechanism by affecting GPCR conformation leading to masking or unmasking of key amino acids and driving bias (Box 4). Interactions with GRKs and b-Arrestins Assuming that the CLR is phosphorylated at the predicted sites, CLR-based receptors should be phosphorylated by GRKs leading to interactions with b-arrestin. GRK6 has been implicated Box 4. Biased Signalling and the CGRP Receptor Family GPCRs are known to activate a range of signalling networks (Figure IA), which includes arrestin-driven and endosomal signalling. Different ligands or GPCR-interacting proteins yield unique GPCR conformations that create diverse signalling signatures; this is known as biased signalling [87]. The ability to selectively control cellular events through biased signalling has garnered significant interest, because it opens up the possibility of generating drugs that select signalling signatures that direct therapeutic benefit, rather than those that bestow unwanted effects [88]. This is most commonly discussed in the context of biased ligands, which have a modified signalling profile compared to a reference ligand that displays ‘balanced’ signalling (Figure IB). However, receptor protein partners, splice variants, and other factors in the GPCR environment can also alter GPCR conformation to influence receptor signalling (Figure IC). Bias is evident in the CGRP receptor family because each RAMP alters the responsiveness of the CLR and CTR to their ligands [7]. Furthermore, for the CLR-based receptors, both ligand- and RAMP-dependent signalling bias have been observed when measuring G protein signalling [89]. Beyond this, other aspects of these receptor signalling profiles, including regulation, have not been specifically compared in the context of bias [1]. Endosomal signalling adds further complication, as distinct receptor populations could preferentially signal through different pathways [3]. The CTR also couples to multiple signalling pathways, with splice variants (Box 1) displaying differences in signalling [34]. Interestingly, work on the CTR shows that the effects of a biased ligand can be preserved further down the signalling pathway, modulating G protein conformation [90]. Pleiotropic coupling of the CTR is retained in the presence of RAMPs, but does not appear to be drastically altered by the different peptide ligands studied so far (CT, amylin, pramlintide, and CGRP) [91], but bias has not been extensively considered for CTR/RAMP complexes. Understanding of biased signalling in the CGRP receptor family is far from complete. To develop biased drugs, it will be important to determine the signalling signatures that occur in disease states or in physiological situations, which can then prompt the hunt for a suitable biased ligand.

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(A) Receptor bias

(B) Ligand bias

(C) Protein bias

Figure I. Different Types of GPCR Bias. A biased receptor (A) preferentially couples to one signalling pathway over another, regardless of the ligand bound. Biased ligands (B) create unique receptor conformations due to their specific contacts with receptors that drive preferential interactions with some signalling molecules over others. Intracellular proteins or other membrane proteins (C) can also engender unique receptor conformations to affect GPCR ligand preference and/or drive signalling.

in the regulation of the CGRP receptor in response to CGRP, whereas GRK2 and GRK5 did not seem to be important (in HEK293 cells) [48]. GRKs have also been considered in the context of AM receptors. Overexpression of GRKs 2, 3, and 4 with the AM1 receptor enhanced receptor internalisation [43]. The role of GRKs was further investigated by cotransfection of GRK4 or GRK5 with the AM1 receptor, which resulted in decreased AM1 receptor cell surface expression [49]. The data regarding CLR phosphorylation by PKA/PKC is inconclusive. While some studies [50,51] found that CGRP receptors could be desensitised in a PKA-dependent manner, a subsequent study did not observe this [52]. In support of predicted CLR interactions with b-arrestin (Figure 1), yellow fluorescent protein (YFP)-labelled b-arrestin 2 colocalised with CLR and RAMP1 after CGRP stimulation [23]. Dominant-negative dynamin and mutants of b-arrestin 2 were used to demonstrate that the internalisation process for the CGRP receptor was dynamin and b-arrestin dependent [23]. Other studies also support b-arrestin interactions with the CGRP receptor [21,53,54]. However, contradictory evidence towards the role of b-arrestins in CLR internalisation was observed when b-arrestins 1 and 2 were overexpressed with the AM1 receptor [55]. This resulted in decreased CLR internalisation without any effect on AM binding or signalling [55]. Therefore, it is possible that there are differential interactions between CLR and GRKs and/or b-arrestins, depending on which RAMP is expressed. The role of different CLR ligands in driving these processes has not been explored, leaving open the possibility that there is significant ligand bias in driving differential CLR/RAMP/arrestin complex formation (Box 4). In the CTR, phosphorylation of the C terminus could not be blocked by PKA or PKC inhibitors, suggesting the phosphorylation process was GRK dependent [46]. Further evidence of GRK involvement in the regulation of the CTR was provided by a cell line stably expressing dominantnegative GRK2. In this cell line, CT responses were enhanced and the number of receptors were increased compared to control cells as assessed through radioligand binding. An increase in CTR mRNA was also observed [56]. Despite this, it is inconclusive whether CTR interacts with b-arrestin. In a DiscoveRx b-arrestin assay which uses CTR modified at the C terminus, arrestin recruitment was detected in response to salmon CT and human CT [57]. However, when using native CTR or a tag to measure arrestin proximity, no recruitment of b-arrestin 1 or 2 was detected [34]. Therefore, it is unclear whether the CTR couples to

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b-arrestin in physiologically relevant systems. No data is available on the b-arrestin interactions with the AMY receptors, that is, how RAMP may affect CTR in this context. Interactions with Endothelin Converting Enzyme (ECE-1) A less well-known mechanism affects whether the CGRP receptor in endosomes is recycled back to the cell surface following transient stimulation, or is trafficked to lysosomes for degradation (Figure 1) [22]. The recycling process is believed to depend on the ECE-1 which degrades the CGRP peptide into fragments incapable of binding [21]. In these endosomes, b-arrestin 2 dissociates from the receptor and moves to the cytoplasm. While most studies were performed in HEK293 cells, it should be noted that recycling was also observed by transfecting tagged receptors into SK-N-MC cells, indicating that this mechanism may have physiological relevance [22]. However, preliminary studies in blood vessels show contradictory results. In rat mesenteric smooth muscle cells and arteries, inhibition of ECE-1 impaired the resensitisation to CGRP [58]. By contrast, studies in human coronary and middle meningeal arteries showed that ECE-1 did not appear to be involved in CGRP resensitisation [59]. Further studies in primary cell lines and tissues are needed to confirm the role of ECE-1. Interactions with Other Proteins In addition to the post-translational modifications described above, tyrosine residues, dileucine motifs and PDZ motifs within GPCRs or their accessory proteins can also regulate receptor sorting and trafficking through a variety of mechanisms [41,60]. Various endocytotic adapter proteins recognise these sites to facilitate internalisation and endosomal sorting of GPCRs. Analysis of the C termini of the CLR and CTR reveals the presence of a putative acid dileucine motif (amino acids- DIENVLL) in the CLR (Figure 2A) [41]. The role of this motif has not been directly investigated in the CLR, so it is unclear what contribution it makes to receptor internalisation. However, in the b2AR, an acid di-leucine motif was found to sort receptors to an hepatocyte growth factor-regulated tyrosine kinase substrate (HRS)-dependent recycling mechanism [61] (Box 2). Therefore, it is possible this motif has a similar role in the CLR. A PDZ motif has been identified in the C terminus of the CTR [62] (Figure 2B). However, it remains to be functionally characterised. It has also been reported that the actin binding protein filamin A may play a role in the regulation of CTR recycling, through interactions with the CTR C terminus [33]. Reduced CTR degradation was observed in cells expressing filamin A, which was determined to be due to reduced recycling of the CTR. It was suggested that salmon CT treatment led to reduced CTR degradation by inhibiting filamin A degradation [33]. However, there are no obvious filamin A binding motifs present within the CTR C terminus [63] (Figure 2B), and therefore the precise binding site is unclear. While the C termini of RAMPs are short, they appear to be capable of interacting with other proteins. The C terminus of RAMP3 contains a PDZ recognition motif (amino acids- DTLL) allowing it to interact with proteins containing PDZ domains (Figure 2C) [19]. Whether there are other motifs present within RAMP1 or RAMP2, that contribute to the differences in trafficking behaviour and ubiquitination state observed, has not yet been established. The role of proteins interacting with the PDZ motif was investigated at the AM2 receptor [19,20]. In HEK293 cells, the AM2 receptor was not resensitised, but when N-ethylmaleimide-sensitive factor (an ATPase involved in receptor recycling [64]) was coexpressed, the receptor was recycled to the cell surface [19]. Coexpression of Na+/H+ exchanger regulatory factor (which regulates the recycling of several GPCRs) [65] blocks receptor internalisation but not desensitisation by tethering the receptor complex to the actin cytoskeleton [20]. In addition to these known interactions, the PDZ motif in RAMP3 is predicted to interact with the SNX27 complex [66] Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy

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(Box 2), therefore, it is possible that the AM2 receptor may recycle through the ASRT pathway. RAMPs may therefore play a direct role in regulating receptor trafficking, by directly interacting with endocytotic machinery or via their allosteric effects [67–70].

Concluding Remarks and Future Perspectives CGRP has reported actions in many systems, and has high affinity for the CGRP and AMY1 receptors [7,14]. The regulatory processes of these receptors therefore modulate CGRP action and have implications for understanding the behaviour of drugs that target CGRP or its receptors. Although we have some understanding of the regulation of the CLR/RAMP1 CGRP receptor, there are many outstanding questions (see Outstanding Questions). For example, CGRP levels are generally reported to be higher in migraine sufferers, but it is not known whether this translates to chronic CGRP elevation, and what impact this might have on CGRP receptor populations, that is, does this create more endosomal receptors? Furthermore, this raises the question of whether these receptors are active or inactive. Any drug developed to block CGRP could have unintended consequences on receptor function – CGRP receptors could be upregulated under conditions of acute or chronic ligand depletion (anti-CGRP antibodies) or receptor antagonism (anti-CGRP receptor antibody, small molecule receptor antagonists). If there are pools of receptors in endosomes, they could be less accessible by CGRP and CGRP blocking drugs. Differences in the physical properties of small molecule antagonists compared to receptor antagonist antibodies could lead to different effects by targeting distinct receptor populations. Interestingly, an antiCGRP receptor antibody AA58 that is related to erenumab, does not appear to be internalised [71]. Hypothetically, an antagonist drug that was cell-permeable, or was cointernalised with receptor (it is not known if the current molecules have this property), might have greater access to receptors within endosomes, affecting the functional outcome of CGRP signalling in a different way to an antibody that could only be active at the cell surface. It is too early to say whether either possibility would be of greater or lesser benefit, but it highlights the ways in which receptor internalisation and signalling from endosomes could affect drug behaviour. This phenomenon was recently illustrated in neurons where nonpeptide drugs activate opioid receptors in a different subcellular location than endogenous peptide agonists, suggesting that different populations of GPCRs within cells represent novel drug targets [72]. Chol-CGRP8–37 has illustrated that CGRP system targeted antagonists that are able to access endosomal receptor pools can theoretically have greater efficacy, providing support for the idea that there is significant merit in tailoring molecules to affect receptor function in different cellular compartments [3]. Moreover, the potential of any CGRP receptor antibody or small molecule antagonist to be biased ligands has also not been considered (Box 4). All of these perspectives also apply to the AMY1 receptor, which is equally capable of being activated by CGRP. This receptor can be blocked by several CGRP receptor antagonists and is found within the trigeminovascular system. Therefore, the regulation of this receptor must also be considered in the context of CGRP physiology, pathophysiology, and of drugs that affect the CGRP system [15,73–77]. A pertinent question in the field currently, is whether differences in the regulation and endosomal signalling profiles of the two CGRP receptors generate any defining features that could be exploited in future drug discovery efforts. Moreover, other peptide–receptor systems within the CGRP family are also drug targets, the most notable example being the amylin agonists. Further understanding of the internalisation and potential endosomal signalling of CTR, amylin, and AM receptors is therefore also important in understanding the mechanisms of action of these drugs and for developing new ones. The CLR is internalised into endosomes in response to agonist stimulation. Depending on the RAMP subtype expressed, and the length of agonist stimulation, these receptors are recycled 10

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Outstanding Questions What effect do elevated CGRP levels in migraine patients have on CGRP receptor signalling, trafficking, and expression? Are CGRP receptors up- or down-regulated under conditions of acute or chronic ligand depletion (anti-CGRP antibodies), or receptor antagonism (anti-CGRP receptor antibody, small molecule receptor antagonists)? Are there pools of receptors (e.g., in endosomes) that are more or less accessible by CGRP and CGRP blocking drugs? Are there differences in the regulation and endosomal signalling profiles of the two CGRP receptors? Does the AMY1 receptor internalise or signal from endosomes at all? How do RAMPs affect CTR regulation? Are any drugs targeting the CGRP system biased?

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or degraded. By contrast, the data regarding the internalisation of the CTR is less conclusive, and the role of RAMPs in altering the internalisation and trafficking of the CTR has yet to be investigated and is an area for future research. The functional consequences of endosomal signalling at CGRP-family receptors are yet to be fully understood, but it may present a novel target for therapeutic intervention, or shape our understanding of mechanisms that influence the activity of recently approved CGRP receptor and ligand antibodies. Acknowledgments We thank Vivian Ward for assistance with drawing Figure 1. D.L.H. is supported by a James Cook Research Fellowship of the Royal Society of New Zealand. E.R.H. is supported by the Barbara Basham Doctoral Scholarship administered by the Auckland Medical Research Foundation and Perpetual Guardian. The authors acknowledge support from the Marsden Fund, Royal Society of New Zealand. We acknowledge the use of the GPCRdb database (http://www.gpcrdb.org).

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