Understanding Amylin Receptors - Springer Link

Chapter 3

Understanding Amylin Receptors Rasmus Just, John Simms, Sebastian G.B. Furness, Arthur Christopoulos, and Patrick M. Sexton

Abstract Amylin is a 37 amino acid peptide that is co-secreted with insulin from pancreatic b-cells following nutrient ingestion, acting to inhibit gastric emptying, feeding and insulin-stimulated glycogen synthesis. Amylin is a member of the calcitonin (CT) family of peptides, which include CT, CT gene-related peptides (CGRP) and adrenomedullin (AM). The receptors for these peptides comprise the CT receptor (CTR) and the CTR-like receptor (CLR) that may be complexed with one of three receptor activity modifying proteins (RAMPs). Amylin receptors are formed when the CTR is in complex with RAMP1, RAMP2 or RAMP3, forming AMY1, AMY2 and AMY3 receptors, respectively. Each of these receptors, while binding amylin with similar affinity, has a distinct agonist and antagonist pharmacology. Analysis of RAMP chimeras and deletion constructs has provided insight into domains of RAMPs that contribute to ligand and signaling specificity. The N-terminal domain is the principle domain involved in alteration of ligand binding specificity, while the C-terminal domain contributes to the peptide signaling profile of the receptor complexes and could be directly involved in the interaction with G proteins. Keywords Calcitonin receptors • receptor activity modifying proteins • amylin receptors • pharmacology • structure–function • G protein • signaling • regulation Abbreviations AC AM

adenylyl cyclase adrenomedullin

R. Just (*) Zealand Pharma, Smedeland 26B, DK-2600, Glostrup, Denmark e-mail: [email protected] J. Simms, S.G.B. Furness, A. Christopoulos and P.M. Sexton Drug Discovery Biology Laboratory, Monash Institute of Pharmaceutical Sciences and Department Pharmacology, Monash University, Victoria, Australia D.L. Hay and I.M. Dickerson (eds.), The Calcitonin Gene-related Peptide Family: Form, Function and Future Perspectives, DOI 10.1007/978-90-481-2909-6_3, © Springer Science+Business Media B.V. 2010

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AMY AM2 cAMP cDNA CGRP CHO CLR CNS COS CT CTR CT(a) CT(b) ERK GPCR HEK293 IDDM IMD IP IP3 MDCK NHERF1 NIDDM NSF PDZ PKA PLC RAEC RAMP RCP D

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amylin receptor adrenomedullin 2 cyclic adenosine monophosphate complementary DNA calcitonin gene-related peptide chinese hamster ovary cells calcitonin receptor-like receptor central nervous system african green monkey kidney cells calcitonin calcitonin receptor calcitonin receptor a isoform calcitonin receptor b isoform extracellular signal-regulated protein kinase G protein-coupled receptor human embryonic kidney cells insulin dependent diabetes mellitus or type 1 diabetes intermedin, also known as AM2 inositol phosphate inositol 1,4,5-trisphosphate mandin-darby canine kidney cells Na+/H+ exchanger regulatory factor 1 non IDDM or type 2 diabetes N-ethylmaleimide-sensitive fusion protein post-synaptic density-95/discs large/Zone occludens-1 homology protein kinase A phospholipase C rabbit aortic endothelial cells receptor activity modifying protein receptor component protein deletion mutant.

3.1 Amylin and Related Peptides Amylin, also termed diabetes associated peptide (DAP) or islet amyloid polypeptide (IAPP), is a 37 amino acid peptide, synthesized and co-secreted with insulin from pancreatic b-cells following nutrient intake. Amylin was initially discovered and characterized in 1987 as a component of amyloid fibrils in the pancreas of type 2 diabetic patients (Cooper et al. 1987). Amylin belongs to the calcitonin (CT) peptide family, comprising CT, CT generelated peptides (CGRPa and b), adrenomedullin (AM), intermedin (IMD; also known as AM2) and three CT receptor-stimulating peptides (CRSP-1,2,3). These peptides all contain an N-terminal six or seven amino acid cyclic structure, formed

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by a disulfide bridge that is required for biological activity; in amylin this cyclic structure involves Cys2 and Cys7. Amylin shares ~20% sequence identity with human CT and AM, and 43–46% with the CGRPs. The effects of amylin on glucose metabolism and food intake have been characterized and are mediated by peripheral and central mechanisms (Young 2005). Amylin and insulin are co-secreted in a constant molar ratio of approximately 1:20 (reviewed in Martin 2006) and plasma insulin and amylin concentrations follow a parallel pattern after meal intake (Kruger et al. 1999). Insulin secretion after a meal stimulates the peripheral tissues to take up circulating glucose with a concurrent increase in amylin secretion that acts to suppress glucagon secretion from the pancreatic a-cells, which in turn suppresses the mobilization of glucose from liver glycogen stores. Amylin also potently slows gastric motility, decreasing nutrient delivery to the small intestine thereby delaying absorption of glucose into circulation. In addition, amylin mediates signals of satiety to the CNS (reviewed in Martin 2006 and Young 2005). Amylin is an important part of the multi-hormonal regulation of glucose homeostasis. In type 1 diabetic patients (Insulin Dependent Diabetes Mellitus; IDDM), there is an absolute deficiency in insulin and amylin, whereas in late stage non-insulin dependent diabetes mellitus (NIDDM) or type 2 diabetes there is a relative deficiency of insulin accompanied by a relative deficiency of amylin (reviewed in Martin 2006). In diabetic patients failure to suppress glucagon secretion after meal intake, even when administered with exogenous insulin, results in a continuous increase in plasma glucose levels from glycogenolysis in the postprandial period, attributed to the lack of insulin’s “partner hormone” amylin (Martin 2006; Odegard et al. 2006). SYMLIN® (pramlintide acetate) was approved by the United States FDA in March 2005 as an adjunct therapy for type 1 and 2 diabetic patients, who use mealtime insulin therapy and have failed to achieve desired glucose control despite optimal insulin therapy. Human amylin is amyloidogenic (Young 2005), however, Pramlintide is an injectable amylin analogue developed by substituting amino acids in the amyloidogenic native human amylin (25Ala, 28Ser and 29Ser) with corresponding prolines in the non-amyloidogenic rat amylin, and 10Gln for Asn (Fig. 3.1). Clinical studies has evaluated the effects of Pramlintide on glucagon release in type 1 (Fineman et al. 2002a) and type 2 diabetic patients (Fineman et al. 2002b) and in both patient groups post-prandial glucagon secretion was suppressed with Pramlintide treatment as opposed to placebo where an abnormal glucagon rise was observed. Other studies have shown that Pramlintide, similar to native human amylin,

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

Pramlintide K C N T A T C A T N R L A N F L V H D D N N F G P I L P P T N V G S N T Y [-NH2] Human amylin K C N T A T C A T N R L A N F L V H D D N N F G A I L S S T N V G S N T Y [-NH2] Rat amylin K C N T A T C A T N R L A N F L V R D D N N F G P V L P P T N V G S N T Y [-NH2]

Fig. 3.1 Alignment of the amino acid sequence (single letter code) of pramlintide with the sequences of human amylin and rat amylin. Divergent amino acids are depicted in bold and are boxed

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has three primary actions; reducing postprandial glucagon secretion, delaying gastric emptying and mediating satiety to the CNS (Kruger and Gloster 2004).

3.2 Amylin Receptors 3.2.1 Discovery Prior to the discovery of amylin receptors, the related peptides CGRP and CT were known to have abundant binding sites in the CNS and these sites were anatomically distinct in most regions. Evidence of a new class of sCT (salmon CT)-sensitive CGRP receptors came in 1988 when Sexton and colleagues discovered a binding site in rat brain with high affinity for the two otherwise biochemically distinct peptides (Sexton et al. 1988); these were originally termed C3 binding sites. These sites were later shown to be high affinity amylin binding sites, and were first characterized in rat nucleus accumbens (Beaumont et al. 1993). The distribution of amylin binding sites in the rat brain was mapped in detail the following year (Sexton et al. 1994) and later in monkey brain (Paxinos et al. 2004). The high affinity amylin binding site displayed a distinctive pharmacology, not similar to any of the previously characterized CT or CGRP receptors, with a high affinity for sCT and a somewhat lower affinity for CGRP (Beaumont et al. 1993; Sexton et al. 1994). The specificity profile of the high affinity amylin binding site, i.e. the relative potency of peptides in competition binding was sCT ³ amylin > CGRP  CT. However, early evidence for potential heterogeneity in amylin binding sites was provided through differential sensitivity, to CGRP, of amylin binding to rat brain sections (Van Rossum et al. 1994). High affinity amylin binding sites were also identified in kidney (Chai et al. 1998; Wookey et al. 1996) and in skeletal muscle (Beaumont et al. 1995; Pittner et al. 1996).

3.2.2 Molecular Identity of Amylin Receptors While cDNA for various species of CT receptor had been isolated and characterized in the early 1990s (reviewed in Findlay and Sexton 2004; Sexton et al. 1999), initial attempts to unravel the molecular identity of amylin receptors failed to identify a unique cDNA that could account for the characterized phenotype. However, there was accumulating evidence of a potential relationship between amylin receptors and CT receptors. Amylin binding was usually associated with presence of CT receptors and indeed an amylin receptor phenotype was seen in cell lines such as the MCF-7 human breast carcinoma line (Zimmermann et al. 1997; Perry et al. 1997) and mouse a-TSH thryrotroph cells (Perry et al. 1997). In the latter cell line, C-terminally directed anti-CT receptor antibodies could also precipitate both amylin


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and CT receptors identified by radioligand cross-linking, suggesting a close relationship between these pharmacologically distinct receptors (Perry et al. 1997). The MCF-7 cells express both the major splice variants of the human CT receptor (Chen et al. 1997; Zimmermann et al. 1997), now termed hCT(a) receptor (CTRa) and hCT(b) receptor (CTRb) (Poyner et al. 2002), however, expression of these receptor isoforms in cell lines did not consistently lead to expression of high affinity amylin receptors (Chai et al. 1998; Perry et al. 1997). Likewise, the levels of the hCT(a) and hCT(b) isoforms were similar between MCF-7 cells that expressed an amylin receptor phenotype and T47D breast cancer cells that did not (Chen et al. 1997). Nonetheless, it was apparent that in certain cell lines, over-expression of CT receptors induced a variety of different potencies/affinities for amylin (e.g. Chen et al. 1997). These data implied a link between expression of CT receptors and specific cellular backgrounds for the generation of amylin receptor phenotypes. The enigma surrounding the molecular nature of amylin receptors was solved in 1998 following the discovery of receptor activity modifying proteins (RAMPs). Indeed, following the discovery of RAMPs (see below), most of the previous observations could be reconciled with the different endogenous background levels of RAMPs expressed in the cells.

3.2.3 Receptor Activity Modifying Proteins The discovery of RAMPs by the lab of Steven Foord, as a family of single pass transmembrane spanning accessory proteins that are required for the expression of functional AM and CGRP receptors was a breakthrough (McLatchie et al. 1998). RAMPs were found to be required for transport of the CTR-like receptor (CLR) to the cell surface, and to regulate the ligand specificity of the mature receptor in a RAMP isoform specific manner. Co-expression of RAMP1 with CLR generated a receptor complex with CGRP receptor pharmacology (CGRP1 receptor), whereas co-expression of RAMP2 or RAMP3 with CLR generated a receptor complex with AM receptor pharmacology (AM1 and AM2 receptors respectively) (McLatchie et al. 1998; Buhlmann et al. 1999; Fraser et al. 1999). Neither RAMPs expressed alone or CLR expressed alone enabled a phenotype in naïve mammalian cells and it was only the combination of the two that engendered the CGRP and AM receptors. In part, the lack of phenotype for the individual proteins was due to the intracellular retention of the individually expressed proteins (McLatchie et al. 1998), although cell surface expression of CLR is, by itself, insufficient to generate CGRP or AM receptors, and it is only the RAMP/CLR complex that gives rise to functional receptors. For CLR, RAMP interaction enhances transit through the Golgi and leads to terminal glycosylation of the receptor (McLatchie et al. 1998; Hilairet et al. 2001a). The three RAMPs share ~30% amino acid sequence identity, and all have a similar basic structure consisting of a relatively large extracellular N-terminal domain containing a signal peptide and four conserved cysteines thought to be important for secondary structure (RAMPs 1 and 3 have an additional pair of cysteines that are not

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present in RAMP2), a single transmembrane domain and a short intracellular C-terminal domain of ~10 amino acids. RAMP2 is the least conserved, being 26 amino acids longer than the 148 amino acid RAMP1 and RAMP3. RAMP tissue distribution was addressed by northern blotting to human tissue samples. The RAMP1 gene was expressed in many tissues, including uterus, bladder, brain, pancreas and gastrointestinal tract. RAMPs 2 and 3 had a similar, but not identical, tissue distribution and are expressed strongly in the lung, breast, immune system and fetal tissues (McLatchie et al. 1998). Having established three biological functions of RAMPs; transport of CLR to the cell surface, defining CLR pharmacology/CLR glycosylation state, the structural domains of RAMPs important for generating CGRP and AM receptors was explored using chimeras of RAMP1 and RAMP2 (Fraser et al. 1999). Chimeras of the RAMP2 transmembrane domain and C-terminal tail were generated with the RAMP1 N-terminus and vice versa. Co-expression of the CLR in HEK293T cells with the RAMP1/2 and RAMP2/1 chimeras generated CGRP or AM receptors with same efficiency and glycosylation state as native RAMP1 and RAMP2 respectively, and thus it was concluded that the N-terminal region of RAMP determine the glycosylation state and ligand binding specificity of the CLR. The CTR shares ~55% amino acid sequence identity with the CLR, and the discovery of RAMPs and the nature of CGRP/AM receptors as CLR:RAMP complexes provided the missing link in the conundrum of amylin receptors. Receptors exhibiting an amylin receptor phenotype were revealed when CTRa was coexpressed with RAMP1 or RAMP3 in COS7 cells (Christopoulos et al. 1999) or rabbit aortic endothelial cells (RAECs) (Muff et al. 1999). As observed with the CLR, the amylin receptor phenotype was dependent on the RAMP isoform present. RAMP1 and RAMP3 generated amylin receptors with different CGRP affinities (CGRP affinity for CTR:RAMP3 was markedly reduced compared to CTR:RAMP1), while RAMP2 was relatively ineffective in inducing amylin receptor phenotype in these cellular backgrounds. With the existence of two major isoforms of the human CTR (CT(a) and CT(b)), three RAMP isoforms, and the discovery that RAMPs define amylin receptor pharmacology, there is potential for at least six subtypes of amylin receptor (Fig. 3.2).

3.2.4 Calcitonin Family GPCRs The CTs are 32 amino acid peptide hormones with both peripheral and central actions mediated via specific receptors. The CT family of G protein coupledreceptors (GPCRs) are categorised as Family B or secretin-like GPCRs. These receptors are thought to share the same topology as other GPCRs with seven putative transmembrane segments and interaction with heterotrimeric GTPases (G proteins) to regulate signaling pathways of intracellular second messengers such as Ca2+, cAMP and inositol phosphates. Family B GPCRs are approximately 500 amino acids with a relatively large extracellular N-terminal domain of >150 amino


47 Amylin 2 receptor (AMY2R)

Amylin 3 receptor (AMY3R)

RAMP2 Endoplasmic Recticulum

RAMP3

RAMP1

Amylin 1 receptor (AMY1R)

CTR

Calcitonin receptor

Fig. 3.2 A representative diagram of complex formation between the CT receptor (CTR) and RAMPs 1, 2 and 3 in the endoplasmic reticulum. All three RAMPs are capable of existing as homodimers in the ER, however in the presence of CTR an equilibrium is established in which hetero-oligomers between the receptor and each of the RAMPs are formed. To date, the stoichiometry of the oligomers remains unclear. AMY1R, AMY2R and AMY3R are generated when the CTR is complexed with RAMP1, 2 and RAMP3, respectively. In addition, although the oligomers between the CTR and RAMPs can then be transported to the cell surface, the CTR is able to be transported to the cell surface in the absence of RAMPs. The human CTR has two major splice variants CT(a) and CT(b) that arise from absence or presence of a 16 amino acid insert in intracellular domain 1. The nomenclature for amylin receptors formed by the different receptor isoforms is denoted by (a) or (b) in the name (e.g. AMY1a denotes an amylin receptor formed from the CT(a) receptor and RAMP1)

acids that contains three conserved disulfide bonds for structural stability and a signal peptide for plasma membrane translocation. When the porcine CTR was cloned in 1991 (Lin et al. 1991) it was one of the first family B GPCRs to be characterized, following closely after receptors for parathyroid hormone and secretin. Not long after, the first human CTR was cloned, named CTR1 (current nomenclature hCT(b)) (Gorn et al. 1992); a second CTR (current nomenclature hCT(a)), was also identified (Kuestner et al. 1994). The human CTR gene includes at least 12 coding and 2 non-coding exons and is located on chromosome 7q21.3 (Kuestner et al. 1994). The two major variants of the human CTR are a result of alternative splicing; hCT(a) differs from hCT(b) in the deletion of 48 bp encoding 16 amino acids in the first intracellular loop (Kuestner

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et al. 1994). The CTRa variant is the most commonly expressed and the receptor used in the initial studies identifying amylin receptors, but both receptor isoforms are extensively expressed, with the hCT(a) receptors occurring in all receptor expressing tissues and the hCT(b) in many tissues including particularly high mRNA expression levels in placenta, ovary, lung and bone marrow (Kuestner et al. 1994). The CTR, although being the most similar receptor to CLR, does not require RAMP co-expression for plasma membrane localization, and is highly expressed at the cell surface by itself. CTRs alone exhibit a general relative potency of sCT ³ pCT ³hCT> amylin, CGRP, AM, with little differences in relative potencies between CTRa and CTRb. CTRs couple promiscuously to G-proteins and activate multiple intracellular signal transduction pathways. This includes Gas proteins and subsequent activation of the adenylyl cyclase (AC)/cAMP-protein kinase A (PKA) pathway, Gaq proteins and subsequent activation of the phospholipase C (PLC) pathway leading to inositol phosphate (IP) hydrolysis and mobilization of Ca2+ from intracellular stores (Kuestner et al. 1994; Moore et al. 1995; Sexton et al. 1993). Activation of the cAMP stimulating pathway was the most potent response to ligand binding, while the agonist potency for PLC stimulation observed was at least 100-fold lower than seen for cAMP, when studied in equivalent cell backgrounds (Moore et al. 1995). The presence of the 16 amino acid insert in the first intracellular loop of hCT(b) receptor has quite profound consequences for the receptor’s ability to activate intracellular signaling pathways, with loss of Gaq mediated PLC activation, attenuation of Gas mediated cAMP stimulation and reduced agonist-induced internalization (Moore et al. 1995; Nussenzveig et al. 1994; Raggatt et al. 2000). Both hCT(a) and hCT(b) receptors have been reported to exhibit constitutive signaling activity to the cAMP pathway when transiently expressed in COS-1 or Mardin-Darby canine kidney (MDCK) cells (Cohen et al. 1997), however this constitutive activity may be related to the polymorphic CTR variant studied. Nakamura and colleagues reported a CTR polymorphism in amino acid position 447 (463 of CTRb) of either a leucine (Leu) or proline (Pro) (Nakamura et al. 1997).

3.2.5 Amylin Receptor Pharmacology Amylin receptors are composed of the CT(a) or CT(b) receptor in complex with RAMP1, RAMP2 or RAMP3, forming AMY1a, AMY1b, AMY2a, AMY2b, AMY3a and AMY3b receptors, respectively. Each of these receptors, while binding amylin with similar affinity, has a distinct agonist and antagonist pharmacology. The ability of RAMPs to switch CTRs from CT receptor phenotype to amylin receptor phenotype is dependent on the receptor isoform (Tilakaratne et al. 2000) and cellular background (Christopoulos et al. 1999; Muff et al. 1999; Tilakaratne et al. 2000; Armour et al. 1999). In COS7, HEK293 and RAEC, RAMPs 1 and 3 induce distinct amylin receptor phenotypes when co-expressed with hCT(a). The CTR expressed alone has high affinity for mammalian CTs and low affinity for amylin and CGRP,


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whereas the AMY1a receptor has high affinity for both amylin and CGRP, and AMY3a receptor has lower affinity for CGRP. RAMP2 did not induce a strong amylin receptor phenotype in these systems (Christopoulos et al. 1999; Muff et al. 1999), nonetheless, RAMP2-based AMY2a receptors in COS7 cells also have reduced affinity for CGRP (Zumpe et al. 2000). In contrast, all three RAMPs are capable of generating AMY receptors in CHO cells when expressed with the hCT(a) receptor. However, co-expression of all three RAMPs with CTRb isoform generated strong amylin receptor phenotype in both COS7 and CHO-P cells (Tilakaratne et al. 2000), thus the efficiency of RAMP2 to induce amylin receptor phenotype, seems to be particularly dependent on CTR isoform and host cell environment. The amylin receptors generated from hCT(b) receptors in COS7 cells displayed a twosite binding profile (high and low affinity), in contrast to the single site binding observed with hCT(a)/RAMP co-expression, in this cell background. Low affinity sites of AMY1b or AMY3b constituted the majority of the binding sites and were equivalent to the single affinity sites observed with AMY1a or AMY3a receptors, respectively (Tilakaratne et al. 2000), and similar to previous observations with CTRa in COS7 cells; RAMP1 generated amylin receptors had increased affinity for both amylin and CGRP, whereas the RAMP3 derived phenotype had relatively poor CGRP affinity. The AMY1a receptor profile with high CGRP affinity, observed in these co-expression studies, corresponds in its pharmacology to the amylin binding sites in the nucleus accumbens core and amygdala, and the AMY3a receptor profile with lower CGRP affinity correspondingly resembles the amylin binding sites in the dorsomedial and arcuate hypothalamic nuclei (Van Rossum et al. 1994). The relatively high CGRP affinity of AMY1 receptors probably accounts for some of the earlier reported CGRP2 receptor pharmacology (Poyner et al. 2002; Hay et al. 2005; Kuwasako et al. 2004). CGRP2 receptors are characterized by weak antagonism by CGRP8–37 (CGRP antagonist fragment) and a similar result is seen with AMY1a (Hay et al. 2005). 3.2.5.1 Agonist Pharmacology To distinguish the pharmacology of AMY1, AMY2 and AMY3 receptors, Hay and colleagues used a range of agonists and antagonists, measuring cAMP accumulation in COS7 cells expressing hCT(a) receptor alone or with RAMPs (Hay et al. 2005). Similar to previous observations, the AMY2 was not well expressed in the COS7 cellular environment, and thus the authors focused on pharmacological discrimination of AMY1 and AMY3. The agonist pharmacology was investigated using hCT, rat amylin, hCGRPa, hCGRPb, Tyr0-hCGRPa, (Cys(ACM)2,7)hCGRPa, (Cys(Et)2,7)hCGRPa, hAM and hAM2 short (40 amino acids). Amylin had similar high potency at AMY1a and AMY3a receptors, whereas AM had only equivalent weak potency at CT(a) and AMY receptors. The CGRPs and analogues thereof displayed higher potency at AMY1a than AMY3a receptors, but had lower potency for CT(a) receptors for both a− and b−CGRP. The linear analogues of CGRP were weak partial agonists of AMY1a receptors but were

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effectively inactive at AMY3a receptors (Hay et al. 2005). AM2 short, which has similar potency across CLR-based AM and CGRP receptors, displayed relative potencies that tracked with the CGRP peptides, being more potent at AMY1a versus AMY3a receptors. Nonetheless, AM2 short was only a weak agonist relative to CGRP at each of these receptors. The potency of hCT was similar for CT(a) receptors alone and for AMY1a receptors derived from CTRa and RAMP1 cotransfection, whereas potency was reduced when CTRa and RAMP3 were co-transfected (Hay et al. 2005). As the affinity of hCT is similar between AMY1a and AMY3a receptors (Christopoulos et al. 1999), the decreased potency of hCT was interpreted as likely representing greater depletion of free CT(a) receptors by RAMP3. This was supported by lack of antagonism of hCT responses by CGRP8–37 at any of the receptor subtypes, while blocking amylin and CGRP mediated responses from AMY receptors (Hay et al. 2005). AMY2a pharmacology has yet to be fully defined due to complications with expression of phenotype in naïve cell lines such as COS7 (Christopoulos et al. 1999; Muff et al. 1999; Hay et al. 2005), although binding competition studies indicate that the AMY2a receptor may have higher affinity for hCT than AMY1a or AMY3a receptors (Tilakaratne et al. 2000; Zumpe et al. 2000). 3.2.5.2 Antagonist Pharmacology Antagonist pharmacology was investigated using sCT8–32, acetylated sCT8–32/rat amylin chimeras (AC187 and AC413), hCGRPa8–37, rat amylin8–37 and hAM22–52. The antagonist peptide fragment of salmon CT (sCT8–32) was the most effective antagonist with a pKB of ~8 across all examined receptors, but it did not discriminate strongly between CT and AMY receptors. However, sCT8–32 does not interact with CLR-based CGRP receptors and can thus be used to discriminate between AMY and CGRP receptor-based CGRP responses. AC187 bound with high affinity to AMY1 and AMY3 receptors (approximately tenfold over CTR) and AC413 additionally exhibited modest selectivity for AMY1 over AMY3 receptors, with pKB values of 7.92 and 7.10, respectively. The CGRP1 receptor antagonist hCGRPa8–37 was selective for AMY1 and AMY3 over CTR, with no antagonism of agonist responses at CTR, however hCGRPa8–37 is only a relatively weak antagonist at AMY1 and AMY3 receptors. Rat amylin8–37 and hAM22–52 was essentially devoid of antagonist effect on any of the receptors tested (Hay et al. 2005). The small molecule antagonist BIBN4096BS has high affinity for the CGRP1 receptor (Doods et al. 2000), and acts in the interface between the CLR and RAMP1, with RAMP1 Trp74 having a key role in the high affinity BIBN4096BS binding (Mallee et al. 2002). Amylin receptors of RAMP1 and hCT(a) (AMY1a) were also found to be sensitive to BIBN4096BS antagonism, albeit being 150-fold less sensitive than CGRP1 receptors (Hay et al. 2006), in contrast neither AMY3a or AM2 receptors with RAMP3 in the receptor heterodimer, displayed any sensitivity to BIBN4096BS antagonism. Consistent with the CLR/RAMP1 based CGRP receptor, BIBN4096BS affinity was found to be dependent on the RAMP1 Trp74 residue, and reduced at


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AMY1a and CGRP1 receptors; when Trp74 was mutated to lysine or alanine. Mutation of the equivalent residue in RAMP3 (Glu74) to tryptophan conferred BIBN4096BS sensitivity at AMY3a and AM2 receptors, supporting the importance of the RAMP1 Trp74 in inducing BIBN4096BS sensitivity at AMY1a and CGRP1 receptors (Hay et al. 2006).

3.2.6 Structure–Function RAMPs have a relatively large extracellular domain, a transmembrane domain and a short intracellular domain of ~10 amino acids. Analysis of RAMP chimeras and deletion constructs has provided insight into domains of RAMPs that contribute to ligand binding and signaling specificity. Previous studies by Fraser and colleagues using RAMP chimeras to study the structural basis of RAMP activity (Fraser et al. 1999) suggested that the RAMP N-terminus was the primary domain involved in generating CGRP and AM receptor phenotypes from CLR. Later Fitzsimmons and colleagues demonstrated that the N-terminal domain of RAMP1 by itself is sufficient to generate functional CL receptors (Fitzsimmons et al. 2003). Furthermore, expression of the RAMP1 extracellular domain as a chimera with the transmembrane domain of platelet-derived growth factor receptor generated fully functional CGRP receptors with only ~10 fold loss in potency, compared to wild type receptors (Fitzsimmons et al. 2003). Zumpe and colleagues investigated the structural basis of RAMP activity in generating amylin receptors. In COS7 cells RAMP1 is more potent than RAMP2 in generating amylin receptor phenotype when co-expressed with hCT(a) receptors and using chimeras of the RAMP2 N-terminus attached to RAMP1 transmembrane domain and C-terminus and vice versa (RAMP1/2), revealed highest specific I125-rat amylin binding with RAMP1 or RAMP2/1, indicative of the transmembrane and C-terminal domains playing an important role in the degree of amylin receptor expression. As seen for the CLR, the N-terminal domain of RAMP determined the binding phenotype of the expressed amylin receptor with similar amylin receptor phenotypes observed, with RAMP1 and the RAMP1/2 chimera engendering higher hCGRPa and lower hCT affinities than the RAMP2 and RAMP2/1 induced phenotypes (Zumpe et al. 2000). C-terminal deletion studies have added further support for the importance of this short intracellular fragment of RAMPs (Udawela et al. 2006a). RAMPs without the last eight amino acids in the C-terminus, RAMP1Dc, RAMP2Dc and RAMP3Dc, respectively, had little effect on the signaling profile and plasma membrane trafficking of CLR-derived receptors, whereas its importance in generating functional amylin receptors was marked. Significant attenuation of AMY receptor phenotype induction, measured by I125-rat amylin binding, was observed with all three truncated RAMPs, with RAMP1Dc and RAMP2Dc based receptors being more impaired than those formed by RAMP3Dc. Interestingly, the loss of I125-rat amylin binding could be partially recovered with co-expression of Gas indicating that the C-terminal deletion leads to loss of G protein coupling. Deletion of the RAMP1 C-terminus also led

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to attenuation of CTR-dependent translocation of this RAMP to the cell surface (Udawela et al. 2006a), leading the authors to speculate that G proteins may be important for stabilization of the RAMP–CTR complex, and that this may, at least in part, contribute to the loss of I125-amylin binding. A role for the RAMP C-terminus in defining AMY receptor signaling is also supported by RAMP chimera studies, where only the C-terminus is exchanged between RAMPs 1 and 2 (Udawela et al. 2006b). In that study, presence of the RAMP1 C-terminus in the RAMP2/1 chimera specifically potentiated CGRP signaling, whereas CGRP responses were attenuated for the RAMP1/2 chimera. Thus, in contrast to the CLR, it seems the RAMP C-terminus is of vital importance to the G protein coupling of CTR-based AMY receptors, and that there are significant differences in signaling by CLR- and CTRbased receptors. This difference could be related to the role of receptor component protein (RCP) in CLR generated receptors. RCP is important in CGRP and AM receptor-mediated signaling, as antisense knock-down of RCP expression leads to significant attenuation of cAMP signaling (Evans et al. 2000); currently there is no evidence supporting a link between RCP and AMY receptor signaling. One could speculate that the RAMP C-terminus is of vital importance for amylin receptor G protein-coupling because there is no interaction with RCP, whereas for the CLR based receptors association with RCP promotes the G protein-coupling function, making the RAMP C-terminus of less importance for this function. In summary, the N-terminal domain is the principle domain involved in defining ligand binding specificity, whilst the combined transmembrane and C-terminal domain contribute to the degree of functional amylin receptor expression. The C-terminal domain itself contributes to the peptide signaling profile of the receptor complexes and may be directly involved in the interaction with G proteins.

3.2.7 Second Messenger Activation To date, only limited studies on AMY receptor signaling have been performed. Where investigated, AMY receptor stimulation has resulted in robust elevations in cAMP, including L6 myocytes (Zhu et al. 1991), hepatocytes (Houslay et al. 1994) and a-TSH thyrotrophs (Perry et al. 1997). The a-TSH cell line has an AMY1a-like receptor, having similar affinity for CGRP and amylin, in addition to an endogenous CTR. In this cell line both cAMP and intracellular calcium mobilization were measured, however, amylin only evoked responses via the cAMP pathway, suggesting relatively weak coupling to calcium mobilization (Perry et al. 1997). In recombinant systems, where there is molecular definition of the receptor phenotype, analysis of signaling has been limited to stimulation of cAMP production. In all cells examined to date, there is a robust increase in cAMP accumulation following stimulation of AMY receptors, including HEK293, COS7 and RAEC (Christopoulos et al. 1999, 2003; Muff et al. 1999; Hay et al. 2005; Kuwasako et al. 2004; Udawela et al. 2006a, b, 2008)


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As discussed above, AMY receptors are comprised of CTR/RAMP complexes and CTRs are known to pleiotropically couple to multiple G proteins, including Gas, Gaq and Gai/o to modulate pathways including generation of cAMP, mobilization of intracellular calcium, and various kinases including ERK1/2 and FAK (reviewed in Findlay and Sexton 2004; Sexton et al. 1999; Purdue et al. 2002). As such, AMY receptors have the capacity to also pleiotropically couple to multiple pathways through the CTR component of the complex, however, the effect of RAMP on signal preference has not been explored. Nonetheless, preliminary data suggests that AMY1a and AMY3a, at least in COS7 cells, may preferentially signal to Gas without parallel increases in signaling via either calcium or ERK1/2 activation (Morfis et al. 2008). This remains to be fully explored but may imply that RAMPs, in addition to determining binding phenotype, may also alter the profile of signal pathway activation. A similar action has been proposed for the VPAC1 receptor, where RAMP2 specifically augmented IP3 signaling (Christopoulos et al. 2003).

3.2.8 Regulation of Receptors The regulation and trafficking of amylin receptors has not, to date, been extensively studied, with current knowledge of regulation of RAMP containing receptors inferred from studies of the CLR–RAMP complex. Hilairet and colleagues investigated the regulation of the RAMP1 and CLR–RAMP1 complex in HEK293T cells (Hilairet et al. 2001b). When expressed alone RAMP1 was retained in the endoplasmic reticulum and Golgi, predominantly as a disulfide-linked homodimer, whereas when co-expressed with CLR, RAMP1 was primarily plasma membranelocated, in complex with the CLR. Upon agonist stimulation with CGRP, the CLR is phosphorylated and the CLR–RAMP1 complex internalized together in a dynamin and b-arrestin dependent manner (Hilairet et al. 2001b). Similarly, RAMP1 is also translocated to the cell surface by CTR (Christopoulos et al. 1999), although the trafficking of the receptor components following agonist stimulation has not been studied to date. More recent studies of RAMP trafficking properties have revealed that the PDZ binding domain in the C-terminal tail of RAMP3 (-DTLL motif, not conserved in RAMP1 or 2), may confer novel receptor regulation to RAMP3 complexed receptors, in a cell-dependent manner (Bomberger et al. 2005a, b). AM1 (CLR/RAMP2) receptors are targeted to the degradative pathway following internalization, whereas, AM2 (CLR/RAMP3) receptors can display altered, agonist-dependent trafficking, leading to receptor recycling (Bomberger et al. 2005a), or lack of internalization (Bomberger et al. 2005b), depending upon the presence of specific PDZ containing proteins; NSF promotes receptor recycling, while NHERF1 inhibits internalization (Bomberger et al. 2005a, b). This altered behavior was entirely dependent on the RAMP3 PDZ binding motif. By analogy, it is probable that regulation of AMY3 receptors may also be altered depending on the types/levels of PDZ containing accessory proteins present in the cells.

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3.2.9 Summary and Future Perspectives AMY receptors are comprised of complexes between CT receptors and one of three RAMPs. Each of these receptors has a unique pharmacology, although the AMY2 receptor has only had limited study. AMY receptors can couple strongly to cAMP-mediated signaling, but the full extent of signal engagement for each of these receptors remains to be elucidated. Physiologically amylin is a potent regulator of gastric emptying and feeding. It is currently used in the treatment of diabetes and is under investigation for the treatment of obesity. Further studies are needed to dissect the effects of CTR-RAMP oligomerization, on receptor regulation and signaling, as the functional significance of the AMY receptor subtypes remains unclear. One major challenge of the future will be to understand the in vivo rationale of the CT and AMY receptor diversity, and, if possible, exploit this knowledge for development of new pharmaceutical inventions and better specificity in targeting diseased organs. Development of non-peptide drugs for oral administration will likely require novel modes of screening if separation of action from the non-complexed CT receptor is desired and if selectivity for specific AMY receptor subtypes is required.

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