GPCR modulation by RAMPs

Pharmacology & Therapeutics 109 (2006) 173 – 197 www.elsevier.com/locate/pharmthera

Associate editor: J. Wess

GPCR modulation by RAMPs Debbie L. Hay a, David R. Poyner b, Patrick M. Sexton c,* a

c

School of Biological Sciences, University of Auckland, Symonds Street, Auckland, New Zealand b School of Life and Health Sciences, Aston University, Birmingham, B4 7ET, UK Howard Florey Institute, Level 2, Alan Gilbert Building, The University of Melbourne, 161 Barry Street, Carlton South, Victoria 3053, Australia

Abstract Our conceptual understanding of the molecular architecture of G-protein coupled receptors (GPCRs) has transformed over the last decade. Once considered as largely independent functional units (aside from their interaction with the G-protein itself), it is now clear that a single GPCR is but part of a multifaceted signaling complex, each component providing an additional layer of sophistication. Receptor activitymodifying proteins (RAMPs) provide a notable example of proteins that interact with GPCRs to modify their function. They act as pharmacological switches, modifying GPCR pharmacology for a particular subset of receptors. However, there is accumulating evidence that these ubiquitous proteins have a broader role, regulating signaling and receptor trafficking. This article aims to provide the reader with a comprehensive appraisal of RAMP literature and perhaps some insight into the impact that their discovery has had on those who study GPCRs. D 2005 Elsevier Inc. All rights reserved. Keywords: Calcitonin receptor; CL receptor; Family B GPCR; GPCR; RAMP; VPAC1 receptor Abbreviations: AM, adrenomedullin; AMY, amylin; CGRP, calcitonin gene-related peptide; CL, calcitonin receptor-like receptor; CT, calcitonin; GPCR, Gprotein coupled receptor; NHERF, Na+/H+ exchange regulatory factor; NSF, N-ethylmaleimide-sensitive factor; PI, phosphatidylinositol; PTH, parathyroid hormone; RAMP, receptor activity-modifying protein; RCP, receptor component protein; VPAC1, vasoactive intestinal peptide/pituitary adenylate cyclase activating peptide 1; VSMC, vascular smooth muscle cells.

Contents 1. 2. 3.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Receptor activity-modifying proteins. . . . . . . . . . . . . . . . . . 3.1. Primary sequence . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Post-translational modifications; disulphide bond formation and 3.3. Receptor and ligand interactions . . . . . . . . . . . . . . . . 3.4. Motifs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Genomic organization . . . . . . . . . . . . . . . . . . . . . . Receptor activity-modifying proteins and pharmacology. . . . . . . . 4.1. Calcitonin gene-related peptide/AM receptor paradigm . . . . . 4.2. Amylin receptor paradigm . . . . . . . . . . . . . . . . . . . 4.3. Receptor activity-modifying proteins as drug targets . . . . . . Localization/distribution of receptor activity-modifying proteins. . . . 5.1. General summary of distribution . . . . . . . . . . . . . . . . 5.2. Central nervous system distribution . . . . . . . . . . . . . . .

* Corresponding author. Tel.: +61 3 8344 1954; fax: +61 3 9348 1707. E-mail address: [email protected] (P.M. Sexton). 0163-7258/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pharmthera.2005.06.015

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5.3.

Correlation of receptor activity-modifying protein distribution with pharmacology . . 5.3.1. Calcitonin gene-related peptide and adrenomedullin. . . . . . . . . . . . . . 5.4. Concluding comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Receptor activity-modifying proteins and signaling . . . . . . . . . . . . . . . . . . . . . . 6.1. Calcitonin receptor-like receptor and calcitonin receptor-based receptors. . . . . . . . 6.2. Vasoactive intestinal peptide/pituitary adenylate cyclase activating peptide 1 receptor . 7. Receptor activity-modifying proteins and trafficking . . . . . . . . . . . . . . . . . . . . . 7.1. Receptor activity-modifying proteins as chaperones . . . . . . . . . . . . . . . . . . 7.2. Receptor activity-modifying proteins and receptor internalization/recycling . . . . . . 8. Interaction of receptor activity-modifying proteins with other proteins . . . . . . . . . . . . 8.1. G-protein coupled receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. Receptor activity-modifying protein dimers. . . . . . . . . . . . . . . . . . . . . . . 8.3. Other proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Receptor activity-modifying protein specificity . . . . . . . . . . . . . . . . . . . . . . . . 10. Receptor activity-modifying protein regulation (in disease/pathophysiological states) . . . . . 10.1. Receptor activity-modifying protein regulation in pregnancy . . . . . . . . . . . . . 10.2. Receptor activity-modifying protein regulation in cardiovascular disease . . . . . . . 10.2.1. In vivo models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.2. In vitro models. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3. Receptor activity-modifying protein regulation in hypertension . . . . . . . . . . . . 10.4. Receptor activity-modifying protein regulation in the kidney . . . . . . . . . . . . . 10.5. Receptor activity-modifying protein regulation in diabetes and obesity . . . . . . . . 10.6. Receptor activity-modifying protein regulation in sepsis . . . . . . . . . . . . . . . 10.7. Receptor activity-modifying protein regulation in hypoxia . . . . . . . . . . . . . . 10.8. Receptor activity-modifying protein regulation by dexamethasone . . . . . . . . . . 11. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Receptor activity-modifying proteins (RAMPs) form a small family of 3 single-transmembrane spanning proteins that can form complexes with G-protein coupled receptors (GPCRs), altering their trafficking, pharmacology, and/or signaling capabilities. The interactions of RAMPs with the calcitonin (CT) receptor-like receptor (CL) are the most extensively studied. These experiments have identified that CL/RAMP complexes are stable dimers that form and are processed in the endoplasmic reticulum/Golgi and stay in this form during transport to the cell surface, peptide interaction and receptor activation, internalization, and subsequent recycling or degradation. However, the RAMP subtype appears to affect the trafficking properties of the receptor complex, being governed by specific protein – protein interactions with the RAMP intracellular C-terminus. While RAMPs provide a mechanism for inducing diversity in receptor repertoire for the calcitonin family of peptides, there is now enticing evidence to suggest that these proteins have a much broader role. Indeed, they are ubiquitous proteins and may be found where calcitonin peptide family receptors are not. Investigation of the functional consequences of the novel interaction of RAMP2 with the vasoactive intestinal peptide/pituitary adenylate cyclase activating peptide 1 (VPAC1) receptor has indicated a novel role for RAMPs in the modulation of receptor signaling.

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RAMPs also interact with other family B GPCRs, but the significance of these interactions has yet to be determined.

2. Discovery RAMPs were first identified during attempts to expression clone a receptor for the neuropeptide calcitonin generelated peptide (CGRP; McLatchie et al., 1998). Historical evidence had suggested that CGRP acted through a GPCR, as its binding had proven sensitive to GTP analogues and stimulation of various tissues and cells led to the accumulation of cAMP, suggesting activation of a Gs-coupled GPCR. However, attempts to clone such a receptor proved difficult. A putative canine CGRP receptor, RDC-1, was identified in 1995, but the original findings have not been replicated and current IUPHAR guidelines do not consider this receptor a genuine CGRP receptor (Kapas & Clark, 1995; Poyner et al., 2002). Shortly afterward, a further orphan receptor (CL, a close homologue of the calcitonin receptor) was shown to be activated by CGRP when transfected into HEK293 cells (Aiyar et al., 1996). This finding posed something of a conundrum since earlier attempts to examine the function of this receptor (or its rat homologue) in Cos 7 cells had not given positive results with CGRP (Njuki et al., 1993; Fluhmann et al., 1995). Given the apparent functionality of the human CL receptor

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in HEK293 cells, the rat homologue was also transfected into this cell type and now responded to CGRP (Han et al., 1997). The authors speculated that there was a factor present in HEK293 cells that conferred high affinity for CGRP on the receptor. In 1998, McLatchie and colleagues confirmed this speculation and provided new insights into the way that GPCRs and their pharmacology can be regulated (McLatchie et al., 1998). It was discovered that a novel family of single transmembrane domain proteins, termed RAMPs, was required for functional expression of CL at the cell surface, explaining why it had been so difficult to observe CGRP binding or function when CL was transfected into cells lacking RAMP expression (Fluhmann et al., 1995; Han et al., 1997; McLatchie et al., 1998). RAMPs were first identified from a library derived from SK-N-MC cells, cells known to express CGRP receptors. An expression-cloning strategy was utilized, whereby an SK-N-MC cDNA library was transcribed and the corresponding cRNA was used for injection into Xenopus oocytes. Cystic fibrosis transmembrane regulator chloride conductance, a reporter for cAMP formation, was strongly potentiated by a single cRNA pool (in the presence of CGRP). Subsequently, a single cDNA encoding a 148-amino-acid protein comprising RAMP1 was isolated. The structure of the protein was unexpected, as it was not a GPCR and it did not respond to CGRP in mammalian cells. Thus, it was postulated that RAMP1 might potentiate CGRP receptors. A CL/RAMP1 co-transfection experiment supported this hypothesis, whereby robust CGRP-mediated cAMP responses and binding were observed in HEK293T cells expressing both proteins. It has been demonstrated that some HEK293 cell lines contain endogenous RAMP1, explaining the original findings of Aiyar et al. (1996). In fact, neither CL nor RAMP1 alone is expressed effectively at the cell surface and both are required for effective transport of a CGRP-binding complex to the cell surface. In contrast, RAMP1 did not interact with RDC-1 (McLatchie et al., 1998). The concept that an accessory protein was required for expression of a GPCR was not completely novel; in Caenorhabditis elegans, ODR4 had been shown to control the expression of olfactory receptors and the cyclophilin NinaA aided the expression of opsins in Drosophila (Baker et al., 1994; Dwyer et al., 1998). More recently, it has been shown that the response of frizzled receptors to their ligands (Wnts), appears to involve low-density lipoprotein receptorrelated proteins (Wehrli et al., 2000) or the modified receptor tyrosine kinase Ryk (Lu et al., 2004). It is also now known that GPCRs can heterodimerize with each other (Bouvier, 2001). The revolutionary finding of McLatchie and colleagues was the observation that the 2 further RAMP1-like sequences that they had identified could also interact with CL, but instead of forming CGRP receptors, high-affinity receptors for the related peptide, adrenomedullin (AM), were generated. Indeed, the long extracellular N-terminus of the RAMPs suggested that these proteins may

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have some interaction with the ligands for the receptors, that is, CGRP/AM on the outside of the cell and did not simply act as anchoring/chaperone proteins for CL. RAMPs therefore provide a novel mechanism for modulating receptor– ligand specificity. The unique pharmacological profiles supported by RAMPs are discussed in later sections.

3. Receptor activity-modifying proteins 3.1. Primary sequence All RAMPs are built around a common structure and form a small family of proteins, each possessing a single transmembrane a-helix, an extracellular amino terminus, and a short intracellular C-terminus. Three human RAMPs have been cloned, RAMP1, RAMP2 (both cloned from SKN-MC cells), and RAMP3 (cloned from human spleen; McLatchie et al., 1998). The sequences of the known RAMPs are illustrated in Fig. 1. Full RAMP sequences are now available from 9 species, with partial sequence data from another 2. As well as mammals, sequences are available from the zebra fish (Danio rerio, RAMP1) and chick (i.e., red Guinea fowl, Gallus gallus; RAMPs 1, 2, and 3). A predicted RAMP-like protein has also been identified from the fish Tetraodon nigroviridis; this shows only weak homology to the other RAMPs (Fig. 1). A search of available T. nigroviridis genomic databases reveals that there are several potential sequences that show significant homology with RAMPs, some with higher homology to RAMPs 1, 2, and 3 than the predicted RAMP protein. A similar pattern can be seen in the Takifugu rubripes genome. Until these genomes are fully annotated, it is difficult to know how far this apparent diversity is due to the presence of pseudogenes. However, given the adaptive radiance shown by members of the CGRP/AM family in teleosts (in the T. rubripes genome, there are 6 AM homologues, 2 CGRP homologues, 2 calcitonin homologues, and 1 amylin [AMY] homologue), it would not be surprising if there was extra diversity also in the RAMP family. There is no evidence for RAMPs in any nonchordate genome. However, family B GPCRs are much older, being present in nematodes. GPCRs with noticeable homology to the calcitonin and CL receptors have been found in arthropods and mollusks (Dubos et al., 2003; Johnson et al., 2005). Most interestingly, the Drosophila equivalent, CG17415, shows an enhanced response to the hormone DH31 when coexpressed with human RAMP1 or 2, or human or Drosophila receptor component protein (RCP; Johnson et al., 2005) in NIH3T3 or HEK293 cells. RCP is a hydrophilic 148-amino-acid peripheral membrane protein that is required for efficient coupling of activated RAMP1 (or 2)/CL to Gs (Prado et al., 2001). DH31 shows weak homology to calcitonin, particularly at its C-terminus, although it lacks the archetypal N-terminal disulphide bond (Coast et al., 2001). It is curious that while there is a

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MaR1 HR1 CR1 RmR1 RR1 MR1 GPR1 PR1 GFR1 ZFR1

AAM15872 NP005846 XP516183 AAW48292 NP113833 Q9WTJ5 Q8R4C6 NP999364 XP421873 AAH76448

---------------------------------------------------------ALLQKLCLTQFQVDMEAVGETL MARALCR--LPRRGLWLLLAHH--LFMTTA---------------------CQEANYGALLRELCLTQFQVDMEAVGETL MARALCR--LPRRGLWLLLAHH--LFMATA---------------------CQEANYGALLQELCLTQFQVDMEAVGETL ---------------------------------------------------------ALLQKLCLTQFQVDMEAVGETL MVRVLRG--LPWRGLWLLLAHQ--LFLVTA---------------------CQDAHYGTLMQELCLSRFQKDMEAMERTL MAPGLRG--LPRCGLWLLLAHH--LFMVTA---------------------CRDPDYGTLIQELCLSRFKENMETIGKTL MAPGLRG--LPRRGLWLLLAHH--LFMVTA---------------------CRDPDYGTLIQELCLSRFKEDMETIGKTL MARGLRG--LPRRGLWLLLVNH--LFLATA---------------------CQDTDHAALLRKYCLPQFQVDMEAIGKAL MAL------LPRRFLCFVLAHH--FIAATA---------------------CHEAEYGRQIRERCLRPFKLSMEGIGQRL MASVSWRHAL------FVLVAVNLLVLARA---------------------CSS-HYGSAIEEFCMAKFKLDMEVLDQRQ

MaR1 HR1 CR1 RmR1 GPR1 RR1 MR1 PR1 GFR1 ZFR1

WCDWGKTIRSYRDLADCTWQVTEKLGCFWPNAEVDRFFLAVHGHYFRSCPVSGRAVRDPPSS------------------------------WCDWGRTIRSYRELADCTWHMAEKLGCFWPNAEVDRFFLAVHGRYFRSCPISGRAVRDPPGSILYPFIVVPITVTLLVTALVVWQ-SKRTEGIV WCDWGRTIRSYRELADCTWHMAEKLGCFWPNAEVDRFFLAVHGRYFRSCPISGRAVRDPPGSILYPFIMVPITVTLLVTALVVWQ-SKRTEGIV WCDWGRTIGSYRELADCTWHMAEKLGCFWPNAEVDRFFLAVHGHYFRACPISGRAVRDPPGS WCDWGKTIGSYGELTDCTRNLAERLGCFWPNVEVDRFFVAVHRHYFRSCPASGRALGDPPSTILCPFVVLPITVTLLVTALVVW-RSKRAESIV WCDWGKTIGSYGELTHCTKLVANKIGCFWPNPEVDKFFIAVHHRYFSKCPVSGRALRDPPNSILCPFIVLPITVTLLMTALVVW-RSKRTEGIV WCDWGKTIQSYGELTYCTKHVAHTIGCFWPNPEVDRFFIAVHHRYFSKCPISGRALRDPPNSILCPFIALPITVTLLMTALVVW-RSKRTEGIV WCDWDKTIGSYKDLSDCTRLVAQRLDCFWPNAAVDKFFLGVHQQYFRNCPVSGRALQDPPSSVLCPFIVVPILATLLMTALVVWQRSKRPEGIV WCDWDETMGTYGELTNCTVAVAENLTCYWPNRLVDEFFVAVHSHYFRNCSPSGRALRDPPNSILCPFILVPILVTLLMTALVVW-RSKRSEGIV WCSWEDTVESYGELTNCTFLVALKMNCFWPNRMVDEFFIRVHRHYFHDCSLSGRLLHDPPNRILGPFIVVPILVTLLMTALVVW-RSKRSEGIV

HR2 CR2 RR2 MR2 GPR2 PR2 DR2 GFR2

NP005845 XP511520 NP113834 AAH69992 Q8R4C5 NP999247 XP537636 XP418143

HR2 CR2 RR2 MR2 GPR2 PR2 DR2

WCDWAMISRPYSTLRDCLEHFAELFDLGFPNPLAERIIFETHQIHFANCSLVQPTFSDPPEDVLLAMIIAPICLIPFLITLVVW-RSKDSEAQA WCDWAMISRPYSTLRDCLEHFAELFDLGFPNPLAERIIFETHQIHFANCSLVQPTFSDPPEDVLLAMIIAPICLIPFLITLVVW-RSKDSEAQA WCNWTLISRYYSNLRYCLEYEADKFGLGFPNPLAESIILEAHLIHFANCSLVQPTFSDPPEDVLLAMIIAPICLIPFLVTLVVW-RSKDGDAQA WCNWTLISRHYSDLQNCLEYNADKFGLGFPNPLAENIILEAHLIHFANCSLVQPTFSDPPEDVLLAMIIAPICLIPFLVTLVVW-RSKDSDAQA WCNWTVISRPYSALRDCLEVEAEVFSLGFPNPLAERVIFETHQLHFSNCSLEQPTLCDPPEDVLLAMIIAPICLIPFFVTLVVW-RSKGTELKT WCDWALISRPYSILQECLEQKADAFKLGFPNPWAERIIFEAHRIHFANCSLVQPTFSDPPEDVLLAMIMVPICLIPFLVTLVVW-RSKDPEAQT WCDWAMISRPYSDLQYCLEHFAEAFGLGFPNPWAEEVIFETHQIHFANCSLVQPTLSDPPEDVLLAMIIAPICLIPFLVTLVVW-RSKDNEAQA

MASLRVERAGGP-RLPRTRVGRP------------------AALRLLLLLGAVLNPHEALA--------QPLPTTGTPGSEGGTVK-NYETAVQFCWNHYKDQMDPIEK-D MQ-RRREAAGAPSPLPLSDPGRPWRVLGATRPSPRNRSRFRGSGSCPAS-RAVLNPHEALA--------QPLPTTGTPGSEGGTVK-NYETAVQFCWNHYKDQMDSIEK-D MAPLRVERAPGGSQLAVTSAQRP----------AALRLPPLLLLLLLLLLGAVSTSPESLN------- QSHPTEDSLLSKG-KME-DYETNVLPCWYYYKTSMDSV-K-D MAPLRVERAPGGSRLGVTRAQRPTALCLPP-----------LLLLLLLLLGAVSASPESLNQSLPESQNQSHPTEDSLVSKG-KME-DYETHVLPCWYEYKSCMDSV-K-D MA----------ARLRP--------------------------LLALLALAAL-CPQETLA--------QPLPTTDTWKSEGQVVD-SYEASAQLCWADYREHMDLLEK-D MASLRAERAAGGPRLPATRAGRP------------------AALRLLLLLGAVLKPQESLA--------QHFPTPDYLNLEGKTLEENYETDAQLCWHDYKDYMDSIKK-D MASLRAERAASGPRLPATRAGRP------------------AALRLLLLLGAVLKPQESLA--------QLLPTEGSLKLEGNKMA-DYETSVQFCWQSYKEQMDSIPK-D MAPCAQMGSGHLS--------RG----------------LLLLCALLGHQFCHVGATAEGFRQEA-RTSPPVSAYNHTGKLVEEIYTNLTHYCWESFVKVMQNVTGAQ

GFR2 LCEWKVISRPYSSLQKCLEDHADLLNYSYPNALAESYIFQSHHHYFQNCSAGSQAYFDPPEDVLLAMIIAPICLIPFLVTLVIW-RSKDGKAQP

HR3 RR3 MR3 GPR3 PR3 GFR3

AAH53852 NP064485 CAI24221 Q8R4C4 NP999254 XP418881

ME-T--GALRRPQLL----PLLLLLCG-GCPRAGG---------------------CNETGMLERLPL-CGKAFADMMGKVDVWK MA-T---PAQRLHLL----PLLLLLCG-ECAQVCG---------------------CNETGMLERLPR-CGKAFAEMMQKVDVWK MK-T---PAQRLHLL----PLLLLLCG-ECAQVCG---------------------CNETGMLERLPR-CGKAFADMMQKVAVWK MG T---RSRRPQLL-W----LLLLCG-TCARVCG---------------------CNETRMLERLPR-CGKTFAERMREVAVWK MEATAPRRRHLLPLLL----LLLLLCG-ECPPVSG---------------------CNEKRMLAMLPR-CGKTFAEMMKKVEVWK MEASGRCRRHLPVLLLWVNGLMMLGLAGVHQQINL---------------------CNESLMLERLPA-CGKFFEEMMKKVDSKK

HR3 RR3 MR3 GPR3 PR3 GFR3

WCNLSEFIVYYESFTNCTEMEANVVGCYWPNPLAQGFITGIHRQFFSNCTVDRVHLEDPPDEVLIPLIVIPVVLTVAMAGLVVW-RSKRTDTLL WCNLSEFIVYYESFTNCTEMETNIVGCYWPNPLAQSFITGIHRQFFSNCTVDRTHWEDPPDEVLI-LIAVPVLLTVAMAGLVVW-RSKHTDRLL WCNLSEFIVYYESFTNCTEMETNIMGCYWPNPLAQSFITGIHRQFFSNCTVDRTHWEDPPDEVLIPLIAVPVVLTVAMAGLVVW-RSKHTDRLL WCDLSQFIVFYESFTNCTEEETVVVGCYWPNPLAQGFITGVHRQFFSNCTVDRTHWEDPPDEVLIPLIAVPILLTVAMTGLVVW-RSKRTDQLP WCNLSEFIVYYESFTNCTEVETNVVGCYWPNPLAQSFITGVHRRHFHNCSVDRQQWQDPPDEILIPLIVVPILLTLAMTGLVVW-RSKRAAQVV WCNLTEFITYYNIFTQCTEREANDASCFWPNPLAESFITGIHKQFFLNCTLDNVHWEDPPDEILIPLILIPVMLTCAMIMLVVW-CSKRSDILG

TN CAF98963 TN

MLLTALPVV-------LLICS-GTAVKINIPP-----------------CDQ-HMFNSNVEDCLSDFNTSLEMSDHQY SCPWPAVKPMYYKLKLCVDNLAITSSCMGHRSLGD-FFLKVHNMYFSSCG----EVRDPPLTTVILLIGPLTLLTLVLPHFCVF-LTTRDP

Fig. 1. Comparison of RAMP sequences. GenBank accession numbers are given for reference. Dark grey shading: sequence identity amongst all RAMPs. Light grey shading: sequence identity within 1 RAMP family. (TN RAMP has been compared with both RAMP1 and RAMP2). Italics, putative signal peptides. Ma, marmoset (partial); H, human; C, chimp; RM, Rhesus monkey (partial); GP, guinea pig; R, rat; M, mouse; P, pig; D, dog; GF, guinea fowl; ZF, zebra fish; TN, Tetraodon nigroviridis. R1, RAMP1; R2, RAMP; R3, RAMP3.

Drosophila equivalent of RCP, there is no equivalent of the RAMPs; in spite of this, CG17415 can interact with RAMPs. It may be suggested that in Drosophila (and presumably in other nonchordate species that have CL or calcitonin receptor homologues), chaperones and scaffold proteins are required for efficient expression and signaling, but in these species, they do not alter ligand binding. It may be that there is a membrane protein similar to NinaA that acts as a chaperone for CG17145. According to this model, the characteristic N-terminus of the RAMPs evolved in chordates. This went with the evolution of CGRP and related peptides; the N-terminus of the RAMPs could interact with the N-terminus of the GPCR to increase receptor diversity, allowing recognition of the new CGRPrelated peptides.

The degree of residue identity varies with both the type of RAMP and the position in the sequence. The transmembrane regions are highly conserved. The N-terminus shows less conservation, particularly at its extreme amino terminus. Within the mammalian RAMPs, there is around 70% identity over the N-terminus using the human sequences as the base. The 1 exception to this is RAMP3, where the mouse and rat proteins show 92% identity with their human counterpart. The variability seen in the RAMPs is greater than that observed in either the corresponding CL receptors or in the sequences of CGRP and AM themselves, which are around 90% identical. Where known, there also appears less conservation of RAMPs compared with CL receptors or ligands in fish and the chick. As the CL receptor has to interact with several

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different ligands and RAMPs, it may be expected to be more structurally constrained than the RAMPs. The sequence differences between RAMPs may be of significance for receptor pharmacology, where RAMPs from 1 species are combined with receptors from another; the resulting complexes may show lower affinities for ligands than same-species combinations. This has been observed for human and rat RAMP2, where human RAMP2/rat CL receptor had a lower affinity for CGRP8 – 37 and AM22 – 52 than the all-rat or all-human combinations (Hay et al., 2003a). An even more dramatic effect was observed with salmon CL, which failed to respond to human AM when coexpressed with human RAMP2 in Cos 7 cells, although an AM response could be generated when it was coexpressed with human RAMP3 (Pidoux & Cressent, 2002). The ability of different species combinations to respond to CGRP or AM and produce cAMP will depend both on the Kd of the resulting RAMP/CL heterodimer for the agonist and also on its ability to recognize Gs and RCP. Differences in coupling efficiencies are also likely to manifest themselves as differences in EC50s for CGRP and AM. Thus, the potency of an agonist will depend not only on the species of the RAMPs and CL receptor, but also on the cell line. Predicting in vivo behavior from transfected cells is particularly problematic for such complicated systems. Depending on the species, there is a predicted Nterminal signal peptide of between 22 and 35 amino acids (RAMPs 1 and 3) or 24 and 60 amino acids (RAMP2; Fig. 2), although it should be noted that the prediction of signal peptide cleavage sites is not straightforward. There then follows an extracellular amino terminus of about 90 residues in RAMPs 1 and 3; in RAMP2, sequences there are normally an extra 13 amino acids. There is a transmembrane domain in the range of 22 amino acids and finally a C-terminal domain, usually consisting of 9 residues. All 3 RAMPs have 4 highly conserved cysteine residues that presumably form disulphide bonds; in RAMPs 1 and 3, an extra pair of cysteines is found. The known mammalian RAMP1s appear not be not glycosylated, although potential glycosylation sites are present in chick and zebrafish. RAMP2 and RAMP3 have multiple glycosylation sites. The majority of the known RAMP3s have 4 potential glycosylation sites (N29, N58, N71, and N104 in man), although only the fourth site (N104) is absolutely conserved. The equivalent of the fourth glycosylation site is also found in all known RAMP2s; in some

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mammalian species, there is also an equivalent of the second site (i.e., N58). 3.2. Post-translational modifications; disulphide bond formation and glycosylation No structures or models for any RAMP have yet been published. However, it is possible to identify a number of important structural elements (Figs. 1 and 2). The disulphide bonding pattern must play a key part in the folding of the Nterminus. As the first and fifth cysteines in RAMPs 1 and 3 are missing in RAMP2, it is likely that these form a disulphide bond in molecules where they are present. Steiner et al. (2003) demonstrated that the deletion or alanine substitution of these residues in human RAMP1 was without effect on CGRP receptor function; in contrast, modification of the other 4 cysteines greatly reduced or abolished cell-surface expression. Alanine substitution of the second or fourth cysteines reduced the maximal cAMP production to 14% and 33% of normal, but with only small changes (< 4-fold) in the EC50 of CGRP. By contrast, alanine substitution of the third and sixth cysteines rendered the complex with CL unresponsive to CGRP concentrations of at least 1 AM. Thus, the likely disulphide bond pattern is 2 –4 and 3 – 6, although this is admittedly based on indirect evidence. This was recently confirmed in abstracted work using mass spectrometry-based analysis of the isolated Nterminal domain of RAMP1 (Chang et al., 2005). Cysteines 1 and 5 probably form a disulphide bond, but clearly, this is not essential for the stability of the CL/RAMP1 complex. The 3 – 6 bond must be essential for correct processing of the RAMP. The 2 – 4 bond is likely important for processing, but it appears that a proportion of the RAMPs can still fold correctly in its absence and that these proteins can associate with CL and that the resulting complexes are able to interact with CGRP in a normal fashion. So far, only a single abstract has been published that addresses the role of cysteines in RAMP2 (Kuwasako et al., 2003c). In this study analyzing human RAMP2, it appears that the mutation of any 1 of the 4 cysteines to alanine prevents cell surface expression even in the presence of CL receptors. Thus, the formation of disulphide bonds appears even more important than in RAMP1. A different pattern appears in mouse RAMP3 (Flahaut et al., 2003). A mutant where all 6 cysteines had been changed to serine was still able to associate with CL and be expressed at the cell

1 10 20 30 40 50 RAMP1 MA--RALCR--LPRRGLWLLLAHH--LFMTTA-----------------CQEANYGALLRELCLTQFQVDMEAVGETL RAMP3 METGALRRPQLL----PLLLLLCG-GCPRAGG-----------------CNETGMLERLPL-CGKAFADMMGKVDVWK RAMP2 MASLRVERAGGP-RLPRTRVGRP-------------AALRLLLLLGAVLNPHEALA----QPLPTTGTPGSEGGTVK-NYETAVQFCWNHYKDQMDPIEK-D

RAMP1 RAMP3 RAMP2

60 70 80 90 100 110 120 130 140 WCDWGRTIRSYRELADCTWHMAEKLGCFWPNAEVDRFFLAVHGRYFRSCPISGRAVRDPPGSILYPFIVVPITVTLLVTALVVWQ-SKRTEGIV WCNLSEFIVYYESFTNCTEMEANVVGCYWPNPLAQGFITGIHRQFFSNCTVDRVHLEDPPDEVLIPLIVIPVVLTVAMAGLVVW-RSKRTDTLL WCDWAMISRPYSTLRDCLEHFAELFDLGFPNPLAERIIFETHQIHFANCSLVQPTFSDPPEDVLLAMIIAPICLIPFLITLVVW-RSKDSEAQA

Fig. 2. Structural features of human RAMPs. Dark shading, gylcosylation sites and conserved cysteines; light shading, regions implicated in ligand binding; italics, putative signal peptides.

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surface (albeit at about half the level of the normal protein), although it was no longer able to bind AM. Mutation of individual cysteines had little effect on the formation of a functional complex with CL; all complexes were expressed at the cell surface at > 70% the levels of normal CL receptor– RAMP3 and there was no more than a 4-fold reduction in AM potency. Thus, at least for mouse RAMP3, any individual disulphide bond is dispensable, although their total removal renders the protein nonfunctional. The lower impact of loss of disulphide bond formation for RAMP3 may, in part, be due to its higher level of glycosylation, which may, in turn, facilitate correct folding of RAMP3. Although cysteines in RAMPs almost certainly form intramolecular disulphides when the proteins form heterodimers with receptors, at least in RAMP1 their function appears to differ in homodimers. In the absence of receptors, RAMP1 accumulates as homodimers within the endoplasmic reticulum and Golgi; the stability of the dimers, however, is diminished by reducing agents, implying that these homodimers are stabilized to some extent by intermolecular disulphide bonds (Hilairet et al., 2001a). Unlike RAMPs 2 and 3, RAMP1 in mammals lacks glycosylation sites and is unable to reach the cell surface in the absence of a partner (but see Section 3.4, ‘‘Motifs’’). The introduction of N-glycosylation sites into mouse RAMP1 at positions corresponding to those of the second (N58), third (N71), and fourth glycosylation (N104) sites of RAMP3 allows RAMP1 to reach the cell surface by itself (Flahaut et al., 2002). Significantly, the latter 2 are potential glycosylation acceptor sites in chick RAMP1. The role of the 4 potential glycosylation sites in (mouse) RAMP3 has been studied by mutating the acceptor asparagines to serines. These experiments suggested that the third and fourth sites were glycosylated most efficiently. When all the cysteines were mutated to serines, then all 4 asparagines were glycosylated with equal efficiencies. Thus, the tertiary structure of the protein acts to inhibit glycosylation (Flahaut et al., 2003). It is significant that the glycosylation sites are all next to conserved cysteines. The mutant RAMP3 lacking all glycosylation sites could still associate with CL, although the cell surface expression of the complex was reduced by about 50%. However, the affinity for AM as measured in radioligand binding was apparently decreased about 300fold. This result implies that glycosylation, either directly or indirectly, is important in AM binding. As only the fourth glycosylation site (N104) of human RAMP3 is absolutely conserved in all RAMP3s, this is the residue that is most likely to be important. If the 3 – 6 cysteine disulphide bond is formed, it would be adjacent to a site implicated in AM binding (see Section 3.3). In functional assays, mutation of N104 alone to serine caused only about a 2-fold decrease in the potency of AM. However, mutation of all the glycosylation sites produced only a 6-fold decrease in potency, making it difficult to draw any conclusions about the relative importance of any single glycosylation site from

the data. Of note, in Cos 7 cells overexpressing RAMP3, a heterogeneous pattern of glycosylation is observed for monomeric RAMP3. In contrast, only 1 species of homodimer occurs, implying that only 1 of the glycosylated species forms the mature, functional RAMP3 (Sexton et al., 2001). Further studies are needed to address the role of glycosylation. Although both RAMP2 and RAMP3 are glycosylated and so can reach the plasma membrane in the absence of an accompanying receptor (Flahaut et al., 2002), the extent to which this happens in vivo is unclear, as the process is influenced by the nature of the signal peptide and any epitope tag that has been attached to the protein (Hilairet et al., 2001b; Christopoulos et al., 2003). As there are few, if any, reliable antibodies to native RAMPs, studies on subcellular/cellular RAMP distribution inevitably rely on tagged proteins. It seems likely that heterodimerization with an appropriate receptor facilitates the surface expression of all RAMPs, and this property provides a convenient assay for receptor– RAMP interaction (Christopoulos et al., 2003). In the early literature on RAMPs, there was much interest in the increase in CL receptor glycosylation noted when the receptor was expressed with RAMP1. There was speculation that this increase in glycosylation might explain the increase in CGRP affinity of the heterodimer (McLatchie et al., 1998). It is now recognized that the increase in glycosylation is unlikely to explain the change in pharmacology. While RAMP1 increases the rate of terminal glycosylation of CL, this occurs in the presence of RAMPs 2 and 3, and high affinity AM binding only occurs to heterodimers containing the fully glycosylated CL and it is only this form of the receptor that is present at the cell surface (Hilairet et al., 2001b). 3.3. Receptor and ligand interactions The precise nature of the interaction of RAMPs with their receptor partners is of considerable interest as RAMPs appear to produce their biological activities by interacting with receptors. Although it is now realized that they can associate with a range of family B GPCRs (see Sections 4.2, 6.2, 8), most structural information comes from studies on the CL receptor. Early studies involving RAMP1/2 chimeras indicated that the N-terminus of the RAMP is the major determinant of receptor pharmacology, with the transmembrane region contributing to the affinity of interaction between the receptor (CL or calcitonin) and the RAMP (Fraser et al., 1999; Zumpe et al., 2000). More recently, RAMP1 constructs have been examined in which the transmembrane domain has either been truncated (Steiner et al., 2002) or entirely deleted (Fitzsimmons et al., 2003). In both cases, there was impairment of function of the resulting complex with CL. The truncation studies suggested that as the transmembrane region was progressively reduced in size, coupling to Gs, then CGRP binding and, finally, CL/RAMP1 heterodimerization were reduced

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(Steiner et al., 2002). This is broadly consistent with the findings of Fitzsimmons et al. (2003), who showed that although it was possible to produce an active heterodimer between the CL receptor and just the N-terminus of RAMP1, CGRP was 4000-fold less potent than with fulllength RAMP1 (although the E max was, if anything, increased). Both studies also agreed that the isolated Nterminus (or a version with only 2 amino acids from the transmembrane domain) was able to produce the characteristic shift in the glycosylation of CL but that the heterodimer was unstable. Replacement of the transmembrane region of RAMP1 with that of the platelet derived growth factor receptor produced a partially functioning receptor, where the affinity for CGRP was reduced 10-fold but with no change in E max (Fitzsimmons et al., 2003). In summary, the data suggests that the transmembrane domain of the RAMP is not essential for the production of a CGRP receptor phenotype. However, in its absence, the heterodimer is less stable and does not properly interact with CGRP (in the case of RAMP1). Loss of stable interaction between CL and RAMP1 in these studies may be due, in part, to effects on the interaction of the RAMP/CL complex with RCP, as knockdown of RCP expression prevents co-immunoprecipitation of RAMP1 (or 2) with CL (Ian Dickerson, personal communication). A number of studies have looked at the role of different regions of the extracellular domain of the RAMPs, chiefly by deletion mutants or through chimeric exchange between RAMP1 and RAMP2. In human, RAMP2 residues 86 –92 are required for high affinity AM binding (Fig. 2); in human RAMP3, it is residues 59– 65 (homologous to 86 –92 in RAMP2; Kuwasako et al., 2001). In RAMP1, these residues are also needed for high affinity interactions with CGRP and AM (Kuwasako et al., 2003a). Alanine scans of these regions in RAMP2 showed that no single amino acid substitution caused a significant perturbation of cAMP production; therefore, more regions are likely to be involved in promoting AM interactions in RAMPs 2 and 3. Data from chimeric RAMPs suggested that the main AM-binding epitope in RAMP2 was located in a region from residues 77 to 101, but several deletion mutants within this stretch gave poor cell surface expression, precluding further analysis. In human RAMP1, residues 41 –45, 59– 65, 67– 71, and 91 – 103 were needed for high affinity CGRP interactions with the CL/RAMP heterodimer. The deletion of residues 78 –80 and 88 –90 allowed cAMP production by CGRP but not AM (Kuwasako et al., 2003a). Residues 91 –103 were subject to an alanine scan; no single residue was identified as important for CGRP binding, but L94A caused an increase in CL/RAMP1 complex formation as determined by ELISA of cell surface expression and maximum CGRP induced cAMP production, while F93A, Y100A, and F101A (and to a lesser extent H97A) led to decreased cell surface expression of the complex but no change in EC50 values for CGRP stimulation of cAMP accumulation (Kuwasako et al., 2003a). It is likely that the epitopes

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identified in these studies are, at least in part, responsible for stabilizing the interaction between the RAMPs and CL. The result with L94A is particularly interesting, as it suggests that the large leucine may sterically hinder the formation of a high affinity RAMP – receptor complex. A recent study has shed some light on the regions of the CL receptor that interact with RAMP1. The N-terminal amino acids 23 – 60 of mouse CL are required for its association with mouse RAMP1 (Ittner et al., 2005). Replacement of these residues with those of the parathyroid hormone (PTH) receptor (which does not interact with RAMP1) abolished RAMP1 trafficking to the cell surface, suggesting that RAMP1 interacts with the extreme Nterminus of CL (Ittner et al., 2005). In the absence of structural data, it is still only possible to speculate how RAMPs interact with CL and their respective ligands in broad terms. The original hypothesis that the TM domain of the RAMPs determined the affinity of interaction with the receptor while the N-terminus determined pharmacology and ligand binding needs to be qualified. The fact that substitution of the N-terminus of CL prevents stable association with RAMP1 demonstrates the importance of interactions between the N-termini for heterodimer formation. On the other hand, as substituting a completely different transmembrane domain into RAMP1 leads to a 10-fold reduction in affinity for CGRP, it appears that the transmembrane region has some role in stabilizing a form of the heterodimer that can recognize ligands with high affinity. It is probably over-simplistic to think of spatially distinct receptor and ligand interaction domains existing on RAMPs; while these may exist, it is possible that some parts of the RAMPs serve both functions. A RAMP – receptor interaction may also allosterically alter the conformation of the complex to increase affinity for a ligand. The delineation of a 6-amino-acid region in RAMPs 2 and 3 required for high affinity AM interactions almost certainly masks the fact that there is multipoint contact between the ligand and the RAMP – receptor complex; the multiple domains defined in RAMP1 are probably a good guide as to what is taking place in the other RAMPs. The inability to identify any single amino acid as being crucial for ligand binding also supports the idea of multiple-point contact. A major unresolved issue is whether the ligands are in direct contact with the RAMPs or whether the RAMPs act indirectly by altering the conformation of the receptor. These are not mutually exclusive and it may be best to envisage ligand contact sites occurring at the RAMP –receptor interfaces. Cross-linking has demonstrated that the RAMPs are in close proximity to CGRP or AM when bound to their respective receptors, but the technique lacks the resolution to show if there are specific residue– residue contacts (McLatchie et al., 1998; Hilairet et al., 2001b). Whatever the mechanism of action, it is not surprising that given an interaction involving 2 protein domains each of about 100 amino acids and ligands of 37

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to 52 amino acids, single amino acid substitutions have so far failed to identify key contact points. While ‘‘contact points’’ between RAMPs and endogenous ligands remain difficult to define, more success has been achieved with BIBN4096BS, a nonpeptide antagonist acting at CL/RAMP1 receptors (CGRP1 receptors; Doods et al., 2000; Fig. 3). BIBN4096BS has ¨ 100-fold selectivity in favor of primate versus rat CGRP1 receptors. The basis of this is the presence of a tryptophan at position 74 in primate RAMP1 sequences; in the rat (and other nonprimates), it is a lysine (Mallee et al., 2002). It remains unclear whether the tryptophan interacts directly with the drug molecule or whether it acts indirectly by modifying the conformation of either some other part of the RAMP1 or CL receptor. Recent data using chimeras of CL and the calcitonin receptor suggest that BIBN4096BS also requires the presence of CL before it will bind with high affinity (Salvatore et al., 2004; Fig. 3), and this is supported by data from calcitonin receptor– RAMP1 complexes where BIBN4096BS has only weak affinity (Debbie Hay, George Christopoulos, David Poyner, Patrick Sexton, unpublished data). 3.4. Motifs The C-termini of the RAMPs, while short, are potential interaction sites with other proteins. Two motifs have been identified. McLatchie et al. (1998) initially speculated that the 2 basic residues in the C-terminal tail of RAMPs 1 and 3 had the appearance of an endoplasmic retrieval signal. Subsequently, Steiner et al. (2002) demonstrated that in RAMP1, these were part of a 5-residue motif (QSKRT; Fig.

2) immediately adjacent to the plasma membrane that did indeed act as an endoplasmic reticulum retention signal. Even though RAMP1 is not glycosylated, when these residues were deleted, it was transported to the cell surface when transfected alone into Cos 7 cells. It remains to be established if this is seen in all cell lines. In human RAMP3, the final 4 amino acids, DTLL, form a type 1 PDZ1 recognition site. McLatchie and coworkers suggested a possible interaction of this site with the Na+/H+ exchange regulatory factor (NHERF); this would give a G-protein independent link to another intracellular signaling pathway. Such an interaction has recently been demonstrated both in HEK293 cells transfected with bovine CL and human RAMP3 and in human proximal tubule cells that endogenously express CL and RAMP3 (Bomberger et al., 2005b). The interaction of RAMP3 with NHERF-1 blocked the internalization of the receptor complex while desensitization of the receptor itself was unaltered. RAMPs 1 and 2, which do not have a PDZ recognition site, were internalized in the presence of NHERF-1 (Bomberger et al., 2005b). There is also good evidence that RAMP3 in humans and rats can interact with N-ethylmaleimide-sensitive factor (NSF) or an equivalent protein following internalization of CL/RAMP3 heterodimers, leading to efficient recycling (Bomberger et al., 2005a). Again, this is mediated by the PDZ-recognition site in human RAMP3. Although the motif is not fully conserved in RAMP3 from other species, other potential PDZ recognition sites are present in some of these (RLL in rat and mouse; Harris & Lim, 2001). RAMPs 1 and 2 do not recognize NSF, and heterodimers involving these

NH 2

H N

CGRP

O N N H N

N

W

N

O

74

N N H

O

O Br

OH Br

BIBN4096 BS

RAMP1

CL receptor

Fig. 3. CGRP1 receptor-specific small molecule antagonists. The small molecule antagonist BIBN4096 BS (brown) is a specific antagonist of the CGRP1 receptor, acting at the interface between RAMP1 and the CL receptor to inhibit CGRP action. At least part of the binding affinity for BIBN4096 BS arises from interaction with Trp74 (red) of RAMP1. In contrast, antagonists that bind principally to the CL component of the complex will not discriminate between different CL/RAMP complexes.

D.L. Hay et al. / Pharmacology & Therapeutics 109 (2006) 173 – 197

181

proteins are degraded following internalization (Kuwasako et al., 2000; Hilairet et al., 2001a; Bomberger et al., 2005a).

which RAMPs are expressed at any given time in cells that can express more than 1 RAMP subtype.

3.5. Genomic organization

4. Receptor activitymodifying proteins and pharmacology

In humans, the RAMP1 gene is present on chromosome 2 at 2q36– 37.3, RAMP2 is on chromosome 17 at 17q12 – 21.2, and RAMP3 is on chromosome 7 at 7p13– 12 (Derst et al., 2000). The gene for the CL receptor is also on chromosome 2, although not particularly close to RAMP1 (2q31 –32.1; Poyner et al., 2002). The calcitonin receptor is on the opposite arm of chromosome 7 from RAMP3 (7q21.3). The genes for the known mammalian RAMPs all have similar structures; those for RAMPs 1 and 3 have 3 exons, while that for RAMP2 has 4 exons (Sexton et al., 2001). In all cases, the first exon corresponds to the bulk of the putative signal peptide (as far as L17 in human RAMP1, G32 in human RAMP2, and C19 in human RAMP3). In all 3 human RAMPs, the final exon begins in the equivalent place, in the middle of the N-terminus (S65 in RAMP1, P72 in RAMP2, and Y65 in RAMP3). In human RAMP2, the junction between the second and third exons occurs between E54 and G55. The genes for chick RAMPs 2 and 3 both have 4 exons (NCBI; homologene 4274, 4275, and 4276). Little is known about the upstream regulatory elements in RAMP genes. However, there is evidence of a binding site for a negative regulatory factor in the mouse RAMP1 gene; presumably, in tissues that express RAMP1, the activity of this factor is suppressed (Pondel & Mould, 2005). The sequence is located between bases 343 and 782; this 480-base segment has potential binding sites for 2 transcriptional repressors, RBPJn/CBF and E4BP4. The sequence upstream of 343 contains a promoter region sufficient to support nearly half of normal transcriptional activity, although full transcriptional activity requires a 4.7 kbase upstream fragment (Pondel & Mould, 2005). Such mechanisms may also exist for the other RAMPs, and the relative suppression of the regulatory factors may govern

4.1. Calcitonin gene-related peptide/AM receptor paradigm The classic function attributed to RAMPs is their ability to switch the pharmacology of CL, thus providing a novel mechanism for modulating receptor specificity. Thus, the CL/RAMP1 complex is a high affinity CGRP receptor, but in the presence of RAMP2, CL specificity is radically altered, the related peptide AM being recognized with the highest affinity and the affinity for CGRP being reduced ¨ 100-fold. CL/RAMP3 receptors are interesting in that while AM is the highest affinity peptide, CGRP is recognized with moderate, rather than low affinity. Indeed, depending on the species and the form of CGRP (h vs. a), the separation between the 2 peptides can be as little as 10fold (Hay et al., 2003a). This may particularly be true if receptor components of mixed species are used. The detailed pharmacology of the CGRP and AM receptors formed by RAMP interaction with CL has recently been reviewed (Born et al., 2002; Poyner et al., 2002; Hay et al., 2004; Kuwasako et al., 2004). A summary is provided in Table 1. As described in Section 3.2, it was initially thought that differential glycosylation of the CL/RAMP products might account for the differences in CL pharmacology; however, this has now been rejected as a likely mechanism (Hilairet et al., 2001a). Although significant amounts of immature core glycosylated CL are present in RAMP2/CL transfected HEK293 cells, 125I-AM only binds to the fully glycosylated, mature form of CL in common with RAMP1/CL transfected cells, and this is the form expressed at the cell surface (Hilairet et al., 2001a). This notion is further supported from experiments where the expression of these RAMP/CL combinations in Drosophila Schneider 2 cells gave AM

Table 1 Pharmacological profile summary for human CGRP, AM, AMY and calcitonin receptors Receptor subtype

Calcitonin

AMY

CGRP

AM

CT(a)

AMY1(a)

AMY2(a)

AMY3(a)

CGRP1

AM1

AM2

Molecular composition Rank order of agonist potency

Calcitonin receptor sCT  hCT  rAMY, aCGRP > AM sCT8 – 32 > AC187 = AC413

Calcitonin receptor + RAMP1 sCT rAMY  aCGRP > hCT > AM

Calcitonin receptor + RAMP2 Poorly defined pharmacology

Calcitonin receptor + RAMP3 rAMY > aCGRP > AM

CL + RAMP1

CL + RAMP2

CL + RAMP3

aCGRP > AM  AMY  sCT

AM O aCGRP > AMY > sCT

AM > aCGRP > AMY > sCT

sCT8 – 32 = AC187 = AC413 > CGRP8 – 37

ND

sCT8 – 32 = AC187 = AC413 > CGRP8 – 37

BIBN4096BS > CGRP8 – 37 > sCT8 – 32 = AC187

AM22 – 52  CGRP8 – 37

CGRP8 – 37  AM22 – 52

Rank order of antagonist affinity

AM, adrenomedullin; AMY, amylin; CGRP, calcitonin gene-related peptide; CL, calcitonin receptor like receptor; CT, calcitonin; h, human; ND, not determined; RAMP, receptor activity modifying protein; r, rat; s, salmon.

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and CGRP receptor pharmacology with the appropriate RAMP despite having identical glycosylation patterns (Aldecoa et al., 2000). The original work by McLatchie and colleagues has been replicated in many laboratories for receptor components from human, rat, mouse, and pig, and the pharmacology is broadly consistent across these mammalian species (Husmann et al., 2000; Oliver et al., 2001; Kikumoto et al., 2003). In transfected cell systems, the concept that CL/ RAMP1 forms a CGRP receptor and CL with RAMP2 or 3 forms an AM receptor has been consistently reproduced (Muff et al., 1998; Kamitani et al., 1999; Chakravarty et al., 2000; Kuwasako et al., 2000). These complexes also function in a similar manner in yeast (Miret et al., 2002). The pharmacology of the CL/RAMP1 complex and its sensitivity to the antagonist CGRP8 – 37 closely matches an extensive literature relating to a ‘‘CGRP1’’ receptor (as reviewed in Hay et al., 2004). Indeed, IUPHAR nomenclature has now ratified CL/RAMP1 as the CGRP1 receptor and CL/RAMP2 and CL/RAMP3 as AM1 and AM2 receptors, respectively (Poyner et al., 2002; Fig. 4). Recent data from transgenic mice provides further support for a relevant relationship of RAMP2 to AM signaling, whereby RAMP2 overexpression potentiated vascular responsiveness to AM (Tam et al., 2004a, AM2

AM1

CGRP1

1

2004b). As expected, responses to CGRP were unaffected by the presence of additional RAMP2. 4.2. Amylin receptor paradigm The widespread distribution of RAMPs (see Section 5) and their presence in tissues or cells which do not express CL suggested that these proteins have other functions or may interact with additional receptors. This is indeed the case; most notably, RAMPs modulate the pharmacology of the calcitonin receptor (Armour et al., 1999; Christopoulos et al., 1999; Muff et al., 1999; Leuthauser et al., 2000; Hay et al., 2005). In common with the difficulty in reconstituting CGRP receptors, the receptors for the related peptide, AMY, also proved difficult to isolate. While AMY binding was usually associated with the presence of calcitonin receptors and certain calcitonin receptor antibodies bound to both AMY and calcitonin receptors, calcitonin receptor transfection into cell lines did not consistently yield high affinity AMY receptors (Perry et al., 1997). However, it was apparent that in certain cell lines, the calcitonin receptor tended to induce a variety of different affinities/potencies for AMY. Indeed, soon after the discovery of RAMPs, it was identified that these observations could probably be explained by different endogenous background levels of RAMPs in these cells. It CT

AMY3

2

AMY2

2

AMY1

3

1

3 4

4 CL-R

RAMP3

RAMP1

CTR

CalS-R

VPAC1-R PTH2-R

4

PTH1-R

RAMP2

Gluc.-R

4

Fig. 4. The broadening spectrum of RAMP – receptor interactions. RAMPs can interact with multiple receptor partners. All RAMPs interact with the calcitonin receptor-like receptor (CL-R), the calcitonin receptor (CTR), and the VPAC1 receptor, while the glucagon and PTH1 receptors interact with RAMP2, the PTH2 receptor with RAMP3, and the calcium sensing receptor (CalS-R) with RAMP1 or RAMP3. The consequence of RAMP interaction varies. For the CL and CalS receptors, RAMPs play a chaperone role, allowing cell surface expression (1). For the CL and calcitonin receptors, RAMP interaction leads to novel receptor binding phenotypes (2). There is also evidence that RAMP interaction will modify signaling, and this has been seen for the VPAC1 – RAMP2 heterodimer and for calcitonin receptor/RAMP complexes (3). In many instances, however, the consequence of RAMP interaction has yet to be defined (4).

D.L. Hay et al. / Pharmacology & Therapeutics 109 (2006) 173 – 197

is now evident that calcitonin receptors have strong interactions with RAMPs and are able to form heterodimers which bind AMY with high affinity. The pharmacological nature of the AMY receptor that is generated by RAMP and calcitonin receptor co-transfection appears to be quite variable and dependent upon host cell environment, perhaps as a consequence of G-protein and endogenous CL/RAMP complement (Christopoulos et al., 1999; Tilakaratne et al., 2000). However, the current IUPHAR consensus is that there are 3 AMY receptors (AMY1 – 3) formed by the interaction of the calcitonin receptor with RAMPs 1 to 3, respectively (Fig. 4). The subtypes may be further subdivided according to the splice variant of the calcitonin receptor present in the heterodimer (Poyner et al., 2002). Several of these subtypes have now been relatively well characterized. In terms of AMY affinity, the results are reasonably consistent across studies showing that AMY only binds with high affinity to RAMP-complexed calcitonin receptors. Similarly, human calcitonin only appears to bind with high affinity to ‘‘free’’ calcitonin receptors (Hay et al., 2005). For AM, there are marked differences in its apparent ability to bind to AMY receptors across studies. In HEK293 cells, Kuwasako and colleagues showed that all 3 RAMPs were able to induce AM sensitivity to calcitonin receptors (Kuwasako et al., 2003b). In contrast, in Cos 7 cells, AM does not bind to or stimulate AMY receptors (Christopoulos et al., 1999; Hay et al., 2005). As both studies used human RAMPs and the same insert negative human calcitonin receptor variant, the influence of cellular background is likely to make more than just a trivial contribution to this observation. Receptors expressed in Cos 7 cells are only weakly coupled to Gas, and this reveals differences in the relative efficacy of peptides. In contrast, the HEK293 cells used by Kuwasako and colleagues are ‘‘highly coupled’’, leading to apparent differences in potency (Kuwasako et al., 2004). As alluded to in Section 3.1, the influence of species may be important for the observed phenotype. A study that has been presented in conference proceedings showed that while porcine CL with porcine RAMPs produced the expected CGRP or AM phenotype when transfected into Cos 7 cells, porcine calcitonin receptor with porcine RAMPs did not have a higher affinity for AMY than the porcine calcitonin receptor alone (Kikumoto et al., 2003). This work is supported by earlier studies showing that porcine calcitonin receptors appear to have high innate affinity for AMY (Sexton et al., 1994b), although this may be a cell typedependent phenomenon (Christmanson et al., 1994). Some calcitonin receptors, when co-expressed with RAMPs (e.g., AMY1(a)), have relatively high affinity for CGRP and may be at least partially responsible for some aspects of reported non-CGRP1 receptor pharmacology (Poyner et al., 2002; Kuwasako et al., 2004; Hay et al., 2005). Indeed, there is some support for the hypothesis that the AMY1(a) receptor may underlie many reports of CGRP2like pharmacology (Kuwasako et al., 2004; Hay et al.,

183

2005). CGRP2 receptors are characterized by weak antagonism by the CGRP antagonist fragment, CGRP8 – 37, and the AMY1(a) exhibits this characteristic, at least in transfected cells (Hay et al., 2005). Furthermore, as there is a high level of AMY binding in the vas deferens, a prototypical CGRP2-receptor expressing tissue, there may be additional support for this hypothesis (Poyner et al., 1999). The pharmacological studies may now provide a basis for the separation of CGRP1 and AMY1(a)-based CGRP receptors with careful use of a spectrum of agonists and antagonists. However, in order to make robust correlations with possible molecular entities, the key receptor components need to be localized in tissues. This matter will be discussed in more detail in Section 5. 4.3. Receptor activity-modifying proteins as drug targets The potential of RAMPs as drug targets is exemplified by the CGRP antagonist, BIBN4096BS (Doods et al., 2000). A single residue within the N-terminus of RAMP1 (tryptophan 74) is responsible for the strong species selectivity of the CGRP receptor antagonist BIBN4096BS (Mallee et al., 2002; Fig. 3). The interaction with this RAMP1 residue is also likely to have implications for the strong selectivity of this compound for CGRP rather than AM receptors. Although CL is also required for the high affinity interaction of BIBN4096BS with the CGRP1 receptor, the lack of significant antagonism of AM receptors which also contain CL suggests that RAMP1 is the key predictor of BIBN4096BS antagonism (Hay et al., 2003a; Salvatore et al., 2004). Thus, the rational design of AM receptor-specific (and AM1- and AM2-specific) ligands will require a consideration of the characteristics of RAMP2, RAMP3, and the unique interface that they generate with CL. However, as the list of RAMP-interacting proteins grows, the specific properties of each RAMP – protein interface become increasingly important. The nature of GPCR/RAMP heterodimers could, in fact, limit the development of selective nonpeptide agonists for complexes of an individual GPCR; the activation of family B receptors is thought to involve a direct interaction of the peptide Nterminus with the receptor core (Pham & Sexton, 2004; Hoare, 2005), and as such, mimetics that target this domain would be likely to activate each GPCR/RAMP complex.

5. Localization/distribution of receptor activity-modifying proteins 5.1. General summary of distribution Overall, RAMPs have a relatively ubiquitous distribution with at least 1 RAMP identified (at least by PCR) in each tissue or cell line that has been studied (Sexton et al., 2001). RAMP1 is expressed in many tissues, including the heart, uterus, brain, bladder, pancreas, skeletal muscle, and

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D.L. Hay et al. / Pharmacology & Therapeutics 109 (2006) 173 – 197

Table 2 Correlation of calcitonin peptide family pharmacology with RAMPs, CL, and calcitonin receptors in tissues and cells Cell/tissue

Sp

Receptor CT(a) or (b)

CL

1

2

3

Articular chondrocytes

h

ND

(

(

(



Intra-epithelial lymphocytes

r

ND

(

(

(

(

Subfornical organ Area prostrema SK-N-MC neuroblastoma

r r h

(a), (b) (a) ND

ND ND (

(  (

( ( (

 ( 

Rat-2 fibroblast L6 myocyte Col-29 colonic epithelial cells Atria Ventricle Cerebellum Spinal cord Liver Spleen Vas deferens Lung Aortic smooth muscle cells

r r h r r r r r r r r h

ND ND ND ND ND ND ND ND ND ND ND ND

( ( ( ( ( ( ( ( ( ( ( (

 ( ( ( ( ( ( ( ( ( ( 

( ( ( ( ( ( ( ( ( ( ( (

   ? ? ( ( ( ( ( ( 

Aortic endothelial cells

h

ND

(



(



Umbilical vascular endothelial cells Myocardial atrial trabeculae (postmortem)

h

ND

(



(



h

ND

(

(

(

(

Myocardial ventricular trabeculae (postmortem)

h

ND

(

(

(

(

Aortic vascular smooth muscle cells (passage 10)

r

ND

(

(

(

ND

Primary ventricular cardiac myocytes

r

ND

(

(

(

ND

Cerebro microvascular smooth muscle cells

h

ND

ND

(

(



Cerebro microvascular endothelial cells

h

ND

ND

(

(



Brain astroglial cells

h

ND

ND

(

(

(

Left anterior descending coronary artery (heart transplant patients) Cardiomyocytes (some fibroblasts) Epididymus Subcutaneous arteries (obtained in surgery from abdominal wall) Basilar artery

h

ND

(

(

(

(

r r h

ND ND ND

( ( (

ND ( (

( ( (

( ND (

gp

ND

(

ND

ND

ND

r

ND

(

(

h

(

(

(

Caudal tip of caudate putamen, lateral/basolateral amygdaloid nuclei Mesenchymal-derived adipocytes

RAMP

(

(

Pharmacology

References

AM increased cAMP (pEC50 6.7), other agents ND CGRP binding present, other agents ND sCT, rAMY induced c-fos sCT, rAMY induced c-fos CGRP > AM, CGRP8 – 37 sensitive AM but no CGRP binding CGRP and AM binding CGRP but no AM binding AM H CGRP binding (B max) AM \ CGRP binding (B max) AM = CGRP binding (B max) AM > CGRP binding (B max) AM = CGRP binding (B max) CGRP > AM binding (B max) AM > CGRP binding (B max) AM O CGRP binding (B max) Weak AM and no CGRP cAMP response Potent AM and no CGRP cAMP response Potent AM and no CGRP cAMP response CGRP increased contractile force (pEC50 7.8). AMY was weak and AM had no effect CGRP increased contractile force (pEC50 7.8). AMY was weak and AM had no effect AM H CGRP (cAMP production, cell proliferation and viability) 100 nM AM increased CRELuc reporter activity (CGRP ND), CGRP8 – 37 sensitive CGRP potently increased cAMP (AM ND), CGRP8 – 37 and BIBN4096 sensitive CGRP potently increased cAMP (AM ND), CGRP8 – 37, and BIBN4096 sensitive CGRP potently increased cAMP (AM ND), CGRP8 – 37 and BIBN4096 sensitive CGRP HAM > AMY in relaxing the artery; CGRP8 – 37 sensitive AM binding present CGRP and AM binding present CGRP relaxed subcutaneous arteries (no other drugs tested)

Chosa et al., 2003 Hagner et al., 2002 Barth et al., 2004 Barth et al., 2004 McLatchie et al., 1998; Choksi et al., 2002 Choksi et al., 2002 Choksi et al., 2002 Choksi et al., 2002 Chakravarty et al., 2000 Chakravarty et al., 2000 Chakravarty et al., 2000 Chakravarty et al., 2000 Chakravarty et al., 2000 Chakravarty et al., 2000 Chakravarty et al., 2000 Chakravarty et al., 2000 Kamitani et al., 1999 Kamitani et al., 1999 Kamitani et al., 1999 Saetrum Opgaard et al., 2000 Saetrum Opgaard et al., 2000 Makino et al., 2001a

Autelitano & Ridings, 2001 Moreno et al., 2002

Moreno et al., 2002

Moreno et al., 2002

Hasbak et al., 2003

Oie et al., 2005 Hwang et al., 2003 Sheykhzade et al., 2004

CGRP relaxed basilar artery (no other drugs tested) CGRP binding present

Sams et al., 1999

CGRP and AM enhanced glycerol release

Linscheid et al., 2005

Oliver et al., 2001

D.L. Hay et al. / Pharmacology & Therapeutics 109 (2006) 173 – 197

gastrointestinal tract. RAMP2 is highly expressed in the lung and also in the heart, placenta, skeletal muscle, and pancreas. RAMP3 has a widespread distribution in the human but appears to have a more limited distribution in the rat, where highest expression (albeit low compared with RAMP1 and 2) was found in lung, kidney, spleen, and spinal cord (Chakravarty et al., 2000, Nagae et al., 2000). Nagae and colleagues also found that all 3 RAMPs are highly expressed in fat tissue, but their function here is unknown. 5.2. Central nervous system distribution The most detailed examination of RAMP localization has been in the rodent brain (Oliver et al., 2001; Ueda et al., 2001; Li et al., 2004). In the rat, in situ hybridization (ISH) detected intense signals for RAMP1 in a number of regions. These included the olfactory tubercules, nucleus accumbens, caudate putamen, cortex, and amygdala. Lower levels were detected in the claustrum, hippocampus, thalamus, hypothalamus, superior colliculus, and spinal cord. Interestingly, no signal was detected in the cerebellum, a region previously shown to have high levels of CGRP binding (see comments also below; Oliver et al., 2001; Li et al., 2004). There was a similar pattern of expression in mouse brain, but there were also notable differences (Ueda et al., 2001). For instance, in the mouse, an ISH signal was detected for RAMP1 in the Purkinje cell layer of the cerebellum. In the rat, there was moderate to high expression of RAMP2 in a number of regions, including the hippocampus, hypothalamus, amygdala, olfactory tubules, cortex, substantia nigra compacta, and the ventral horn of the spinal cord. There was also low level expression in the granule cell layer of the cerebellum. In contrast, in the mouse, RAMP2 expression was more restricted. While RAMP2 ISH signals were also detected in the hippocampus and olfactory bulb of the mouse, expression in the cerebellum was present in the Purkinje cell layer of the cerebellum. RAMP2 was expressed in the choroid plexus of the third, fourth, and lateral ventricles and blood vessels of the pia mater of the mouse (such distribution was not reported in the rat). There is good agreement in RAMP3 distribution in mouse and rat, being most abundant in the thalamic nuclei, where RAMPs 1 and 2 are not highly expressed. RAMP3 was also expressed in the granule cell layer of the cerebellum in both species. In contrast to RAMP2, RAMP3 mRNA was absent from the ventricular choroid plexus and blood vessels. The distribution of RAMP mRNA in the brain provides both overlap with and distinction from the distribution of mRNA for CL and calcitonin receptors (Nakamoto et al.,

185

2000; Oliver et al., 2001). For example, the localization of RAMP1 mRNA in the accumbens nucleus, caudate putamen, and amygdala correlates well with the expression of both calcitonin receptor and CL mRNA, while the same is true for RAMP3 and CL expression in the thalamus. Although no direct parallel is available at a cellular level, the overlapping distribution of RAMP and receptors correlates with known sites of CGRP and AMY binding (Sexton et al., 1986, 1994a). In contrast, RAMP expression at sites such as the cortex and hippocampus do not (or only weakly) overlap with sites of CL/calcitonin receptor expression. Therefore, it is unlikely that RAMP expressed in these regions functions as a partner to CL or the calcitonin receptor. Of note, the cortex, hippocampus, and cerebellum express mRNA (at least developmentally) for the VPAC1 receptor (Basille et al., 2000), which in vitro interacts strongly with RAMPs (see Section 6.2). This anatomical data provides at least correlative, circumstantial evidence that such interactions may occur in vivo. 5.3. Correlation of receptor activitymodifying protein distribution with pharmacology While profiling of RAMPs and their associated receptors in heterologous expression systems is informative in terms of pharmacology and mechanism of interaction, ultimately, it is desirable to understand the contribution of the resulting heterodimers to peptide physiology. This is not a trivial task, given the complexity that might ensue when several RAMPs and receptor partners are expressed in the same tissue or cell. Indeed, resolving the presence of RAMPs and receptors at the cellular level is crucial for determining the important physiological interactions, as a tissue is likely to express many more components than an individual cell. Moreover, mRNA expression does not always correlate with protein (Choksi et al., 2002) and locality may also be important. For example, the absence of a component’s mRNA at the synapse may not be reflective of its expression in a distant neuronal cell body. These factors provide researchers with a major challenge in determining the relative importance of each receptor complex. Therefore, despite the requirement for detailed examination of the localization of the various proteins, it is mRNA analysis that predominates given inherent issues with antibodies for RAMPs and family B GPCRs. In particular, the lack of high affinity, selective antibodies means that RAMP – receptor complexes have yet to be immunoprecipitated from tissues. Nevertheless, inroads have been made in correlating peptide function with the expression of relevant receptor components. Many of these studies are summarized in Table 2.

Notes to Table 2: ( = present;  = absent; ? = weak signal, inconclusive. AM, adrenomedullin; AMY, amylin; CGRP, calcitonin gene-related peptide; CL, calcitonin receptor like receptor; CT, calcitonin; CT(a) or (b), calcitonin receptor isoforms; gp, guinea-pig; h, human; ND, not determined; r, rat; s, salmon; Sp, species.

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5.3.1. Calcitonin gene-related peptide and adrenomedullin To date, most studies have focused on the relationship between RAMPs and CL. These data show that RAMP/CL complexes are the likely candidates responsible for AM and CGRP binding in native cells and tissues (Sams & JansenOlesen, 1998; Chakravarty et al., 2000). For instance, a very strong correlation between CGRP binding and RAMP1 and CL mRNA was found in rat tissues (Chakravarty et al., 2000). This study was important because it was the first to quantify the relationship of binding with expression using a stringent statistical analysis. A key type of analysis is one where expression studies are compared with functional analysis in the same study. For example, it was shown that platelet-derived growth factor stabilized RAMP3 mRNA in rat mesangial cells. This led to increased expression of RAMP3 at the cell surface and enhanced cell signaling in response to AM (Nowak et al., 2002). In cardiomyocytes, it was shown that CL and RAMP2 were required for AM signaling (Autelitano & Ridings, 2001). In rat vascular smooth muscle cells (VSMC), an increase in RAMP2/CL as the cells were passaged led to the formation of functional AM receptors (Makino et al., 2001a). A cell passage-dependent effect has also been observed in the human neuroblastoma cell line, SK-N-MC (Choksi et al., 2002), and supports the hypothesis that RAMP/CL complexes form CGRP and AM receptors. Potent CGRP-stimulated cAMP responses and CGRP binding were present at the same passage as abundant CL and RAMP1 mRNA (Choksi et al., 2002). However, continuous culture of the cells led to a loss of CGRP binding, functional response, and mRNA for both receptor components. In fact, this study showed a positive correlation between appropriate RAMP/CL component mRNA (and CL protein) and peptide pharmacology in several cell lines that are commonly used as models for CGRP and AM receptors (Choksi et al., 2002; Table 1). Of most interest, perhaps, was the apparent expression of RAMP2 mRNA (as well as RAMP1 and CL) in Col-29 and SK-N-MC cells, cell lines that did not bind significant amounts of AM. In another study, while RAMP1 appeared to be strongly expressed by Northern blot in VSMC, there was very little evidence for a CGRP receptor phenotype by way of CGRP-mediated cAMP elevation, even with the concomitant presence of CL, whereas there was strong CL/RAMP2-mediated AM signaling (Makino et al., 2001a). The presence of multiple RAMP mRNA species, yet only apparent pharmacology for 1 peptide, suggests that there may be some form of regulation, allowing functional expression of only 1 type of receptor complex in these cells. Additionally, these RAMP mRNA species that do not seem to have function in terms of CGRP and/or AM biology in such cells may have alternative functions such as partnering with other proteins (see Section 8). There has been analysis of RAMP content in human brain astrocytes, cerebrovascular endothelial cells, and smooth muscle cells (Moreno et al., 2002). Each cell type

expressed mRNA for RAMP1 and RAMP2, but only the astrocytes convincingly expressed RAMP3 mRNA. Pharmacological analysis of each of the cell types was performed, where a-CGRP, h-CGRP, and [Cys(Et)2,7]aCGRP all potently elevated cAMP, an effect that could be blocked by 2 CGRP antagonists (CGRP 8 – 37 and BIBN4096BS), indicating that CGRP receptor pharmacology could be observed where RAMP1 was expressed. Nonetheless, given the known fluctuation of CGRP receptor components, it would be unwise to make correlative assumptions on phenotype on the basis of mRNA expression (Moreno et al., 1999; Choksi et al., 2002). The most informative studies would be those that included an assessment of expression of each component (RAMPs1 –3, CL, calcitonin receptor, RCP [see Section 6.1]; preferably protein expression) along with functional pharmacological analysis. At present, such studies are lacking. Along this line of thought, there is an important message from a study of RAMP and CL expression in human lenticulostriate branches of the middle cerebral artery (Sams et al., 2000). Here, the effects of CGRP and AM were investigated in a series of arterial segments of different size. While CGRP and AM had a similar relative potency across all samples, there was considerable variability in receptor component (i.e., CL receptor and RAMPs) detection between subjects. While the absence of receptor components by RT-PCR from some patient samples might suggest that a particular receptor is not involved in the observed response, it is more likely to reflect the inherent variability and quality of RNA obtained from postmortem tissue. Equally, this may be another example where mRNA does not correlate with protein and stresses the importance of analyzing expressed protein. 5.4. Concluding comments Overall, the distribution data presented so far are supportive of the hypothesis that RAMP and CL or calcitonin receptor combinations are able to account for the observed CGRP, AM, and AMY pharmacology. A salient point for CGRP receptors relates to the cerebellum, where the lack of CL mRNA in some studies despite abundant CGRP binding has prompted speculation of alternative CGRP receptors (Oliver et al., 2001; Chauhan et al., 2003). Nevertheless, this apparent lack is study dependent and CL has been identified in cerebellum in other studies (Chakravarty et al., 2000; Ueda et al., 2001).

6. Receptor activity-modifying proteins and signaling Some consideration has been given to the potential role that RAMPs may have in modifying receptor behaviors other than ligand binding pharmacology. An additional functional consequence might be that of alteration of receptor signaling characteristics.

D.L. Hay et al. / Pharmacology & Therapeutics 109 (2006) 173 – 197

6.1. Calcitonin receptor-like receptor and calcitonin receptor-based receptors Depending on the cell line, the 2 subtypes of AM receptor appear at times to have only a subtle pharmacological distinction. Therefore, it might be expected that the difference in RAMP2 or RAMP3 could have additional significance. However, in broad terms of cAMP and coupling to intracellular calcium, this does not seem to be the case and both receptors behave comparably in response to AM (Kuwasako et al., 2000). Nevertheless, potential signaling differences between AM1 and AM2 have not been investigated thoroughly, and it may just be a case of performing the appropriate experiments. In physiological terms, there are several examples where there are contrasting effects of AM that could relate to differences in receptor subtype and the signaling pathways that they are coupled to (Hinson et al., 2000). As yet, there is no explanation for the ability of AM to promote or inhibit mitogenesis in different tissues and cells and this could relate to distinct coupling of AM1 and AM2 to MAP kinase pathways, although these pathways are notoriously sensitive to cell-specific factors. As described in Section 7.2, there is now evidence for distinct trafficking characteristics of human RAMP2 and 3, when coupled to CL (Bomberger et al., 2005a, 2005b). In addition to the interaction of CL with RAMPs, there is an additional protein required for signaling. RCP is an intracellular peripheral membrane protein that co-immunoprecipitates with CL and appears to assist CGRP and AM

187

receptor coupling to Gs (Luebke et al., 1996; Evans et al., 2000; Prado et al., 2001). It is unclear if there is a direct association of RCP with RAMPs or whether RCP is required for coupling to alternative effectors, but RCP has been shown to interact with the second intracellular loop of CL and may influence the stability of CL – RAMP interactions (Loiseau & Dickerson, 2004). The signaling consequences of calcitonin receptor– RAMP interaction have hardly been studied. However, no changes in maximal capacity for phosphatidylinositol (PI) hydrolysis or cAMP production were observed with different calcitonin receptor –RAMP combinations when compared with the calcitonin receptor alone (Christopoulos et al., 2003). More recently, the relative coupling of AMY receptors to cAMP generation, intracellular Ca2+ mobilization, and ERK1/2 activation in transfected Cos 7 cells has been examined. In contrast to the strong (> 20-fold) increase in potency of AMY to generate cAMP, there was only a weak increase in AMY potency for Ca2+ and ERK1/ 2 signaling (< 5-fold; Maria Morfis & Patrick Sexton, unpublished data). This data suggests that the calcitonin receptor– RAMP-based AMY receptors are relatively lesswell coupled to Gq than the isolated calcitonin receptor. It is also apparent that the generation of high affinity AMY binding for calcitonin receptor – RAMP2 complexes is particularly sensitive to experimental conditions. This relates to the splice variant of the calcitonin receptor used and cellular background (Tilakaratne et al., 2000) and most likely reflects differences in coupling to signal transduction

VPAC1 receptor

VPAC1 receptor

plus RAMP2

alone

Gs – cAMP

Fraction VIP maximum for VPAC1 receptor alone

Gq – PI hydrolysis

3

3

2

2

1

1

0

0 -12

-11

-10

-9

-8

Log [VIP]

-7

-6

-5

-12

-11

-10

-9

-8

-7

-6

-5

Log [VIP]

Fig. 5. RAMP2 modulation of VPAC1 receptor signaling. The VPAC1 receptor couples to multiple signaling pathways. Vasoactive intestinal polypeptide (VIP) causes a potent stimulation of cAMP accumulation and a weaker activation of phosphoinositide (PI) hydrolysis. The coexpression of the receptor with RAMP2 leads to a specific enhancement of agonist-mediated PI hydrolysis without modulating cAMP production.

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D.L. Hay et al. / Pharmacology & Therapeutics 109 (2006) 173 – 197

apparatus, as the overexpression of Ga protein subunits can modulate the induction of the AMY phenotype (Smyth et al., 2001). Thus, there may be much to learn from the AMY receptors generated by different calcitonin receptor subtypes. 6.2. Vasoactive intestinal peptide/pituitary adenylate cyclase activating peptide 1 receptor While there is currently little evidence for signaling modifications of CL-based receptors in association with RAMPs, a completely different paradigm is evident for the VPAC1 receptor. This receptor has strong interactions with all 3 RAMPs, but its pharmacology, in terms of agonist binding, does not appear to be modified by their presence (Christopoulos et al., 2003). On the other hand, there was a clear functional consequence of RAMP2 overexpression with the VPAC1 receptor where PI hydrolysis was specifically augmented relative to cAMP, which did not change. The potency of the response (EC50 of vasoactive intestinal peptide) was not altered, but the maximal PI hydrolysis response was elevated in the presence of RAMP2 (Fig. 5). It has been suggested that this may reflect a change in compartmentalization of the receptor signaling complex (Christopoulos et al., 2003). Such augmentation was not evident for the interaction of the VPAC1 receptor with RAMP1 or RAMP3; in these cases, the outcome of heterodimerization may be more subtle or involve the modification of different receptor parameters such as trafficking.

7. Receptor activity-modifying proteins and trafficking

Xenopus oocytes (Flahaut et al., 2002). An anti-FLAG tag binding assay was used to assess cell-surface expression of FLAG-tagged CL. Unlike studies in mammalian systems, CL appeared to express efficiently at the cell surface without co-injection of RAMPs. Furthermore, CL expression was not further enhanced with RAMP. These data argue against a universal CL chaperone role for RAMPs and the authors excluded the possibility of endogenous RAMPs promoting CL expression (Flahaut et al., 2002). Nevertheless, this study differs in many ways from earlier studies. Microinjection of cRNA into nonmammalian cells plus the type of tag used on the receptor and other methodological differences could all contribute to this observation. Very recently, preliminary data suggest that RAMPs are required for the cell surface expression and terminal glycosylation of the calcium sensing receptor, a family C GPCR. In Cos 7 cells, in the absence of RAMPs, the receptor remains in the endoplasmic reticulum as a 150kDa, partially glycosylated receptor. RAMPs 1 and 3 (but not RAMP2) allow translocation to the Golgi and then to the cell surface, where the receptor now exists as a 175-kDa protein (Tristan Bouchet, Stephane Martin, & Jeremy Henley, personal communication; Fig. 4). This has striking parallels with the effects on CL transport and glycosylation (McLatchie et al., 1998). It will be important to determine if other GPCRs outside of family B also require RAMPs as chaperones. However, the work raises the possibility that this function of RAMPs is more widespread than the modulation of receptor pharmacology or signaling and is consistent with the proposal that RAMPs originally evolved from chaperone proteins. 7.2. Receptor activity-modifying proteins and receptor internalization/recycling

7.1. Receptor activity-modifying proteins as chaperones For CL, heterodimerization with a RAMP is a prerequisite for its efficient transport to the cell surface. In this sense, RAMPs have a chaperone role (McLatchie et al., 1998). Epitope tagging of CL has yielded valuable information regarding its transport to the cell surface and shows that each RAMP is able to elicit translocation of CL from intracellular compartments to the cell surface. Fluorescence-associated cell sorting data and images from confocal microscopy show that there is a much greater proportion of CL at the cell surface when RAMPs are present (McLatchie et al., 1998; Fraser et al., 1999; Kuwasako et al., 2000). Thus, CL/RAMP complexes can be considered obligatory heterodimers. It is unclear why CL does not express at the cell surface without RAMPs, but there is accumulating evidence that dimerization of certain receptors in required for them to pass ‘‘quality-control checkpoints’’ during biosynthesis (reviewed by Bulenger et al., 2005). However, the inability of CL to express alone is not universally consistent given some conflicting data from

In an initial study, it was observed that the type of RAMP did not affect CL internalization or its targeting to a degradative pathway (Kuwasako et al., 2000). CL and RAMPs were colocalized and were internalized together following agonist stimulation. Subsequent investigation of CL/RAMP 1 complexes produced similar observations and further showed that the internalization was probably harrestin and dynamin dependent (Hilairet et al., 2001b). Recent investigations of the trafficking properties of RAMP2 and RAMP3, however, have highlighted the importance of the PDZ-domain in the C-terminal tail of RAMP3. It appears that this domain may be responsible for the unique trafficking properties that have recently been reported for AM2 receptors which are able to recycle as opposed to AM1 receptors, which do not possess a PDZ domain and are targeted to a degradative pathway (Bomberger et al., 2005a; Fig. 6). As previously identified by Kuwasako et al. (2000), in HEK293 cells, all CL/RAMP complexes are subject to targeting for degradation following internalization. However, the overexpression of NSF, selectively switched CL/RAMP3 towards recycling. A

D.L. Hay et al. / Pharmacology & Therapeutics 109 (2006) 173 – 197

189

AM RAMP3

CL

AM2

RAMP2

CL

NSF

AM1

AM2 Recycling endosome

AM1

Early endosome Lysosomes Fig. 6. Modulation of receptor recycling through RAMP-specific interaction with accessory proteins. RAMP2/CL and RAMP3/CL generate specific AM receptors (AM1 and AM2, respectively). Upon agonist (AM) binding, AM1 receptors are rapidly internalized and targeted to lysosomal degradation. In contrast, the internalization and recycling of AM2 receptors can be modified by the interaction of RAMP3 with accessory proteins that bind to its C-terminal PDZ domain. Illustrated is the potential interaction of the AM2 receptor with N-ethylmaleimide-sensitive factor (NSF) leading to rapid recycling of the receptor complex.

similar function of RAMP3 in AM receptor recycling was also shown in rat mesangial call and Rat-2 fibroblasts (Bomberger et al., 2005a). Furthermore, only CL/RAMP3 internalization may be regulated by NHERF-1 binding to the RAMP3 PDZ-domain (Bomberger et al., 2005b; see also Section 3.4, ‘‘Motifs’’). These differential properties of RAMP2 and RAMP3 may explain the relevance of 2 AM receptors which otherwise appear to have many similarities. Furthermore, these or other differences could underlie the reported inconsistency in the literature of whether or not AM receptors are subject to agonist-stimulated desensitization (reviewed by Hay et al., 2003b). In addition, the C-termini of all 3 RAMPs contain combinations of serine and threonine residues that are potential sites of phosphorylation (Sexton et al., 2001; Fig. 2). However, it has been shown in HEK293T cells that RAMP1 is not phosphorylated, unlike CL (Hilairet et al., 2001b). In RAMP3, the mutation of threonine 146 to alanine does not prevent RAMP3/CL desensitization or internalization but prevents recycling of the receptor complex back to the cell surface (Bomberger et al., 2005a). This residue is in the predicted PDZ-binding

domain and the inhibition of recycling appears to be the result of disrupting the interaction of RAMP3 with NSF. As the mutant RAMP3 receptor complex still underwent agonist-stimulated receptor-desensitization, these data suggest that potential phosphorylation of threonine 146 in RAMP3 is not an important determinant of RAMP3/CL regulation.

8. Interaction of receptor activity-modifying proteins with other proteins 8.1. G-protein coupled receptors At present, there is only limited information regarding the interaction of RAMPs with receptors outside of the calcitonin receptor family. The most convincing argument for a broader role for RAMPs than just modulating receptor specificity for the calcitonin peptide family was a recent publication investigating potential interactions of RAMPs with other family B GPCRs (Fig. 4). An inherent property of RAMPs is their inefficient transport to the cell surface in the

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absence of an interacting receptor partner, being particularly marked for the nonglycosylated RAMP1. In terms of screening potential receptor partners, this was used as an advantage and several receptors were co-transfected with tagged RAMPs, whose relative appearance at the cell surface by way of fluorescent antibody was monitored (Christopoulos et al., 2003). Several novel potential interactions were identified. The glucagon and parathyroid hormone (PTH) 1 receptors selectively translocated RAMP2, whereas the PTH2 receptor translocated RAMP3. No interactions were seen with the VPAC2, GLP-1, GLP-2, or GHRH receptors, but the VPAC1 receptor was effective in translocating all 3 RAMPs. The VPAC1/RAMP interactions were further studied and showed that while receptor ligand binding pharmacology was unaltered, PI hydrolysis was selectively augmented (cAMP responses were unchanged; Christopoulos et al., 2003; Section 6.2). In an earlier study, no receptor out of the PTH1, glucagon, V1a, or V2 vasopressin receptors gave evidence for translocation of any RAMP to the cell surface (Flahaut et al., 2002). However, there could be several reasons for this discrepancy in the data between these studies. Flahaut et al. (2002) used FLAG-tagged, mouse RAMPs 2 and 3 with altered glycosylation sites in Xenopus oocytes in contrast to HAtagged or myc-tagged, unmodified human RAMPs in Cos 7 cells used by Christopoulos et al. (2003). It is known that the type of epitope tag has a strong influence on RAMP expression profiles, particularly for RAMP2 and 3 (Christopoulos et al., 2003). As noted above, there is preliminary data to suggest that RAMPs 1 and 3 can associate with the calcium sensing receptor, a family C GPCR, and that this is required for receptor trafficking (Tristan Bouchet, Stephane Martin, & Jeremy Henley, personal communication). There is also preliminary evidence suggesting that RAMP expression may alter h-adrenoceptor pharmacology (Tilakaratne et al., 2002), although no direct evidence for RAMP –receptor interaction was provided in the study. Thus, it is possible that RAMPs may have a more generalized role in regulating the activity of GPCRs. 8.2. Receptor activity-modifying protein dimers In vitro evidence using tagged RAMPs suggests that these proteins are able to form homodimers (Hilairet et al., 2001a; Sexton et al., 2001; Udawela et al., 2004). These homodimers can be maintained in the reducing and denaturing conditions of SDS-PAGE, suggesting that there is a stable interaction between the proteins (for comment on the potential role of cysteine residues in the stabilization of RAMP dimers, see Section 3.2). Under the conditions studied, the homodimers do not appear to exist in association with receptors at the cell surface. For example, CGRP receptors at the cell surface appear to be composed of 1:1 ratio of a CL and a RAMP1 protein (Aldecoa et al., 2000; Hilairet et al., 2001a). In cross-linking studies, 125I-

CGRP labels a band consistent in size with 1 receptor plus 1 RAMP plus ligand (Aldecoa et al., 2000). The phenomenon of RAMP homodimerization has not been extensively investigated, but it is apparent that the quantity of RAMP1 and RAMP3 homodimers is reduced in the presence of a receptor with which the RAMP can interact (Udawela et al., 2004). These dimers might allow dynamic regulation of the amounts of RAMP available to particular receptors. Nonetheless, it remains unclear whether the GPCR component of the complex exists in a monomeric or dimeric form; like many other GPCRs, family B receptors also have the capacity to form at least homodimeric complexes (Ding et al., 2002; Seck et al., 2003; Heroux & Bouvier, 2005). 8.3. Other proteins The full extent to which RAMPs interact with other proteins remains to be determined. As described above, the PDZ motif of RAMP3 allows interaction with at least 2 proteins, NHERF and NSF (Bomberger et al., 2005a, 2005b), and it is likely that other proteins can interact with this domain. Furthermore, as lack of RCP impairs coimmunoprecipitation of RAMP/CL complexes (Ian Dickerson, personal communication), it may be inferred that RAMPs also directly interact with this protein, although allosteric stabilization of a CL/RAMP interface cannot be ruled out at this stage. It seems likely that RAMPs may also influence the coupling of receptors to signaling intermediates such as G-proteins, but evidence for a direct interaction is currently unavailable.

9. Receptor activity-modifying protein specificity The analysis of RAMP interaction with family B receptors provides clear evidence for specificity in the interaction profile of individual RAMPs. CL, calcitonin, and VPAC1 receptors can interact with all 3 RAMPs, whereas glucagon and PTH1 receptors appear to interact only with RAMP2, and PTH2 receptors, only with RAMP3 (Christopoulos et al., 2003). For the family C calcium sensing receptor, there appears to be functional interaction with RAMP1 or RAMP3 but not RAMP2. For those receptors capable of interacting with multiple RAMPs, the determinants for preferential interaction with 1 RAMP over another is not clear. There are a number of observations that may provide a guide to relative specificity of RAMP interaction with CL, but many of these are tempered by the use of cross-species components (e.g., rat CL, human RAMP). UMR106-01 rat osteogenic sarcoma cells express endogenous RAMP2 and exhibit an AM1 phenotype when transfected with rat CL (Buhlmann et al., 1999). The AM response can be further amplified by cotransfection with human RAMP2, but it is inhibited by human RAMP1 in parallel with appearance of a CGRP1 receptor phenotype. However, there is no change to CGRP

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binding by further transfection of human RAMP2, either in UMR106-01 or Cos 7 cells (that lack endogenous CL and RAMP; Buhlmann et al., 1999). Thus, for rat CL, human RAMP1 is bound preferentially over human RAMP2. In rabbit aortic endothelial cells that have an endogenous AM1 receptor, human RAMP1 induces the formation of a CGRP1 receptor, but the endogenous AM response is unaltered by RAMP1 (or RAMP2 or RAMP3; Muff et al., 1998). However, co-transfection of human RAMP3 but not human RAMP2 along with human RAMP1 causes decreased CGRP binding, suggesting that the human RAMP3 interaction with rabbit CL is preferential to that of human RAMP1 (Muff et al., 1998). However, the lack of modulation of the endogenous AM response makes further conclusions difficult. For mouse RAMP with rat CL, each RAMP induces a distinct receptor profile. In this paradigm, RAMP1 and RAMP2 co-transfection leads to a mutual decrease in either CGRP or AM binding, implying a similar level of interaction between each of these RAMPs with CL (Husmann et al., 2000). However, no alteration in the level of binding to CL/RAMP3 was seen following co-transfection with either RAMP1 or RAMP2, suggesting that like the interaction in rabbit aortic endothelial cells, RAMP3 had highest affinity for CL. Perhaps, the most convincing data comes from studies in primary human kidney cells. These cells express CL and both RAMP2 and RAMP3 but display an AM phenotype that is almost exclusively AM 2. Knockdown of the endogenous RAMP3 by RNAi causes a shift to an AM1 phenotype (Bomberger et al., 2005b), indicating that both RAMP2 and RAMP3 proteins were expressed and that the human CL interacts preferentially with RAMP3. As described in Section 5, there are examples of cells that express mRNA for multiple RAMPs, but where only 1 apparent phenotype can be observed. However, in the absence of knowledge of actual protein levels, these observations are difficult to interpret with respect to the selectivity of RAMP –receptor interaction. More studies based on the knockdown of naturally expressed protein are required to unravel how RAMPs physiologically regulate cellular phenotype.

10. Receptor activity-modifying protein regulation (in disease/pathophysiological states) There has been much interest in the dynamic regulation of RAMPs. This has most commonly been investigated in animal models of disease where RAMP mRNA expression may be differentially regulated. Many of these studies have recently been summarized in comprehensive tabular format in 2 reviews (Kuwasako et al., 2004; Udawela et al., 2004). Most of the work so far has focused on the AM receptor system (CL/RAMP2), with particular emphasis on the potentially important cardiovascular role for AM, and thus,

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the regulation of RAMP1 and RAMP3 has not been extensively examined. From these and other studies, it has become clear that the expression of the RAMPs is not static and under conditions of disease (hypertension, heart failure, diabetes, sepsis, and renal impairment), drug treatment (dexamethasone, captopril, angiotensin II, endothelin, and trichlormethiazide), pregnancy, and altered oxygenation status (hypoxia), the RAMPs are subject to differential regulation. While examined in most of the presented studies, the regulation of CL has been excluded in order to focus on RAMPs themselves. 10.1. Receptor activitymodifying protein regulation in pregnancy There is evidence that RAMPs are subject to regulation during pregnancy. In the rat uterus, RAMP1, 2, and 3 mRNA levels were higher during pregnancy, at delivery, and postpartum compared with nonpregnant rats (Thota et al., 2003). In the rat uterine artery, only RAMP1 was investigated but was also shown to have elevated expression in pregnancy when compared with nonpregnant diestrus rats (Gangula et al., 2003). There is also an elevation in mRNA expression of RAMP1 in the rat placenta, where an increase was observed over days 17 to 22 of pregnancy, being maximal at day 22. At labor, expression returned to the level seen at day 17 (Dong et al., 2003). It is evident that the expression of RAMPs in these tissues and in isolated rat uterine smooth muscle cells is subject to regulation by the steroid hormones 17h-estradiol and progesterone (Dong et al., 2003; Thota et al., 2003; Thota & Yallampalli, 2005). There is some recent evidence to suggest that RAMP1 mRNA is less abundant in preeclamptic compared with normotensive human pregnancies (Dong et al., 2005). 10.2. Receptor activity-modifying protein regulation in cardiovascular disease 10.2.1. In vivo models In a model of heart failure, induced by aortic stenosis, RAMP1 and RAMP3 (but not RAMP2) mRNA expressions were elevated in the atria and ventricles 6 months after surgery (Cueille et al., 2002). In another study, RAMP2 mRNA (RAMP1 and RAMP3 were not tested) was found to be up-regulated in the atria and ventricle of rats with congestive heart failure induced by coronary artery ligation (Totsune et al., 2000). A similar increase in RAMP2 was also observed in another study using coronary artery ligation to induce heart failure. RAMP2 mRNA was elevated 2 days postmyocardial infarction and was maintained to 42 days (Oie et al., 2000). This increase in RAMP2 could be reversed using a mixed endothelinA/B receptor antagonist (Oie et al., 2000). In a rat model of myocardial hypertrophy and ischemia, induced by isoproterenol, RAMP2 mRNA levels were increased in the heart and aorta compared with vehicle-treated rats (Qi et al., 2003a). Aortocaval shunt-

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induced cardiac hypertrophy in rats induced an increase in RAMP2 and RAMP3 mRNA in the left ventricle compared with control rats (Yoshihara et al., 2005). Heart failure in rats can also be induced by a high-salt diet and follows hypertension and left ventricular hypertrophy. In such a model, both RAMP2 and RAMP3 mRNA levels were elevated at the left ventricular hypertrophy and congestive heart failure stages compared with age-matched controls that were not sensitive to a high-salt diet (Nishikimi et al., 2003). Cardiomyocytes and noncardiomyocytes isolated from myocardial tissue of rats at 7 days postmyocardial infarction showed a selective increase in RAMP2 mRNA in cardiomyocytes compared with control rats, whereas RAMP3 levels were increased in both cell types (Oie et al., 2005). 10.2.2. In vitro models RAMP regulation has also been examined in primary rat cardiomyocytes (Mishima et al., 2003). In the presence of increasing concentrations of angiotensin II for 24 hr, RAMP1 and RAMP3 mRNA levels were increased. RAMP2 levels did not change in response to drug treatment (Mishima et al., 2003). Calcified VSMC show elevated RAMP2 and RAMP3 levels compared with control cells (Qi et al., 2003b; Pan et al., 2004). 10.3. Receptor activity-modifying protein regulation in hypertension The regulation of RAMPs in hypertension has been investigated using a variety of animal models including saltloaded, spontaneously hypertensive rats (Nishikimi et al., 2001, 2005; Cao et al., 2003; Stachniak & Krukoff, 2003; Tadokoro et al., 2003; Wang et al., 2003) and also in pregnancy-induced hypertension in women (Makino et al., 2001b). While there has been the clear demonstration that RAMP levels do change under conditions of hypertension, the precise nature of the change seems to be somewhat model and tissue dependent. For example, Nishikimi and colleagues demonstrated differential changes in RAMP mRNA levels in the rat renal cortex and medulla, depending on the hypertensive rat model that they used (Nishikimi et al., 2001, 2005). There is, however, a general consistency that elevated RAMP mRNA levels in the kidney and left ventricle of spontaneously hypertensive rats are normalized following the use of antihypertensive drugs (angiotensin converting enzyme inhibitors, diuretics; Wang et al., 2003; Nishikimi et al., 2005). 10.4. Receptor activitymodifying protein regulation in the kidney In a rat model of obstructive nephropathy, RAMP1 and RAMP2 mRNA levels were up-regulated in the kidney, but RAMP3 levels were unchanged. The authors speculated that the up-regulation of these receptor components in the

obstructed kidney may provide a protective mechanism against fibrotic changes or proliferation (Nagae et al., 2000). In rats with 5/6 nephrectomy, a model of acute renal failure with compensative renal hypertrophy, RAMP2 levels were unchanged 4 and 14 days after surgery compared with sham-operated animals, whereas RAMP3 levels were decreased at both time points (Totsune et al., 2001). 10.5. Receptor activity-modifying protein regulation in diabetes and obesity In streptozotocin-treated rats, a model of diabetes, RAMP2 levels were elevated in the kidney, but RAMP3 levels were not different to nondiabetic rats (Hiragushi et al., 2004). The effect of high-fat feeding and, therefore, obesity on RAMP2 expression has been investigated in rats. RAMP2 mRNA levels were up-regulated in epididymal, mesenteric, and retoperioneal adipose tissues but not in subcutaneous fat (Fukai et al., 2005). RAMP2 levels also fluctuated during the process of adipocyte differentiation (Fukai et al., 2005). 10.6. Receptor activitymodifying protein regulation in sepsis In a mouse model of lipopolysaccharide-induced sepsis, there was a time-dependent decrease in lung RAMP2 mRNA, with a greater than 50% reduction 30 min following lipopolysaccharide treatment. In contrast, RAMP3 expression was greatly increased after 12 hr (Ono et al., 2000). 10.7. Receptor activitymodifying protein regulation in hypoxia In chronic hypobaric hypoxia, RAMP1 and RAMP3 but not RAMP2 were shown to be up-regulated in rat lung (Qing et al., 2001). The effect of hypoxia on RAMP2 mRNA expression has been evaluated in 2 human neuroblastoma cell lines (IMR-32, NB69; Kitamuro et al., 2001). Hypoxia itself decreased the amount of RAMP2 mRNA in IMR-32 but not NB69 cells. On the other hand, the hypoxia mimetic agents, cobalt chloride and desferrioxamine mesylate, decreased RAMP2 expression in both cell lines. The reduction of RAMP2 in hypoxia was not due to reduced mRNA stability, as RAMP2 mRNA actually had a greater half-life under hypoxia than normoxia in IMR-32 cells, and therefore, the reduction was most likely the result of decreased transcription. However, cobalt chloride induced a more rapid decay of RAMP2 mRNA in these cells (Kitamuro et al., 2001). 10.8. Receptor activity-modifying protein regulation by dexamethasone It has been shown that RAMP1 and RAMP2 are subject to regulation by dexamethasone in osteoblastic cells. There

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was a time (but not dose)-dependent increase in their expression following dexamethasone treatment, while RAMP3 levels were unchanged (Uzan et al., 2004). In human coronary VSMC, RAMP1 levels were transiently increased, but RAMP2 mRNA levels were stable over time with 10 7 M dexamethasone treatment (Frayon et al., 2000). In contrast to the osteoblastic cells, RAMP2 levels decreased compared with control at 10 10 M dexamethasone (Frayon et al., 2000).

11. Concluding remarks RAMPs transformed our understanding of how receptor pharmacology can be modulated and provided a novel mechanism for generating receptor subtypes within a subset of family B GPCRs. Their role has now broadened and they have been shown to interact with several other family B GPCRs, in 1 case modifying signaling parameters. There is now evidence to suggest that their interactions also reach into family C, and possibly family A, GPCRs, indicating that their function may not be restricted to modulation of a highly specific subset of receptors. Indeed, many aspects of RAMP function remain poorly understood, and the full extent of their action remains to be explored.

Acknowledgments PMS is a Senior Research Fellow of the National Health and Medical Research Council of Australia. DLH is supported by the Lottery Health Commission (New Zealand), Auckland Medical Research Foundation, and the University of Auckland Staff Research Fund. DRP was supported by the BBSRC. Elements of some figures were created using templates from Science Slides (VisiScience, NC, USA).

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