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ACTIVITY MODIFYING DIFFERENTIALLY MODULATE THE G -COUPLING EFFICIENCY OF RECEPTORS

Abbreviated title: RAMPs modulate selectivity

Maria Morfis, Nanda Tilakaratne, Sebastian G. B. Furness, Tim D. Werry, Arthur Christopoulos and Patrick M. Sexton

Drug Discovery Laboratory, Department of Pharmacology, Monash University, Clayton, Victoria 3800, Australia (MM, SGBF, NT, TDW, AC, PMS), Howard Florey Institute and Department of Pharmacology, The University of Melbourne, Victoria 3010, Australia (MM).

Address correspondence to: Prof. Patrick M. Sexton, Department of Pharmacology, Building 13E, Monash University Wellington Road, Clayton, Victoria 3800, AUSTRALIA Phone: 61 3 9905 4849 Fax: 61 3 9905 5953 Email: [email protected]

Non-standard abbreviations: GPCR, G protein-coupled receptor; CT, ; CTR, calcitonin receptor; CL, calcitonin receptor-like; CGRP, calcitonin -related ; AM, receptor; AMY, amylin receptor; PI, phospho-; PLC, phospho-lipase C; PKC, phospho-kinase C; PKA, phospho-kinase A; PI3K, Phosphoinositide-3 kinase; RAMP, receptor activity modifying protein; RCP, receptor component protein

Footnotes: This work was funded by the National Health and Council (NHMRC) of Australia (grant numbers 299810 and 436781 to PMS). PMS is a Principal Research Fellow of the NHMRC. AC is a Senior Research Fellow of the NHMRC. MM is a NHMRC Peter Doherty postgraduate scholar.

ABSTRACT

Receptor activity modifying proteins (RAMPs) 1, 2 and 3 are prototypic G protein-coupled receptor (GPCR) accessory proteins that can alter not only receptor trafficking but also receptor phenotype. Specific RAMP interaction with the calcitonin receptor (CTR) generates novel and distinct receptors for the peptide amylin, however, the role of RAMPs in receptor signalling is not understood. The current study demonstrates that RAMP interaction with the CTRa in COS-7 cells leads to selective modulation of signalling pathways activated by the receptor complex. There was a 20-30 fold induction in amylin potency at CTR/RAMP1 (AMY1) and CTR/RAMP3 (AMY3) receptors, compared with CTR alone, for formation of the second messenger cAMP that parallels the increase in amylin binding affinity. In contrast, only 2-5 fold induction of amylin potency was seen for mobilization of intracellular Ca++ or activation of ERK1/2. In addition, the increase in amylin potency for Ca++ mobilization was 2 fold greater for AMY3 receptors compared with AMY1 receptors and this paralleled the relative capacity of over- 125 expression of Gαq proteins to augment induction of high affinity I-amylin binding. These data demonstrate that RAMP-complexed receptors have a different signalling profile to CTRs expressed in the absence of RAMPs and this is likely due to direct effects of the RAMP on G protein-coupling efficiencies.

1 INTRODUCTION domain. Although RAMPs share a common G protein-coupled receptors (GPCRs) basic structure, including four conserved are the largest family of cell surface receptors; in the N-terminus, they share only a they play roles in virtually every physiological relatively low (~30%) sequence system and are implicated in most major identity. Originally discovered by McLatchie et diseases. Recently, increasing attention has been al during attempts to clone the receptor for focused on the role of accessory proteins in the CGRP, RAMPs were shown to chaperone the modulation of GPCR function (1-3). Receptor calcitonin receptor-like receptor (CL-R) to the activity-modifying proteins (RAMPs) are cell surface to form high affinity CGRP and AM prototypical accessory proteins that interact with receptors (10). Unlike CL-R, CTR when specific receptors to alter their function expressed alone, traffics to the cell surface and including, in a receptor-dependent manner, cell functions as a high affinity receptor for CT surface expression, binding phenotype, . However, when co-expressed with internalization and recycling (4,5). The most RAMP1, RAMP2 or RAMP3 the CTR/RAMP characterized partners for RAMP interaction are complexes generate pharmacologically distinct the calcitonin (CT) and calcitonin receptor-like amylin receptors, AMY1, AMY2 and AMY3 (CL) receptors that, with RAMPs, yield respectively (4). Hence, RAMPs can act as a receptors for calcitonin family peptides, pharmacological switch for these two GPCRs, including CT, amylin, calcitonin gene-related providing a sophisticated and novel mechanism peptide (CGRP) and adrenomedullin. for modulating receptor phenotype. Amylin, a 37 amino acid peptide, is co- To date, there have been only limited secreted with from the pancreatic β-cells studies looking at the potential role of RAMPs after food intake and has a range of effects on a in modulation of CL-R or CTR signalling. Co- number of different tissues to modulate expression of RAMPs with the predominant nutritional status. It is a potent inhibitor of receptor isoform CTRa had no effect on the vagally-mediated gastric emptying and has magnitude of cAMP accumulation or additional effects including reduced appetite, phosphatidylinositol (PI) hydrolysis in COS-7 reduced post-prandial and the cells (11). Despite this early study, it is clear inhibition of insulin-stimulated that the ability of RAMP2 to form functional production in (6,7). The stable amylin receptors is dependent on cellular amylin analogue, , has recently background and on receptor isoform. For received FDA approval for treatment of Type II instance, CTRa expressed with RAMP2 (7). Amylin is structurally related to the generated an amylin receptor in CHO-P cells but other CT peptide family members and shares a only very weakly induced an amylin response in number of actions with the other peptides, COS-7 cells (12). Furthermore, RAMP2 co- particularly CT (4). Nonetheless, CT has a expressed with the CTRb isoform (differing to distinct set of physiological actions, most CTRa by a 16aas insert in the 1st intracellular notably to regulate blood calcium levels by loop) was able to induce amylin receptors in inhibiting osteoclast mediated resorption COS7 cells (12), while the CTRa isoform and stimulating renal calcium clearance. It is effectively cannot. Interestingly, there is no commonly used for the therapeutic treatment of significant difference in peptide binding between hypercalcaemic conditions, including Paget’s the two receptor isoforms, yet the presence of disease and osteoporosis (8). this loop does impair G protein-coupling and Amylin receptors are generated from the signalling in a cell specific manner (13,14). CT receptor (CTR) gene product when co- Differences in the ability or availability of expressed with RAMPs (9). RAMPs constitute a particular G proteins to interact with specific unique family of type-I transmembrane proteins, RAMP-receptor heterodimers may account for comprising RAMP1, RAMP2 and RAMP3. the alterations in pharmacological phenotype Each RAMP possesses a large extracellular N- and cell type specificity. This theory is terminal domain, a single transmembrane α- supported by evidence that over-expression of helix and a small intracellular C-terminal Gαs proteins can modify the formation of

2 functional RAMP/CTRa complexes in COS-7 31-8220 and ET-18-OCH3 were purchased from cells (15). Calbiochem (La Jolla, CA). and The current study demonstrates that RAMP forskolin were purchased from Sigma Aldrich interaction with the CTRa leads to selective (St. Louis, MO). modulation of signalling pathways activated by cDNA constructs— The preparation of the receptor complex. There was a marked cDNA with a double haemaglutinin (HA) induction in amylin potency at AMY1 and epitope tag at the N-terminus of CTa 447 AMY3 receptors for formation of the second receptor ( variant) has been described messenger cAMP that parallels the increase in previously (17). Human RAMP1, RAMP2 and amylin binding affinity. In contrast, only very RAMP3 cDNA constructs were a gift from Dr. weak induction of amylin potency was seen for Steven Foord (GlaxoSmithKline, Stevenage, ++ mobilization of intracellular Ca or activation of UK; 10). cDNA for Gαs(short), Gαi2, GαoA and ERK 1/2. In addition, subtle differences in the Gαq were from the UMR cDNA resource centre effect of RAMP1 versus RAMP3 were observed (www.cdna.org). All constructs had been that were also seen in the relative capacity of subcloned into the pcDNA3.1 mammalian over-expression of Gα proteins to augment (Invitrogen). induction of high affinity amylin binding. These data demonstrate that RAMP-complexed Cell culture and transfection. COS-7 cells receptors have a different signalling profile to were maintained in DMEM supplemented with CTRs expressed in the absence of RAMPs and 5% FBS and maintained at 37°C in a humidified this is likely due to direct effects of the RAMP atmosphere of 5% CO2. For competition on G protein-coupling efficiencies. binding assays, cells were seeded in 48 (1 cm2)- well plates at a density of ~125,000 cells/well. METHODS The following day, when cells were 90-100% Materials. Human calcitonin (hCT) and rat confluent, they were transfected using 0.75 amlyin (rAmy) were purchased from Auspep µl/cm2 of Metafectene with 50ng and 75ng/cm2 (Parkville, VIC, Australia). BSA, isobutyl of CTRa and RAMP cDNA, respectively, as methylxanthine (IBMX) and poly-L-lysine were previously described (18). For analysis of the from Sigma (St.Louis, MO). Amplified effect of over-expression of Gα protein on luminescent proximity homogenous assay RAMP-induced amylin binding, cells were (ALPHA)-screen cAMP kits were purchased seeded into 24-well plates to achieve a final from Perkin Elmer (Boston, MA), ERK Surefire density of ~250,000 cells/well. Cells were assay kits were a kind gift from TGR Bioscience incubated under growth conditions for a further (Adelaide, Australia). Fluo-4 label was 36 h before being used in radioligand binding purchased from Molecular Probes (Invitrogen; assay. For the functional assays, cells were Carlsbad, CA). DMEM and HEPES were from seeded in 75cm2 flasks and grown overnight to Invitrogen; fetal bovine serum (FBS) was from ~90% confluence. Each flask was transfected TRACE Biosciences (Sydney, Australia). Cell with 3.2µg of CTRa and 4.6µg of RAMPs or culture plasticware was from Nunc (Roskilde, pcDNA3 cDNA using 30µl of metafectene per Denmark) and Metafectene was from Biontex flask. Cells were incubated and recovered in supplied by Scientifix (Melbourne, Australia). growth media as described above. Cells were 125 I rat amylin was prepared using Bolter-Hunter harvested 16 h after transfection and seeded for reagent; 2000 Ci/mmol from Amersham use in cAMP, ERK1/2 and Ca++ (Buckinghamshire, UK) and purified by reverse- mobilization assays or for antibody binding phase high performance liquid chromotography experiments. Allowing 16 h incubation for cells 125 as described previously (16). I-labeled goat to adhere, cells were subsequently serum-starved anti-mouse IgG was obtained from Perkin Elmer for a further 24 h prior to use in the functional (Boston, MA, USA). The inhibitors H-89, U- assays. 73122, PD98059, U0126, wortmannin and staurosporine were purchased from BIOMOL Measurement of cAMP. Intracellular cAMP International (USA). Tyrphostin AG1478, Rö- levels were determined using the AlphaScreen

3 cAMP (PerkinElemer Life Sciences). On the CaCl2, 2.2; probenecid, 2 and 0.5% (w/v) BSA. day of assay, cells were harvested and assayed In light-diminished conditions, a 100μl of wash as previously described (18). Each assay point solution was added containing the cell-permeant was performed in triplicate and the quantity of Ca++ fluorophore, Fluo-4/AM (10 µM) and cAMP generated was calculated from the raw incubated for 1 h at 37°C. The fluorophore data using a cAMP standard curve. solution was aspirated from the wells, and cells were washed twice then incubated for 30 min in Radioligand binding. Cells transfected in 48- modified HBSS at 37°C. The assay plate was well plates and incubated for approximately 36 h transferred to a FlexStation (Molecular Devices, 125 were assayed for I-rat amylin binding. Cells Palo Alto, CA, USA), which performed the were incubated in binding buffer (DMEM with robotic addition of ligands (10 × stocks in 0.3% w/v BSA) containing approximately 120 modified HBSS). Receptor-mediated changes in 125 pM I-rat amylin in the absence (total binding) intracellular Ca++ concentration were or presence of increasing concentrations of immediately recorded by the FlexStation using unlabelled peptide. Cells were incubated for 1 h an excitation wavelength of 485 nm and at 37°C before being washed with 250 µl PBS, emission wavelength of 520 nm. Data were and then solubilized with 250 µl of 0.5 M collected for each well every 1.52 s for a total of NaOH. The cell lysate was collected and 135 s. counted in a Packard γ-counter (75% efficiency) to determine bound radioactivity. Experiments ERK phosphorylation assay. Transfected were performed in duplicate, n=4. To assess the cells were seeded in 96-well plates at a density effect of over-expression of Gα proteins on of 50,000 cells/well, and incubated for 16 h prior induction of AMY receptor phenotype, cells to being serum starved overnight. On the day of were plated into 24-well plates and transfected assay, cells were pre-treated with buffer or with CTRa (100ng) and one of the four (Gαs, inhibitors (at the concentrations specified) and Gαi2, GαoA, Gαq) Gα subtypes (150ng) together then stimulated with at 37°C. Time with either pcDNA3.1 empty vector or one of course results demonstrated a peak response at 5 RAMP1, RAMP2 or RAMP3 (100ng). Whole min for all receptor complexes following agonist 125 cells were assayed for I-rat amylin binding 48 stimulation; this time point was subsequently h post-transfection, by incubating transfected used in concentration-response studies. ERK1/2 cells with radioligand (80 pM/well) in the phopshorylation was measured using the absence (total binding) or presence of 10-6 M AlphaScreen based ERK SureFire assay kit as unlabelled rAmy (non-specific binding). previously described (19). Experiments were performed in triplicate, n=3-5. Data Analysis. Competition binding data and Measurement of cell surface expression of cAMP, ERK1/2 phoshporylation and Ca++ HA-CTR by antibody binding. Cell mobilization concentration-response data were surface expression of HA-tagged CTa receptor analyzed via nonlinear regression using PRISM was determined as previously described (18) ~48 version 4.3 (GraphPad Software, San Diego, h post transfection using mouse anti-HA CA). In all instances, data shown are the 125 (12CA5) antibody and I-labeled goat anti- mean ± S.E. Comparisons between data were mouse IgG secondary. performed by one-way analysis of variance; post-hoc testing was via either Dunnet’s test for Calcium mobilization assay. Transfected cells comparisons with control or Tukey-Kramer’s were seeded in poly-L-lysine coated 96-well test for multiple comparisons. Statistical analysis plates at a density of 50,000 cells/well, was performed within the Prism 4.3 incubated overnight and serum-starved for a environment. Unless otherwise stated, values further 24 h. Cells were washed three times with of p< 0.05 were taken as significant. Potency a modified Hanks buffered saline solution data are presented as negative log molar (p) as (HBSS; containing (in mM): NaCl, 150; KCl, errors are log normally distributed. 2.6; MgCl2, 1.18; D-, 10; HEPES, 10;

4 receptors were 6.8 ± 0.2 and 6.4 ± 0.4, RESULTS and DISCUSSION respectively, and this is equivalent to those The CTRa functionally couples to reported previously for cAMP accumulation multiple effector pathways including the (18). Thus, while the change is small, the proximal second messengers cAMP and Ca++ increase in amylin potency observed in ERK1/2 and the more distal effector phosphorylated- activation is via the RAMP/CTR complexes. ERK1/2 (8,13,14). We therefore examined the Direct stimulation of adenylate cyclase with ability of RAMPs to induce amylin receptors forskolin was ineffective at activating ERK1/2 that are linked to these pathways. (data not shown), suggesting that ERK We first compared the relative ability of activation in this system could not be stimulated amylin, acting via RAMP/CTR complexes, to by cAMP alone and must result from coupling induce cAMP accumulation and to stimulate to alternative pathways. Taken together, the ERK1/2 phosphorylation. CTRa was co- above results raise the possibility that the expressed in COS-7 cells with RAMP1, coupling efficiencies of AMY1 and AMY3 RAMP2, RAMP3 or vector control and receptors for different G protein subtypes differs induction of cAMP accumulation or ERK1/2 from that of the CTRa. phosphorylation were assessed in parallel. In We subsequently investigated the ability

agreement with previous findings in this cellular of CTRa and AMY receptors to couple to Gαq by background, RAMP1 and RAMP3 induced a measuring the downstream release of marked increase in amylin potency for cAMP intracellular Ca++. Analogous to cAMP production (27-fold and 21-fold, respectively) responses, hCT-induced intracellular Ca++ compared to CTRa receptor and vector, while mobilization was mostly unaffected by co- RAMP2 had only a weak effect (Fig. 1A and expression of RAMPs, with only a small Table 1) (9,12). Responses to human CT (hCT) decrease in potency seen with co-expression of were not affected by RAMP1 or RAMP2, but RAMP3 (Fig. 1F). CTRa co-expression with decreased (~3-fold) with RAMP3, presumably RAMP1 or RAMP3 led to a weak increase in due to reduction in free CTRa availability (18) amylin potency for Ca++ mobilization of 2 and 4 (Fig. 1B). In contrast to cAMP production, the fold, respectively (Fig. 1E). potency of amylin in stimulating ERK1/2 Together with the data from ERK1/2 phosphorylation in CTRa/RAMP co-expressing activation (Fig. 1C and Table 1), this suggests cells was much smaller (3.8-fold and 4.7-fold that the relative coupling of AMY receptors is

potency increase for RAMP1 and RAMP3, much stronger to Gαs than to the alternative respectively) (Fig. 1C and Table 1). pathways investigated. This large difference in Given the marked divergence in potency coupling efficiency across the three effector between the two signalling pathways we sought pathways measured was not observed at the to establish whether the small increase in Amy CTRa in the absence of RAMPs; when CTRa potency for ERK activation was via the same was expressed alone the agonist potencies in the receptor mediating the larger cAMP response. cAMP, ERK1/2 and Ca++ mobilization were Therefore, we performed concentration response similar for both rAmy and for hCT (Table 1). analysis of amylin-induced ERK1/2 When comparing the potency of amylin across phosphorylation in the presence of CGRP8-37, a the CTRa/RAMP complexes, we also found that selective antagonist that is able to differentiate amylin was more potent on AMY3 than on ++ between CTRa and AMY receptors (18). AMY1 receptors for Ca mobilization (Table 1), CGRP8-37 caused a rightward-shift of the amylin which is the reverse of that observed in cAMP dose-response curve at AMY1 (CTRa + accumulation. RAMP1) and AMY3 (CTRa + RAMP3) To eliminate the possibility that the receptors but not at CTRa (alone), while the changes in amylin potency observed were due to non-selective antagonist AC187 right-shifted differences in receptor expression, we measured both hCT and amylin responses at each of the expression in parallel to the receptor complexes. The pKB values for the functional assays by immuno-detection using antagonist CGRP8-37 at the AMY1 and AMY3 antibodies against the HA- tag on the N-

5 terminus of the CTRa. The presence of RAMPs We next performed ERK1/2 did not significantly alter the amount of CTRa phosphorylation and intracellular Ca++ expressed on the cell surface (Fig. 2A). As a mobilization assays in the presence of selective further control, we used unlabelled rAmy to signalling inhibitors to interrogate the pathway competitively inhibit 125I rAmy binding to cells of activation for differing RAMP/receptor co-expressing CTRa and RAMPs. The data complexes. The use of inhibitors of protein illustrate that rAmy has approximately equal kinase (PK) A (H89; 10μM, 1 h) and Gαi/o affinity for AMY1, AMY2 and AMY3 receptors proteins (pertussis toxin; 100ng/ml, 18 h) (Fig 2B) with pIC50 values of 8.13 ± 0.10, 7.83 established that stimulation of ERK1/2 ± 0.17 and 8.13 ± 0.12, respectively, although phosphorylation was independent of activation the level of induced binding was less with of PKA or Gαi/o proteins; the former being RAMP2; this is in agreement with previously consistent with lack of forskolin-mediated published data in this cellular background activation of ERK1/2 in the COS-7 cells. (12,20). Inhibition of PI 3-kinase (wortmannin; 100nM, The cAMP, ERK1/2 and Ca++ functional 30 min) and PI- C (PI-PLC) responses assessed in this study were measured (U73122; 10μM, 30 min) both led to partial over different time scales. The cAMP assay is an inhibition of response, as did inhibition of the accumulation assay where receptors are exposed epidermal receptor to the agonist for 30 min prior to quantifying the (AG1478; 100nM, 30 min), indicating that each amount of cAMP produced. In comparison, of these pathways could contribute to the ERK ERK1/2 phosphorylation is measured 5 mins response. Inhibition of PKC (staurosporine; ++ after agonist stimulation, while the Ca 1μM, 30 min; Rö-318220; 10μM, 30 min) led to mobilization is measured as peak response that the abolition of signalling, as did inhibition of occurs seconds after ligand exposure. For both mitogen activated protein kinase kinase (MEK) ++ ERK1/2 and Ca assays, the ligand has not had (U0126; 10μM, 30 min; PD-98058; 20μM, 30 sufficient time to equilibrate in the system prior min), indicating that there is convergence on to the measurement of downstream signalling PKC and eventually MEK for activation. ET-18- and this may cause difficulties in comparing OCH3, which can inhibit PI-PLC, PI 3-kinase results of these assays with the cAMP assay, and Raf (100μM, 30 min) also abolished where equilibrium can be reached. To ensure signalling. However, there was no apparent that the differences we observed were caused by difference in the relative contribution of the RAMPs and not by a kinetic non-equilibrium different signalling pathways to ERK activation effect we repeated the cAMP accumulation between the CTRa alone or the AMY1 or AMY3 assay, measuring cAMP production after only 5 receptor complexes (Fig. 4). The inability to min agonist treatment, equivalent to the agonist detect pathway differences in the activation of stimulation time of the ERK1/2 activation assay. ERK is likely to be due, at least in part, to the The results demonstrate that the strong induction strong background phenotype of the CTRa alone of amylin phenotype at the CTRa in the in the RAMP co-transfected cells coupled with presence of RAMP1 and RAMP3 is maintained, only a weak induction of amylin phenotype for with a 31- and 20- fold enhancement of potency, signalling to this pathway. respectively, compared to CTRa alone (Fig. 3). For Ca++ mobilization, the PKA inhibitor Interestingly, the pEC50 values for the 5 min H89 had no effect on responses to either hCT or cAMP assay are all consistently 2-6 fold greater amylin, while the broad spectrum inhibitor ET- than the 30min assay, which is probably due to 18-OCH3 completely abolished signalling at the non-equilibrated status of the system (Table each of the receptors (Fig. 5). In contrast, there 1). The data confirm that the greater induction of were receptor specific differences in the capacity amylin phenotype seen in the cAMP assays of the PI-PLC inhibitor U73122 to inhibit compared to the ERK1/2 phosphorylation and responses. There was strong inhibition of Ca++ mobilization assays is not due to the assay signalling via the AMY1 receptor complex conditions. (~72%), compared with weak inhibition for the

6 CTRa alone or the AMY3 receptor complex (30- the RAMPs were contributing directly to the 40% inhibition). ability of the receptor complexes to interact with The above signalling data implied that G proteins (15). Intriguingly, and consistent with the different RAMP/CTR complexes were the current study, truncation of the C-terminus causing differential modulation of the ability of differentially affected individual RAMPs and the receptor to interact with specific G proteins. there were also differences in the sensitivity of To test this more empirically, we examined the the truncated RAMP/CTR complexes to effect of over expression of different Gα protein recovery by Gαs overexpression. subunits (Gs, Gi2, GoA, Gq) on the ability of In conclusion, this study demonstrates individual RAMPs to induce high affinity 125I- that RAMPs can differentially modulate the rAmy binding. As previously reported in this coupling efficiency of CTRa to various G cell background, RAMP1 and RAMP3 potently proteins. This expands the repertoire of actions induced 125I-rAmy binding, with the greatest that RAMPs have in modulating GPCR function, effect seen with RAMP1, while RAMP2 and likely extends beyond the CTR investigated generated only a low level of induced binding in the current work. This form of fine

(Fig. 6). None of the Gα subunits modulated the manipulation of receptor signalling provides binding to the CTRa expressed alone (Fig. 6A). new opportunities for development of novel Similarly, while there were trends for Gs to therapeutic agents targeting RAMP complexed increase and Go to decrease RAMP1-induced receptors. binding, neither of these was significant (Fig. 6B). In contrast, there was a large increase in RAMP2-induced binding in the presence of Gs but not other Gα proteins (Fig. 6C), while both 125 Gs and Gq led to increased I-rAmy binding with RAMP3 (Fig. 6D). Thus, these data support the proposition that individual RAMPs may each lead to a different profile of signalling from the CTR expressed alone. Structurally, the 3 RAMPs have only a very short intracellular C-terminal tail of ~10 amino acids (10). Evidence for a direct role of this domain in the signalling specificity of RAMP-receptor complexes arises from chimeras of RAMP1 and RAMP2 where the potency of CGRP, a high affinity ligand of RAMP1 complexed CTR but not RAMP2 complexed CTR, to stimulate cAMP production was contextual on the C-terminal domain present; CGRP had increased potency when the RAMP1 C-terminus was present, despite an overt binding phenotype that was primarily influenced by the N-terminal domain present (21). Further evidence for involvement of the C-terminus in G protein interaction arose from studies on C- terminally truncated RAMPs; deletion of the last 8 amino acids led to a marked loss in the capacity of RAMPs to induce high affinity amylin receptors from CTRa receptors. This loss could be, at least partially, recovered by the over-expression of Gαs protein, indicating that

7 REFERENCES

1. Bockaert J, Fagni L, Dumuis A, Marin P 2004 GPCR interacting proteins (GIP). Pharmacol Ther 103:203-221 2. Sato M, Blumer JB, Simon V, Lanier SM 2006 Accessory proteins for G proteins: partners in signaling. Annu Rev Pharmacol Toxicol 46:151-187. 3. Tilakaratne N, Sexton PM 2005 G Protein-coupled receptor-protein interactions: basis for new concepts on receptor structure and function. Clin Exp Pharmacol Physiol 32:979-987. 4. Poyner DR, Sexton PM, Marshall I, Smith DM, Quirion R, Born W, Muff R, Fischer JA, Foord SM 2002 International Union of Pharmacology. XXXII. The mammalian calcitonin gene- related peptides, adrenomedullin, amylin, and calcitonin receptors. Pharmacol Rev 54:233-246. 5. Hay DL, Poyner DR, Sexton PM 2006 GPCR modulation by RAMPs. Pharmacol Ther 109:173-197. 6. Young A 2005 Amylin, Physiology and Pharmacology. In Advances in Pharmacology, Volume 52. San Diego, Academic Press, pp360. 7. Want LL, Ratner RE 2006 Pramlintide: A new tool in . Curr Diab Rep 6:344-349. 8. Sexton PM, Findlay DM, Martin TJ 1999 Calcitonin. Curr Med Chem 6:1067-1093. 9. Christopoulos G, Perry KJ, Morfis M, Tilakaratne N, Gao Y, Fraser NJ, Main MJ, Foord SM, Sexton PM 199) Multiple amylin receptors arise from receptor activity-modifying protein interaction with the calcitonin receptor gene product. Mol Pharmacol 56:235-242. 10. McLatchie LM, Fraser NJ, Main MJ, Wise A, Brown J, Thompson N, Solari R, Lee MG, Foord SM 1998 RAMPs regulate the transport and ligand specificity of the calcitonin-receptor- like receptor. Nature 393:333-339. 11. Christopoulos A, Christopoulos G, Morfis M, Udawela M, Laburthe M, Couvineau A, Kuwasako K, Tilakaratne N, Sexton PM 2003 Novel receptor partners and function of receptor activity-modifying proteins. J Biol Chem 278:3293-3297. 12. Tilakaratne N, Christopoulos G, Zumpe ET, Foord SM, Sexton PM 2000 Amylin receptor phenotypes derived from human calcitonin receptor/RAMP coexpression exhibit pharmacological differences dependent on receptor isoform and host cell environment. J Pharmacol Exp Ther 294:61-72. 13. Moore EE, Kuestner RE, Stroop SD, Grant FJ, Matthewes SL, Brady CL, Sexton PM, Findlay DM 1995 Functionally different isoforms of the human calcitonin receptor result from alternative splicing of the gene transcript. Mol Endocrinol 9:959-968. 14. Raggatt LJ, Evdokiou A, Findlay DM 2000 Sustained activation of Erk1/2 MAPK and suppression by the insert-negative, but not the insert-positive isoform of the human calcitonin receptor. J Endocrinol 167:93-105. 15. Udawela M, Christopoulos G, Morfis M, Christopoulos A, Ye S, Tilakaratne N, Sexton PM 2006 A critical role for the short intracellular C terminus in receptor activity-modifying protein function. Mol Pharmacol 70:1750-1760. 16. Bhogal R, Smith DM, Bloom SR 1992 Investigation and characterization of binding sites for islet polypeptide in rat membranes. 130:906-913. 17. Pham V, Wade JD, Purdue BW, Sexton PM 2004 Spatial proximity between a photolabile residue in position 19 of salmon calcitonin and the amino terminus of the human calcitonin receptor. J Biol Chem 279:6720-6729. 18. Hay DL, Christopoulos G, Christopoulos A, Poyner DR, Sexton PM 2005 Pharmacological discrimination of calcitonin receptor: receptor activity-modifying protein complexes. Mol Pharmacol 67:1655-1665.

8 19. Van der Westhuizen ET, Werry TD, Sexton PM, Summers RJ 2007 The Family Peptide Receptor 3 (RXFP3) activates ERK1/2 through a PKC dependent mechanism. Mol Pharmacol 71:1618-1629. 20. Zumpe ET, Tilakaratne N, Fraser NJ, Christopoulos G, Foord SM, Sexton PM 2000 Multiple ramp domains are required for generation of amylin receptor phenotype from the calcitonin receptor gene product. Biochem Biophys Res Commun 267:368-372. 21. Udawela M, Christopoulos G, Tilakaratne N, Christopoulos A, Albiston A, Sexton PM 2006 Distinct receptor activity-modifying protein domains differentially modulate interaction with calcitonin receptors. Mol Pharmacol 69:1984-1989.

9 FIGURE LEGENDS

Figure 1. Measurement of rat amylin (rAmy) or human calcitonin (hCT) induced signalling activation via the CTa receptor in the absence or presence of RAMPs. COS7 cells were co-transfected with CTRa and either RAMP1 (open circles), RAMP2 (filled triangles), RAMP3 (open inverted triangles) or pcDNA control (filled circle) and cells were assayed 64 h after. The left hand panels represent dose responses to rAmy stimulation and the right hand panels to hCT stimulation. (A) and (B) show agonist mediated cAMP accumulation, measured after 30 min stimulation with rAmy or hCT, respectively. Graphed in (C) and (D) are the changes in ERK1/2 phosphorylation following agonist-stimulation for 5 min; the time-point of peak response determined from time-course assays (data not shown). Graphs (E) and (F) show the Fluo4 measured intracellular Ca++ release. Data are presented as mean ± SEM, n=4-9.

Figure 2. Competition binding and cell surface receptor labelling for HA-CTa receptor in the absence or presence of RAMPs. (A) Cell surface expression of CTRa protein. COS-7 cells are transiently transfected with CTRa and pcDNA3 empty vector (v) or CTRa and either RAMP1, RAMP2, or RAMP3, measured by binding anti-HA antibody to the 2×HA-N terminal-tagged CTRa followed by detection with incubation of a 125I-labeled goat anti-mouse IgG secondary antibody. Data are expressed as a percentage of the binding of 125I-IgG antibody to cells expressing the CTRa protein alone. Data are mean ± SEM from 8 independent experiments. (B) Peptide competition binding for 125I-rAmy binding to CTRa and either RAMP1, RAMP2 or RAMP3. Data were mean ± S.E.M of four separate experiments 125 with duplicate repeats. B, I-rAmy; B0, total binding in the absence of cold peptide; N, nonspecific binding (measured in the presence of 10-6M peptide).

Figure 3. Measurement of rat amylin (rAmy) or human calcitonin (hCT) induced cAMP accumulation over 5 min for the CTRa in the absence or presence of RAMPs. (A) rAmy or (B) hCT stimulation. Data are means ± SEM from 5 independent experiments.

Figure 4. Pharmacological inhibitor based analysis of signaling components involved in activation of ERK1/2. Shown is peak (5 min) ERK1/2 phosphorylation in COS7 cells expressing CTRa and either pcDNA3 (A and B), RAMP1 (C) or RAMP3 (D) following stimulation with 100 nM rAmy (A, C and D) or hCT (B). Prior to stimulation with , cells were pre-incubated with either buffer (untreated) or one of the following inhibitors; H-89 (10μM, 1hr), pertussis toxin (100ng/ml, 18 h), wortmannin (100nM,

30 min), U73122 (10μM, 30min), ET-18-OCH3 (100μM, 30 min), staurosporine (1μM, 30 min) Rö-31- 8220 (10μM, 30 min), PD-98058 (20μM, 30 min), Tyrphostin AG1478 (100nM, 30 min) or U0126 (10μM, 30min). Data are mean ± SEM, n=3- 4.

Figure 5. rAmy-induced Ca++ mobilization is differentially modulated by PLC inhibition in CTRa/RAMP1 versus CTRa/RAMP3 expressing COS-7 cells. Ca++ mobilization in response to increasing concentrations of hCT (A) or rAmy (B-D) in COS-7 cells transiently transfected with CTRa together with pcDNA3 (A, B), RAMP1 (C) or RAMP3 (D). Cells were pretreated with buffer (filled circles) or inhibitors for the times indicated before agonist stimulation in the continued presence of inhibitor. The inhibitors used were: H-89 (open triangles, PKA inhibitor; 10 μM, 1 h); U73122 (open circles, PLC inhibitor; 10 μM, 30 min); ET-18-OCH3 (open inverted triangles, PLC, PI3K, PKC inhibitor; 100 μM, 30min). Data are mean ± SEM, n=3. FOB, fold over basal. Emax values (FOB) for the AMY1 receptor in the absence and presence of U73122 were 4.17 ± 0.27, n=5 and 1.91 ± 0.19, n=3, respectively. Emax values for the AMY3 receptor in the absence and presence of U73122 were 4.34 ± 0.40, n=5 and 3.12 ± 0.20, n=3, respectively. The effect of U73122 was significantly greater for the AMY1 receptor

10 compared with the AMY3 receptor or CTRa alone, p<0.05; one-way ANOVA followed by Bonferri’s multiple comparison test.

Figure 6. Overexpression of Gα subunits differentially modulates induction of amylin receptor phenotypes by RAMPs. 125I-rat amylin (125I-rAmy) binding to COS-7 cells co-transfected with CTRa (100ng) and one of the four (Gαs, Gαi2, GαoA, Gαq) Gα subtypes (150ng) together with either pcDNA3.1 empty vector (A), RAMP1 (B), RAMP2 (C) or RAMP3 (D) DNA (150ng). Whole cells were assayed for 125I-rAmy binding 48 h post-transfection, by incubating transfected cells with radioligand (80 pM/well) in the absence (total binding) or presence of 10-6 M unlabelled rAmy (non-specific binding). Specific binding was determined by subtracting non-specific from total binding. Data are mean ± SEM, n=3-5. *Significantly different from vector control group; one-way ANOVA with Dunnett’s post-hoc test.

11 Table 1. Agonist potency estimates. Shown below are the pEC50 values for peptide-induced cAMP production (for both 30 min and 5 min agonist stimulation), ERK1/2 activation and intracellular Ca++ mobilization. “Fold Δ” gives the fold change in amylin potency when comparing potency for CTRa/RAMP1 complexes to CTRa + pcDNA3. Data are presented as mean ± S.E.M. The number of individual experiments analysed is shown in parentheses.

cAMP (30 min) cAMP (5 min) ERK1/2 (5 min) Ca++ rAmy Fold Δ rAmy Fold Δ rAmy Fold Δ rAmy Fold Δ

CTRa + pcDNA3 7.80 ± 0.11 (5) 8.26 ± 0.11 (5) 7.76 ± 0.11 (7) 7.44 ± 0.06 (9) CTRa + RAMP1 9.23 ± 0.07* (6) 27 9.76 ± 0.15*(5) 31 8.34 ± 0.13* (8) 3.8 7.73 ± 0.07* (9) 2.0 CTRa + RAMP2 8.25 ± 0.10*† (6) 2.8 8.53 ± 0.09†(5) 1.8 7.83 ± 0.10† (8) 1.2 7.66 ± 0.14 (4) 0.7 CTRa + RAMP3 9.12 ± 0.13* (6) 21 9.57 ± 0.14*(5) 20 8.43 ± 0.11* (8) 4.7 8.04 ± 0.08*‡(9) 4.0

hCT hCT hCT hCT CTRa + pcDNA3 8.80 ± 0.12 (5) 9.61 ± 0.16 (4) 8.47 ± 0.09 (8) 8.23 ± 0.04 (9) CTRa + RAMP1 8.64 ± 0.11 (6) 9.28 ± 0.16 (4) 8.35 ± 0.07 (8) 7.98 ± 0.09 (4) CTRa + RAMP2 8.82 ± 0.09 (6) 9.27 ± 0.18 (4) 8.56 ± 0.07 (8) 8.08 ± 0.08 (4) CTRa + RAMP3 8.20 ± 0.09* (6) 9.03 ± 0.13 (4) 8.02 ± 0.08* (8) 7.80 ± 0.08* (4)

*Significantly different from CTRa control. †Significantly different from CTRa/RAMP1 and CTRa/RAMP3. ‡Significantly different from CTRa/RAMP1 and CTRa/RAMP2. One way ANOVA with Tukey-Kramer multiple comparisons post-hoc test (P<0.05).

12

Figure 2

AB

125 100 HA-CTRa + 100 75 RAMP1 75 RAMP2 50 RAMP3 -N x 100) 50 0

I-rAmy bound 25

Antibody binding 25 125 (B-N/B (% control) CTRa+v 0 0

v 1 2 3 0 -11 -10 -9 -8 -7 -6 + MP MP MP Ta A A A Log [rAmy] (M) C R R R + + + Figure 3

A B 5 min rAmy 5 min hCT 100 100

75 75

50 + pcDNA3 + pcDNA3 50 + RAMP1 + RAMP1 25 + RAMP2 + RAMP2 25 + RAMP3 + RAMP3 0 cAMP response (% max) 0 cAMP response (% max)

0 -11 -10 -9 -8 -7 -6 0 -11 -10 -9 -8 -7 -6 Log [rAmy] (M) Log [hCT] (M) Figure 4

CTRa - 100nM hCT CTRa - 100nM rAmy AB2.5 2.5

2.0 2.0

1.5 1.5

1.0 1.0 ERK1/2activation(FOB) ERK1/2 activation(FOB)

0.5 0.5

ted 89 xin nin 22 H3 ine 20 58 78 26 ted 89 xin nin 22 H3 ine 20 58 78 26 a H o n 1 C r 2 0 4 1 a H o n 1 C r 2 0 4 1 73 73 tre s t -O po 1-8 G1 U0 tre s t -O po 1-8 G1 U0 n i tma U 8 s 3 A n i tma U 8 s 3 A U ö- PD-98 u ö- PD-98 uss or T-1 uro uss or T-1 uro t W E a R t W E a R er er P St P St

CTRa/RAMP1 - 100nM rAmy CTRa/RAMP3 - 100 nM rAmy CD2.5 2.5

2.0 2.0

1.5 1.5

1.0 1.0 ERK1/2 activation(FOB) ERK1/2 activation(FOB)

0.5 0.5

e e H89 CH3 H89 CH3 O O treated U0126 treated U0126 n U73122 AG1478 n U73122 AG1478 u PD-98058 u PD-98058 Rö-31-8220 Rö-31-8220 Wortmannin ET-18-taurosporin Wortmannin ET-18-taurosporin Pertussis toxin S Pertussis toxin S Figure 5

A CTRa + pcDNA3 B CTRa + pcDNA3 7 6

6 5 5 4 4 3 3 (FOB) (FOB) 2 2

Calcium response 1 Calcium response 1 0 0 0 -11 -10 -9 -8 -7 -6 0 -11 -10 -9 -8 -7 -6 Log [hCT] (M) Log[rAMY] (M)

wt H-89 U73122 ET-18-OCH3

C CTRa + RAMP1 D CTRa + RAMP3 5 5

4 4

3 3 (FOB) 2 (FOB) 2

1 1 Calcium response Calcium response

0 0 0 -11 -10 -9 -8 -7 -6 0 -11 -10 -9 -8 -7 -6 Log [rAMY] (M) Log [rAMY] (M) Figure 6

AB 12

10 20

8 15 cells) cells) 6 6 6 I-rAmy bound I-rAmy bound 10 125 4 125

(pmol/10 (pmol/10 5 2

Specific 0 Specific 0 2 2 Gs Gs ctor Gi Go Gq ctor Gi Go Gq e + + + + e + + + + v R v R TR T TR TR TR T TR TR C C C C C C C C TR + TR + C C

CD 12 12 * 10 10 * 8 8 * cells) cells) 6 6 6 I-rAmy bound 6 I-rAmy bound 125 4 125 4 (pmol/10 2 (pmol/10 2 Specific 0 Specific 0 2 r 2 Gs Gi Go Gq Gs Gi Go Gq ctor + + + + e + + v R vecto R TR T TR TR + C C C C R CT CTR CTR + CTR + TR + T C C