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Receptor Activity Modifying Proteins (Ramps) Interact with the VPAC2 Receptor and CRF1 Receptors and Modulate Their Function

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Receptor Activity Modifying Proteins (Ramps) Interact with the VPAC2 Receptor and CRF1 Receptors and Modulate Their Function British Journal of DOI:10.1111/j.1476-5381.2012.02202.x www.brjpharmacol.org BJP Pharmacology RESEARCH PAPER Correspondence David R Poyner, School of Life and Health Sciences, Aston University, Birmingham, Receptor activity modifying B4 7ET, UK. E-mail: [email protected] ---------------------------------------------------------------- proteins (RAMPs) interact Current address: *Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, 381 with the VPAC2 receptor Royal Parade, Parkville, Victoria 3052 Australia; †Discovery Sciences, AstraZeneca R&D, and CRF1 receptors and Mölndal, Sweden; ‡R&I iMED, AstraZeneca R&D, Mölndal, Sweden. modulate their function ---------------------------------------------------------------- Keywords D Wootten1*, H Lindmark2†, M Kadmiel3, H Willcockson3, KM Caron3, receptor activity-modifying proteins (RAMPs); RAMP1; J Barwell1, T Drmota2‡ and DR Poyner1 RAMP2; RAMP3; VPAC2 receptor; +/- CRF1 receptor; Ramp2 mice; 1 2 School of Life and Health Sciences, Aston University, Birmingham, UK, Department of Lead G-protein coupling; biased Generation, AstraZeneca R&D, Mölndal, Sweden, and 3Department of Cellular and Molecular agonism Physiology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA ---------------------------------------------------------------- Received 10 July 2012 Revised 15 August 2012 Accepted 28 August 2012 BACKGROUND AND PURPOSE Although it is established that the receptor activity modifying proteins (RAMPs) can interact with a number of GPCRs, little is known about the consequences of these interactions. Here the interaction of RAMPs with the glucagon-like peptide 1 receptor (GLP-1 receptor), the human vasoactive intestinal polypeptide/pituitary AC-activating peptide 2 receptor (VPAC2) and the type 1 corticotrophin releasing factor receptor (CRF1) has been examined. EXPERIMENTAL APPROACH GPCRs were co-transfected with RAMPs in HEK 293S and CHO-K1 cells. Cell surface expression of RAMPs and GPCRs was examined by ELISA. Where there was evidence for interactions, agonist-stimulated cAMP production, Ca2+ mobilization and GTPgS binding to Gs,Gi,G12 and Gq were examined. The ability of CRF to stimulate adrenal corticotrophic hormone release in Ramp2+/– mice was assessed. KEY RESULTS The GLP-1 receptor failed to enhance the cell surface expression of any RAMP. VPAC2 enhanced the cell surface expression of all three RAMPs. CRF1 enhanced the cell surface expression of RAMP2; the cell surface expression of CRF1 was also increased. There was no effect on agonist-stimulated cAMP production. However, there was enhanced G-protein coupling in a receptor and agonist-dependent manner. The CRF1 : RAMP2 complex resulted in enhanced elevation of intracellular calcium to CRF and urocortin 1 but not sauvagine. In Ramp2+/– mice, there was a loss of responsiveness to CRF. CONCLUSIONS AND IMPLICATIONS The VPAC2 and CRF1 receptors interact with RAMPs. This modulates G-protein coupling in an agonist-specific manner. For CRF1, coupling to RAMP2 may be of physiological significance. Abbreviations ACTH, adrenal corticotrophic hormone; CGRP, calcitonin gene-related peptide; CLR, calcitonin receptor-like receptor; CTR, calcitonin receptor; GLP-1, glucagon-like peptide 1; HA, haemagglutinin; RAMP, receptor activity modifying proteins; PACAP, pituitary AC-activating peptide; PHM-27, peptide histidine methionine-27; VIP, vasoactive intestinal polypeptide; WT, wild-type 822 British Journal of Pharmacology (2013) 168 822–834 © 2012 The Authors British Journal of Pharmacology © 2012 The British Pharmacological Society RAMP interactions with VPAC2 and CRF1 receptors BJP Introduction calcium signalling and furthermore, in Ramp2+/– mice, the physiological effects of CRF on adrenal corticotrophic Receptor activity-modifying proteins (RAMPs) consist, in hormone (ACTH) release are reduced. mammals, of three single-pass transmembrane proteins, first identified as essential components of calcitonin gene-related peptide (CGRP) and adrenomedullin receptors (McLatchie et al., 1998). They associate in the endoplasmic reticulum Methods with a GPCR known as calcitonin receptor-like receptor (CLR). The RAMPs and CLR by themselves have poor abilities to reach the cell surface and cannot bind any known endog- Materials Peptides were from Bachem (St. Helens, UK). Unless other- enous ligand. However, the complexes are translocated to the wise specified, chemicals were from Sigma or Fisher (Lough- cell surface where they respond to CGRP, adrenomedullin borough, UK). Cell culture reagents were from Gibco BRL and adrenomedullin 2 via CGRP, AM and AM receptors 1 2 (Paisley, Renfrewshire, UK) or Sigma. Monoclonal anti-HA, (Poyner et al., 2002; Wootten et al., 2010; Hong et al., 2011). mouse clone HA-7, monoclonal mouse anti-FLAG, clone M2 The trafficking of adrenomedullin receptors is also influenced and monoclonal goat anti-mouse antibody containing a con- by the RAMPs (Bomberger et al., 2005). jugated HRP were purchased from Sigma. G-protein antibod- RAMPs have subsequently been shown to interact with a ies (G ,G ,G ,G ) were from Santa Cruz (Santa Cruz, wider range of GPCRs. The best characterized of these inter- s q/11 12/13 i/o/t/z CA). actions is with the calcitonin receptor (CTR), where the RAMPs do not alter receptor cell surface expression but instead change ligand binding and G-protein coupling to give Expression constructs amylin receptors (Hay et al., 2006; Morfis et al., 2008). RAMPs Plasmid DNA was extracted from the cultures using a Wizard- 1 and 3 are needed for cell surface expression of the calcium Prep DNA extraction kit according to the manufacturer’s sensing receptor, a glutamate-like/family C GPCR (Bouschet instructions (Promega, Southampton, UK). The plasmid DNA et al., 2005). However, most interest has focussed on secretin- was eluted in 100 mL sterile distilled water and stored at like/family B GPCRs. By monitoring the ability of GPCRs to -20°C. For all receptors, a pcDNA3.1– template vector was increase the cell surface expression of RAMPs, it has been produced containing a T8 signal peptide and a HA-Tag shown that the vasoactive intestinal polypeptide (VIP)/ (cloned in using NotI and EcoRI). The mature protein pituitary AC-activating peptide 1 receptor (VPAC1) could sequence for each receptor was then cloned in using EcoRI interact with all three RAMPs, the parathyroid hormone and HindIII. The EcoRI site between the tag, and the receptor PTH1 receptor could interact with RAMP2, the parathyroid sequence was removed using Quikchange. The RAMPs were hormone PTH2 receptor could interact with RAMP3 and the modified with a FLAG tag inserted just before residue 24 glucagon receptor could interact with RAMP2. No evidence (RAMP1), 42 (RAMP2) and 25 (RAMP3). A pcDNA3.1+ tem- was found for interactions between RAMPs and the VPAC2 or plate vector was produced containing a CD33 signal peptide either the GLP-1 or GLP-2 receptors (Christopoulos et al., and a FLAG-Tag (cloned in using HindIII and EcoRI). The 2003). Only in the case of the VPAC1 was there any charac- mature protein sequence for each RAMP was then cloned in terization of the effects of RAMP association. The complex using EcoRI and XhoI. The EcoRI site between the tag, and the with RAMP2 had normal expression and pharmacology for RAMP sequence was removed using site mutagenesis with a activation of AC, but the maximum stimulation of phosph- Quikchange kit (Stratagene, Leicester, UK). The untagged oinositide turnover in response to VIP was increased (Chris- constructs were in pcDNA3.1– and were either gifts from topoulos et al., 2003). It would seem unlikely that there is no AstraZeneca (receptors) or Dr Steve Foord, GSK-Wellcome consequence of RAMP association with these different recep- (RAMPs). The CRF1 receptor was isoform 1, which includes +/– tors. Indeed, recent studies of Ramp2 knockdown mice has residues 147–176. shown there was a wide range of phenotypic changes that go far beyond what would be expected for effects mediated by peptides acting through CLR and CTR/RAMP complexes Cell culture and transfection (Kadmiel et al., 2011). Cells were cultured in DMEM supplemented with 10% (v/v) In this study, the ability of three family B GPCRs, the type FBS and 5 % (v/v) penicillin/streptomycin in a humidified 1 corticotrophin releasing factor receptor (CRF1), the GLP-1 95% air/5% CO2 atmosphere. For transfection, the cells were receptor and the VPAC2 receptor to interact with all three plated onto either 12- and 48-well plates or 100 mm dishes. RAMPs have been examined in HEK 293S and CHO-K1 cells. Cells were transfected using a mixture (per 1 mg DNA) of 6 mL These receptors are of potential therapeutic interest. In evo- 10 mM polyethyleneimine and 45 mL 5% glucose solution lutionary terms, the CRF1 is the closest family B GPCR to the incubated for 30 min at room temperature and added to an CTR and CLR (Fredriksson et al., 2003), but its ability to appropriate final volume of full media. Twelve- and 48-well interact with RAMPs has not previously been investigated. plates were treated with 1 mg DNA per well, and 100 mm The VPAC2 and GLP-1 receptor are on the other two main dishes were treated with 10 mg DNA per dish. The ratio of branches that make up secretin-like/family B GPCR family. RAMP to receptor cDNA was 1:1 unless otherwise stated; The results show that the VPAC2 can interact with all three where receptor or RAMP alone was transfected, the balance to – RAMPs, and the CRF1 can interact with RAMP2. These inter- 1or10mg was made up with empty pcDNA3.1 vector. Char- actions differentially modulate G-protein coupling; in the acterization of expressed receptors was performed 48–72 h case of the CRF1, it is shown that this alters the pattern of after transfection. British Journal of Pharmacology (2013) 168 822–834 823 BJP D Wootten et al. Real-time quantitative PCR incubated overnight. Cells were washed with PBS, loaded Cells from confluent flasks were washed briefly with 1 mL with 100 mL per well of loading buffer (1X HBSS/20 mM cold PBS, detached with versene and pelleted by spinning at HEPES/2 mM CaCl2, pH 7.4) containing 5 mM probenecid 350¥ g for 5 min.
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