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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2014 Sep 5;171(19):4425–4439. doi: 10.1111/bph.12808

Molecular basis of agonist docking in a human GPR103 homology model by site-directed mutagenesis and structure–activity relationship studies

C Neveu 1,2,3,*, F Dulin 3,4,*, B Lefranc 2,3, L Galas 2,3, C Calbrix 2,3, R Bureau 3,4, S Rault 3,4, J Chuquet 1,2,3, J A Boutin 5, L Guilhaudis 3,6, I Ségalas-Milazzo 3,6, D Vaudry 1,2,3, H Vaudry 1,2,3, J Sopkova-de Oliveira Santos 3,4, J Leprince 1,2,3,✉
PMCID: PMC4209149  PMID: 24913445

Abstract

Background and Purpose

The neuropeptide 26RFa and its cognate receptor GPR103 are involved in the control of food intake and bone mineralization. Here, we have tested, experimentally, the predicted ligand-receptor interactions by site-directed mutagenesis of GPR103 and designed point-substituted 26RFa analogues.

Experimental Approach

Using the X-ray structure of the β2-adrenoceptor, a 3-D molecular model of GPR103 has been built. The bioactive C-terminal octapeptide 26RFa(19–26), KGGFSFRF-NH2, was docked in this GPR103 model and the ligand-receptor complex was submitted to energy minimization.

Key Results

In the most stable complex, the Phe-Arg-Phe-NH2 part was oriented inside the receptor cavity, whereas the N-terminal Lys residue remained outside. A strong intermolecular interaction was predicted between the Arg25 residue of 26RFa and the Gln125 residue located in the third transmembrane helix of GPR103. To confirm this interaction experimentally, we tested the ability of 26RFa and Arg-modified 26RFa analogues to activate the wild-type and the Q125A mutant receptors transiently expressed in CHO cells. 26RFa (10−6 M) enhanced [Ca2+]i in wild-type GPR103-transfected cells, but failed to increase [Ca2+]i in Q125A mutant receptor-expressing cells. Moreover, asymmetric dimethylation of the side chain of arginine led to a 26RFa analogue, [ADMA25]26RFa(20–26), that was unable to activate the wild-type GPR103, but antagonized 26RFa-evoked [Ca2+]i increase.

Conclusion and Implications

Altogether, these data provide strong evidence for a functional interaction between the Arg25 residue of 26RFa and the Gln125 residue of GPR103 upon ligand-receptor activation, which can be exploited for the rational design of potent GPR103 agonists and antagonists.

Table of Links

TARGETS LIGANDS
β2-adrenoceptor
GPR103, QRFP receptor Neuropeptide 26RFa, QRFP
Neuropeptide Y Y2 receptor
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This Table lists the protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and the Concise Guide to PHARMACOLOGY 2013/14 (Alexander et al., 2013).

Introduction

The neuropeptide 26RFa, which belongs to the RFamide superfamily, was originally isolated from the frog brain (Chartrel et al., 2003) and subsequently cloned in rats and humans (Chartrel et al., 2003; Fukusumi et al., 2003). Analysis of the human 26RFa precursor indicates that pre-pro26RFa may generate several additional peptides including an N-terminally extended form, 43RFa (now known as QRFP) and a truncated form, 26RFa(20–26) (GGFSFRF-NH2,) that is strongly conserved across vertebrate species (Chartrel et al., 2011; Leprince et al., 2013). In the human hypothalamus and spinal cord, processing of the precursor generates both 26RFa and 43RFa (Bruzzone et al., 2006), while in the rat and chicken brain, the mature form is 43RFa (Takayasu et al., 2006) and 26RFa (Ukena et al., 2010) respectively.

The search for a receptor target for 26RFa has led to the pairing with the former orphan receptor GPR103 (Fukusumi et al., 2003; Jiang et al., 2003). GPR103 is a class A (rhodopsin-like) 7-transmembrane (TM) domain GPCR that shares some sequence similarities with other RFamide receptors such as the neuropeptide FF-2 receptor, and with several neuropeptide receptors including the Y2 neuropeptide Y receptor, the GAL1 galanin receptor, the orexin and cholecystokinin receptors (Lee et al., 2001; Jiang et al., 2003). GPR103 activation with 26RFa or 43RFa induces an inhibition of cAMP formation (Fukusumi et al., 2003), and an increase in intracellular calcium concentration ( [Ca2+]i) in a Pertussis toxin (PTX)-independent manner, indicating that GPR103 is coupled to a Gi/0 and/or Gq protein (Fukusumi et al., 2003). While only one GPR103 gene is found in the human genome, two isoforms of the receptor have been cloned in rodents (Kampe et al., 2006; Takayasu et al., 2006). 26RFa binds mouse GPR103A and GPR103B with the same affinity (Takayasu et al., 2006) and stimulates inositol trisphosphate via both rat receptors with similar efficacy (Kampe et al., 2006).

In the CNS, 26RFa mRNA is expressed in discrete hypothalamic nuclei in rats (Chartrel et al., 2003; Fukusumi et al., 2003; Jiang et al., 2003), mice (Takayasu et al., 2006) and humans (Bruzzone et al., 2006). 26RFa binding sites are widely distributed in the rat CNS (Bruzzone et al., 2007) and GPR103 isoforms mRNAs are differentially expressed in the rat (Kampe et al., 2006) and the mouse brain (Takayasu et al., 2006). In humans, the highest concentrations of GPR103 are observed in the cerebral cortex, hypothalamus and vestibular nuclei (Jiang et al., 2003). In human peripheral organs, the GPR103 gene is expressed in the retina, pituitary, heart, kidney, testis and bone (Lee et al., 2001; Fukusumi et al., 2003; Baribault et al., 2006).

GPR103-mediated 26RFa/43RFa effects include modulations of energy homeostasis, nociceptive transmission, locomotion, BP, heart rate, gonadotropin and aldosterone secretion, and bone mineralization (see Chartrel et al., 2011; Leprince et al., 2013). The wide spectrum of biological functions of 26RFa has made GPR103 a candidate target for therapeutic approaches concerning, for instance, feeding disorders and osteoporosis.

Elucidation of the structures of GPCRs and characterization of the mechanisms controlling ligand/receptor binding are required for rational drug design. An ideal method to address these issues would be the determination of the 3-D structure of the ligand/receptor complex by X-ray diffraction methods. However, the inherent characteristics of GPCRs, such as their hydrophobic nature, their flexibility and the requirement for membrane-like environments to insure proper folding, make crystal preparation particularly difficult. Thus, computer-assisted molecular modelling is an alternative approach to acquire information on the architecture of GPCRs. Indeed, homology modelling methods allow to construct an atomic resolution model of the target protein from the solved 3-D structures of homologous proteins (Mobarec et al., 2009; Simms et al., 2009; Levit et al., 2012).

In order to study GPR103–26RFa interactions, we have built a 3-D model of human GPR103 (hGPR103), based on the structure of the human β2-adrenoceptor, that has subsequently been used to dock a biologically active human 26RFa (h26RFa) C-terminal fragment. A candidate residue in the binding pocket has been identified as interacting with a 26RFa amino acid. We have experimentally validated the hGPR103/h26RFa model using receptor site-directed mutagenesis and synthetic h26RFa analogues, and characterized this interaction as a GPR103 activation switch.

Methods

Molecular modelling

Receptor model

First, the sequence of hGPR103 was retrieved from the UniProt Knowledgebase (UniProtKB) (Jain et al., 2009) (accession number: Q96P65). Taking into account the relatively high sequence homology of hGPR103 with the β2-adrenoceptor, as well as the high resolution (2.4 Å) of the β2-adrenoceptor-T4 lysozyme chimera bound to the partial inverse agonist carazolol (accession number: 2RH1), we have built a first hGPR103 model on this template. hGPR103 and human β2-adrenoceptor sequences have been aligned using different algorithms in the @tome server (Labesse and Mornon, 1998). Then, the alignment between the two sequences has been manually optimized and evaluated with the TITO program (Labesse and Mornon, 1998). The disulfide bond (C118–C201) between the TM3 domain and extracellular loop (ECL) 2 of hGPR103 was conserved. This alignment was used as the basis for the homology modelling with the Modeller software (Eswar et al., 2008), an automatic comparative modelling program. The folding quality of the model was considered to be good according to the 3-D evaluation tools, that is Verify3D (Eisenberg et al., 1997) and Eval23D (Gracy et al., 1993). Sequence alignment figure was made with the ESPript software (Gouet et al., 2003).

Docking studies

Docking of 26RFa(19–26) into hGPR103 was carried out using the NMR structure of the C-terminal part (26RFa(19–26)) of h26RFa in methanol (Thuau et al., 2005) by means of the GOLD program with the default parameters (Jones et al., 1995; 1997). This program applies a genetic algorithm to explore conformational spaces and ligand-binding modes. To evaluate the proposed ligand poses, the GoldScore fitness function was applied. The binding site in the hGPR103 model was defined as a 15 Å sphere centred on a point (−33.671, 6.986, 8.164) located in the centre of the cavity. During this docking procedure, we paid special attention to some amino acids in the binding site, which were kept flexible such as the Gln125 residue.

Model refinement (energy minimization)

The selected hGPR103/26RFa(19–26) complexes were optimized using the CHARMM software (Brooks et al., 1983) with parameter set-22 (MacKerell et al., 1988). The van der Waals interactions were truncated using a switching function, and the electrostatic ones using a force switching function (between 8 and 13 Å). The vacuum dielectric constant was used during all calculations. The SHAKE constraint was used for the hydrogen atoms. As we would optimize only the side chain orientations, the complexes were solvated by TIP3P (Jorgensen et al., 1983) water molecules (water molecules clashing with the protein were removed), and surrounded by identical translated images of itself using periodic boundary conditions. To allow only the side chains optimization, the backbone atoms of TM helices were constrained. The two complex models were first minimized to an RMS energy gradient <10−4 kcal mol−1/Å and after a quick dynamic simulation using Verlet algorithm was performed. The system was heated to 300 K at 6 ps and then temperature-equilibrated during 50 ps.

Site-directed mutagenesis

Mutant receptor construct

The entire coding region of hGPR103, containing the Flag M2 epitope sequence (DYKDDDDK) at its 3′-end, was subcloned into the pcDNA3.1(+) mammalian expression vector (Invitrogen, Life Technologies, Saint-Aubin, France) as previously described (Le Marec et al., 2011). The pcDNA3.1(+)/hGPR103_M2 plasmid, inserted into a TOP-10 dam+ Escherichia coli strain for methylation of parental DNA, was isolated by a rapid plasmid extraction procedure using the QIAprep Spin Miniprep kit (Qiagen, Courtabœuf, France). Standard-purified mutagenesis primers were designed following the QuickChange Site-directed Mutagenesis kit guidelines (Stratagene, Agilent Technologies, Massy, France). The sequences of the primers were (mutagenic positions underlined): forward 5′-AAGATGGTGCCATTTGTCGCGTCTACCGCTGTTGTGAC-3′, reverse 5′-GTCACAACAGCGGTAGACGCGACAAATGGCACCATCTT-3′. Mutations were introduced into the hGPR103 sequence using the QuickChange II Site-directed Mutagenesis kit (Stratagene, Agilent Technologies) according to the manufacturer’s protocol. The mutagenesis reactions were carried out in 50 μL total volume using 50 ng of plasmid DNA, 2.5 U of PfuTurbo DNA polymerase and 125 ng of each primer. The cycling conditions were a 30 s initial denaturation at 95°C, 18 cycles with 30 s denaturation at 95°C, 1 min annealing at 55°C and 6.5 min extension at 68°C. The product was restricted with 10 U of DnpI and incubated for 1 h at 37°C to digest selectively the parental, methylated strands. Then, 1 μL of the reaction medium was used for transformation of XL1-Blue cells by thermal shock and cells were plated for selection of ampicillin resistant clones. Single transformant clones were grown overnight at 37°C in 5 mL LB-Broth completed with 80 μg mL−1 ampicillin. A rapid plasmid extraction was carried out from a 4 mL culture using the QIAprep Spin Miniprep kit (Qiagen).

Sequencing

Sequencing was carried out following the GenomeLab Dye Terminator cycle Sequencing Quick Start kit (Beckman Coulter, Villepinte, France) to assess whether the mutation had been incorporated. Forward primers were: 5′-TAATACGACTCACTATAGGG-3′; 5′-CCTGCATTGCTGTGGAAAGGCAC-3′; and reverse primers were: 5′-GCCTCGACTGTGCCTTCTA-3′; 5′-TGCCTTTGTGCTGGAGAGAAGGT-3′. The PCR reaction was performed in a 20 μL total volume using 450 ng (100 fmol) of dsDNA, 1.6 μM of primers and all other reagents of the kit. The cycling conditions were 30 cycles with 20 s at 96°C, 20 s at 50°C and 4 min at 60°C. The PCR product was precipitated with ethanol and templates were loaded into a capillary sequencer SEQ8000 (Beckman Coulter).

Cell culture and transfection

Non-transfected host CHO cells and stably transfected human GPR103 CHO (shGPR103-CHO) cells were obtained as previously described (Rodriguez et al., 2001; Le Marec et al., 2011). The cells were maintained in Ham-F12 medium supplemented with 10% (v/v) FBS (inactivated at 56°C for 30 min), 2 mM glutamine, 500 IU mL−1 penicillin and 100 μg mL−1 streptomycin. Expression of Gα16 was maintained using selection antibiotic hygromycin B (200 μg mL−1) and that of hGPR103 using geneticin G418 (500 μg mL−1) (Gibco, Life Technologies). Native pcDNA3.1(+)/hGPR103_M2, mutant pcDNA3.1(+)/Gln125Ala-hGPR103_M2 and empty pcDNA3.1(+) plasmids were transfected into CHO-Gα16 cells by nucleofection using the Amaxa cell line nucleofector T kit as described by the manufacturer (Lonza, Levallois-Perret, France). CHO cells were used at 80% confluency. One million cells per sample were resuspended at room temperature in 100 μL nucleofector solution, combined with 2 μg plasmid DNA. Cells were electroporated with use of the nucleofection programme U-023. Transfected cells were transferred into the culture plate containing culture medium.

RT-PCR

Expression of hGPR103 or Gln125Ala human GPR103 mutant (Q125AhGPR103) was verified by RT-PCR. Cells (5 × 105) were lysed and total RNA was extracted using the TRI Reagent kit (Sigma-Aldrich, Saint-Quentin Fallavier, France) (Chomczynski and Sacchi, 1987). mRNA (1.25 μg) was reverse-transcribed using the ImProm-II kit (Promega, Charbonnières-les-Bains, France). PCR reactions were performed in a volume of 25 μL containing 5 μL buffer, 0.5 μL dNTP, 1 μL forward and reverse primers, 5 μL cDNA and 0.5 μL GoTaq DNA polymerase. Primer sequences used for hGPR103 and Q125AhGPR103 amplification were: forward 5′-TTAACATTACCCCGGAGCAG-3′ and reverse 5′-TAATCGGTACCATGCCCACT-3′. The cycling conditions were: 1 cycle at 94°C during 5 min, followed by 35 cycles of 30 s at 94°C, 30 s at 52°C and 1.5 min at 72°C, and terminated by 1 cycle at 72°C during 5 min. The amplified cDNAs were then analysed after gel migration (agarose 1%, Tris-acetate-EDTA 0.5X, 2 μg/100 mL de BET- 100 V during 20 min).

Immunocytochemistry

Non-transfected host CHO, shGPR103-CHO, transiently transfected human GPR103 CHO (thGPR103-CHO) and tAhGPR103-CHO cells were incubated for 3 days into 24-well culture plates containing cover glasses (15 mm diameter), previously coated with poly-L-lysine (10 ng mL−1), at a density of 100 000 transiently transfected cells per well or of 30 000 stably transfected cells per well. Cells were incubated with medium alone or with cycloheximide (3 × 10−4 M) (Sigma-Aldrich) during 90 min and h26RFa (10−6 M) during 2 min, and fixed with 2% paraformaldehyde for 10 min. After rinsing in PBS, cells were permeabilized by 0.1% saponin during 20 min and incubated for 1 h with PBS containing 0.1% saponin, 1% BSA, 1/50 (v/v) normal goat serum, before adding a mouse anti-Flag M2 antibody (1/300) (Sigma-Aldrich) during 40 min. Cells were rinsed and incubated with an Alexafluor 488-conjugated goat anti-mouse IgG antibody (1/250) (Molecular Probes, Life Technologies) during 30 min. Nuclei were stained with DAPI. The cover glasses were fixed on the slides by Mowiol (Sigma-Aldrich) and the fluorescence was examined using an inverted confocal microscope (Leica TCS SP5, Nanterre, France).

Calcium mobilization assays

Changes in intracellular Ca2+ concentrations induced by h26RFa in non-transfected host CHO, transiently pcDNA3.1-transfected CHO (tpCHO), shGPR103-CHO, thGPR103-CHO and tAhGPR103-CHO cells were measured on a benchtop scanning fluorometer Flexstation III (Molecular Devices, Sunnyvale, CA, USA) as previously described (Le Marec et al., 2011). Briefly, 96-well assay black plates with clear bottom (Corning International, Avon, France) were seeded at a density of 100 000 transiently transfected cells per well 36 h prior to assay or 20 000 stably transfected cells per well 48 h prior to assay. Cells were loaded with 2 μM fluo-4AM (Invitrogen) during 1 h, rinsed three times and incubated 30 min with standard HBSS containing 2.5 mM probenecid and 5 mM HEPES. HBSS, h26RFa (at final concentrations ranging from 10−8 to 10−6 M) and ATP (10−4 M) were successively added 17, 60 and 180 s after the onset of acquisition and the fluorescence intensity was recorded over a 5 min period. A xenon lamp was used as an excitation source. The wavelengths of excitation (485 nm) and emission (525 nm) of fluo-4AM were selected by two monochromators included in the device equipped with a bottom reading probe.

Peptide synthesis

Reagents

All Fmoc-amino acid residues, O-benzotriazol-1-yl-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU) and 1-hydroxy-benzotriazole (HOBt) were purchased from PolyPeptide (Strasbourg, France) or Christof Senn Laboratories (Dielsdorf, Switzerland). The Rink amide 4-methylbenzhydrylamine resin was from VWR International (Fontenay-sous-Bois, France). N,N-diisopropylethylamine (DIEA), piperidine, trifluoroacetic acid (TFA) and triisopropylsilane (TIS) were supplied by Acros Organics (Geel, Belgium). N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM) and other reagents were from Sigma-Aldrich. Acetonitrile was from Fisher Scientific (Illkirch, France).

Synthesis

h26RFa, 26RFa(19–26) and 26RFa(20–26) analogues were synthesized (0.1 mmol scale) by the solid phase methodology on a Rink amide 4-methylbenzhydrylamine resin using a 433A Applied Biosystems peptide synthesizer (Applera-France, Courtaboeuf, France) and the standard Fmoc manufacturer’s procedure as previously described (Le Marec et al., 2011). All Fmoc-amino acids (1 mmol, 10 eq.) were coupled by in situ activation with HBTU/HOBt (1.25 mmol: 1.25 mmol, 12.5 eq.) and DIEA (2.5 mmol, 25 eq.) in NMP. Peptides were deprotected and cleaved from the resin by adding 10 mL of the mixture TFA/TIS/H2O (99.5:0.25:0.25, v/v/v) for 120 min at room temperature. After filtration, crude peptides were precipitated by addition of tertbutylmethylether (TBME), centrifuged (4500 rpm), washed twice with TBME, and freeze-dried. The synthetic peptides were purified by reversed-phase HPLC on a 2.2 × 25 cm Vydac 218TP1022 C18 column (Grace, Epernon, France) using a linear gradient (10–50% over 45 min) of CH3CN/TFA (99.9:0.1; v/v) at a flow rate of 10 mL min−1. Analytical HPLC, performed on a 0.46 × 25 cm Vydac 218TP54 C18 column (Grace), showed that the purity of all peptides was >99.9% (Table 2). The purified peptides were characterized by MALDI-TOF mass spectrometry on a Voyager DE PRO (Applera-France) in the reflector mode with α-cyano-4-hydroxycinnamic acid as a matrix.

Table 2.

Chemical data for Arg25-substituted 26RFa(20–26) analogues

Peptide Code HPLC MS
tR (min)a Purity (%) Calcdb Obsdc
h26RFa LV-2002 27.3 100 2830.45 2831.26
26RFa(20–26) LV-2021 19.3 100 815.41 816.41
[Lys25]26RFa(20–26) LV-2108 18.3 100 787.92 788.38
[Orn25]26RFa(20–26) LV-2109 18.2 100 773.89 774.48
[Cit25]26RFa(20–26) LV-2174 18.6 100 816.92 817.32
[ADMA25]26RFa(20–26) LV-2185 19.1 100 843.98 844.46
[SDMA25]26RFa(20–26) LV-2199 18.7 100 843.98 844.46
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aRetention time determined by RP-HPLC. bTheorical monoisotopic molecular weight. cm/z [MH+] value assessed by MALDI-TOF-MS. Cit, citrulline; Orn, ornithine.

Results

hGPR103 model

Among the solved GPCR structures that were available in the PDB database when we started this study, hGPR103 exhibited 20, 23, 22, 23, 26, 24, 23 and 22% sequence identity with bovine and squid rhodopsins, bovine opsin, avian (turkey) β1- and human β2-adrenoceptors, human A2A adenosine receptor, human CXCR4 and human dopamine D3 receptor respectively. Manual adjustments of important structural and functional features of class A GPCRs have been incorporated to the initial alignment such as the conserved disulfide bridge between the N-terminal extremity of the TM3 domain (Cys118) and the ECL2 (Cys201). Sequence alignment showed substantial homology within TM helices ranging from 45 to 75% for TM4 and TM7, respectively (Figure 1). Loops connecting the TM helices were modelled on the basis of the template loops except for intracellular loop (ICL) 3, which was replaced by lysozyme T4 in the β2-adrenoceptor to facilitate its crystallization. The modelled structure of hGPR103 remained very close to the X-ray structure of human β2-adrenoceptors. Indeed, root mean square deviation on Cα TM residues was 0.495 Å. All residues of the hGPR103 model were in favoured regions of the Ramachandran plot. Like the structure of the β2-adrenoceptor, the hGPR103 model exhibited two short non-TM helices, in ECL2 (extracellular helix, EH; Ile188 to Lys196; Figure 1) and in the intracellular C-terminal tail (intracellular helix, IH; Glu337 to Val347; Figure 1), IH being present in all rhodopsin-like GPCRs (Katragadda et al., 2004). The hGPR103 model encompassed a relatively narrow and deep binding pocket on the extracellular face of the protein for harbouring endogenous or synthetic ligands.

Figure 1.

Figure 1

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Amino acid sequence alignment of hGPR103 (hQRFPR) and human β2-adrenoceptor (h2RH1). TM (TM1–7) helices are delimited by horizontal dark blue arrows. ICLs (1–3) and ECLs (1–3) are indicated by horizontal purple and pink lines respectively. The EH in ECL2 and the IH in the C-terminal tail are delimited by horizontal light blue arrows. The disulfide bridge between the Cys118 and Cys201 residues of hGPR103 is also indicated. Fully conserved amino acids are highlighted with red boxes and highly conserved amino acids with white boxes.

Molecular docking of 26RFa(19–26) into the hGPR103 model

The C-terminal octapeptide 26RFa(19–26) was docked in the hGPR103 model. Among the proposed docking poses, only two appeared to be realistic, corresponding to the highest score fits from GoldScore fitness function. In both poses, the C-terminal Phe-Arg-Phe-NH2 part was oriented inside the binding cavity whereas the N-terminal Lys19 residue of the octapeptide remained outside (Figure 2A and B). The two hGPR103/26RFa(19–26) complexes were energy minimized, keeping the TM helix backbones fixed, to optimize the orientation of all side chains within the cavity. A complete description of the intermolecular interactions between 26RFa(19–26) and hGPR103 is listed in Table 1. In the two poses, 26RFa(19–26) interacted with all TM domains except the TM1 and TM2 helices (Figure 2A and B). The major differences between these two solutions occurred in the position of the N-terminal and C-terminal regions. Indeed, in the central region of 26RFa(19–26), Ser23 and Phe24 were rather well superimposed in both solutions (Figure 2C). In the N-terminal part, the Phe22 residue of each solution was superimposed with the Gly21 residue of the other solution, steering Lys19 and Gly20 in opposite directions. In the C-terminal part, the Arg25 and Phe26 residues were also oriented poles apart with a superimposition of Arg25 of one solution with the Phe26 of the other (Figure 2C). Among the two complexes, we have selected the second one, as it presented the highest interaction energy between the two partners ( |ΔE| ∼ 340.2 kcal mol−1 compared with |ΔE| ∼ 210.8 kcal mol−1 for the first solution, Table 1). In this selected solution, the strongest interactions, according to interaction energy per residue, were established between the guanidine function of the Arg25 residue of the peptide and the amide side chain of the Gln125 moiety situated on the top of the TM3 helix (|ΔE| ∼ 101.5 kcal mol−1), and between the ε-amine group of the Lys19 residue and the side chain of the Asp307 residue of ECL3 (|ΔE| ∼ 120.4 kcal mol−1) (Table 1; Figure 2D). Furthermore, in this solution, the Phe26 residue of 26RFa(19–26) interacted with a hydrophobic region of the binding pocket (Ile218, Leu222, Val129 and Val130) strengthening the stability of the complex (Table 1; Figure 2E). Indeed, the hydrophobic contribution in the interaction energy at this point was 18.8 and 20.5 kcal mol−1 in poses 1 and 2, respectively (Table 1).

Figure 2.

Figure 2

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26RFa(19–26) heptapeptide docked in the hGPR103 receptor model. The 26RFa(19–26) backbone and side chains are highlighted by sticks (solution one is coloured in orange and solution two in cyan), ECLs 1, 2 and 3 are represented by pink ribbons and TM domains by dark grey ribbons (A) or schematic bars (B). (C) Focus of the superimposition of the two solutions. The amino acids of 26RFa(19–26) are labelled. The middle part of the hexapeptides where amino acids of the two solutions are almost superimposed is dash-circled in dark blue (Ser23 and Phe24). (D,E) Detailed views of the interactions between 26RFa(19–26) in the position observed in solution two. Amino acids of hGPR103, which established H-bonds (green dash-line) with 26RFa(19–26) are highlighted by grey sticks (D) and hydrophobic amino acids are represented in green. The Glu297 residue of hGPR103 is displayed by pink sticks. The orientation of the complex is maintained in views A–C and slightly shifted for better viewing in panels D and E. Figures were drawn with PYMOL, version 1.1eval (DeLano Scientific, 2002, San Carlo, CA, USA).

Table 1.

List of 26RFa(19–26) and hGPR103 residues in close contact and associated energy

26RFa(19–26) hGPR103a,b |ΔΕ| (kcal mol−1)
Pose 1 Pose 2 Pose 1 Pose 2
Lys19 bb Ser207 sc Lys212 sc 21.7 120.4
Lys19 sc Asp307 sc
Gly20 5.9 7.1
Gly21 6.4 9.1
Phe22 bb Ser207 sc 26.1 28.2
Phe22 sc Ile296 sc Ile296 sc, Phe314 sc
Ser23 sc Glu204 sc, Gln318 sc Glu204 sc 29.8 34.5
Phe24 bb Gln318 sc 18.5 14.5
Phe24 sc Leu202 sc, Val121 sc, Phe322 sc, Pro122 sc
Arg25 bb Gln125 sc 70.9 101.5
Arg25 sc Val176 bb, Gly177 bb, Ser126 sc Gln125 sc, Glu132 sc
Phe26 bb His290 sc 31.5c 24.9d
Phe26 sc Phe289 sc, Phe322 sc, Val129 sc, Trp286 sc Ile218 sc, Leu222 sc, Val129 sc, Val130 sc
210.8 340.2
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aResidues with H-bond interacting atom are indicated in italic. bResidues with hydrophobic/aromatic interacting atoms are indicated in bold. cHydrophobic contribution counts for 18.8 kcal mol−1. dHydrophobic contribution counts for 20.5 kcal mol−1. bb, backbone; sc, side chain (sc).

Expression of the hGPR103 mutant

The proposed molecular model 2 (Figure 2D and E) was challenged by probing the molecular partner of the Arg25 residue of 26RFa(19–26). The Q125AhGPR103 has been engineered and tested for its activation by h26RFa. Expression of hGPR103 and its mutant in transiently transfected cells was verified by RT-PCR (Figure 3). shGPR103-CHO, thGPR103-CHO and transiently transfected Q125A-GPR103 CHO cells (tAhGPR103-CHO) were positive for a wild-type or mutant hGPR103 expression, whereas non-transfected host CHO and tpCHO cells exhibited neither wild-type nor mutant hGPR103 transcripts (Figure 3). Expression and targeting at the cell surface of hGPR103 or Q125AhGPR103 proteins have been verified by immunofluorescence microscopy using antibodies against the Flag M2 epitope, which was incorporated at the C-terminal extremity of wild-type and mutant receptors (Figure 4). Confocal microscope analysis of shGPR103-CHO, thGPR103-CHO and tAhGPR103-CHO cells in resting conditions showed that all three receptors were primarily localized in the cytoplasm in the absence (Figure 4 A1, A2 and A3) as in the presence of cycloheximide (Supporting Information Figure S1). After treatment with cycloheximide and h26RFa, as previously described (Iturrioz et al., 2010), only the cell lines stably and transiently transfected with the wild-type receptor, that is shGPR103-CHO and thGPR103-CHO cells, respectively, displayed intense immunofluorescence staining at the plasma membrane (Figure 4B1, B2, C1 and C2). In contrast, in tAhGPR103-CHO cells, the fluorescence remained localized in the cytoplasm (Figure 4B3 and C3). Finally, non-transfected host CHO cells were not stained, and non-permeabilized shGPR103-CHO cells did not exhibit any immunofluorescence (data not shown).

Figure 3.

Figure 3

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Expression analysis of hGPR103 and Q125AhGPR103 mRNA. RT-PCR was performed on pcDNA3.1/hGPR103 plasmid, thGPR103-CHO cells, tAhGPR103-CHO cells, tpCHO cells, non-transfected host CHO cells and shGPR103-CHO cells. Total RNAs were retrotranscribed and used for PCR amplification of hGPR103 and Q125AhGPR103 cDNA fragments with the same specific primer. As expected, the amplified products, analysed on 1% agarose gel corresponded to 1294 bp. PCR amplification products of shGPR103-CHO and pcDNA3.1/hGPR103 plasmid were used as positive controls. M, markers.

Figure 4.

Figure 4

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Confocal images showing localization of the Flag M2-tagged hGPR103 and Q125AhGPR103 in stably and transiently transfected CHO cells in the absence or presence of cycloheximide and h26RFa. Immunocytochemistry was performed with a primary antibody against the Flag M2 and a secondary antibody labelled with green-fluorescence Alexafluor 488. Nuclei were stained with DAPI (blue). (A) In the absence of cycloheximide and h26RFa, the Flag M2-tagged receptors were detected in shGPR103-CHO cells (A1), in thGPR103-CHO cells (A2) and in tAhGPR103-CHO cells (A3). Scale bars = 10 μm. (B) shGPR103-CHO cells (B1), thGPR103-CHO cells (B2) or tAhGPR103-CHO cells (B3) were incubated with cycloheximide (3 × 10−4 M) for 90 min and h26RFa (10−6 M) for 2 min. Scale bars = 10 μm. (C) Higher magnification showing the occurrence of the wild-type receptor (C1 and C2) and the absence of the mutant receptor (C3) at the cell surface. Scale bars = 5 μm.

Effect of h26RFa on mutant hGPR103

In order to confirm the importance of the Gln125 residue, located inside the binding cavity, in hGPR103 recognition and/or activation processes, the ability of h26RFa to activate the mutant receptor Q125AhGPR103 was determined by assessing the Ca2+ response of transfected cells (Figure 5). As a negative control, it was verified that administration of culture medium alone did not cause an increase in [Ca2+]i in any of the cell types studied that is host CHO, tpCHO, shGPR103-CHO, thGPR103-CHO and tAhGPR103-CHO cells (Figure 5, arrow 1). As a positive control, ATP (10−4 M) was shown to induce a 200–300% [Ca2+]i rise in all cell types (Figure 5, arrow 3). h26RFa (10−6 M) had no effect on calcium mobilization in non-transfected host CHO and tpCHO cells (Figure 5A, arrow 2), whereas it enhanced [Ca2+]i by 200 and 150% in shGPR103-CHO and thGPR103-CHO cells, respectively (Figure 5B and C, arrow 2). In contrast, h26RFa (10−6 M) failed to increase [Ca2+]i in tAhGPR103-CHO cells (Figure 5D, arrow 2).

Figure 5.

Figure 5

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Effect of h26RFa on [Ca2+]i in wild-type or mutant hGPR103 transfected CHO cells. Representative curves showing variations of [Ca2+]i induced by administration of buffer (negative control) at 17 s (arrow 1), h26RFa (10−6 M) at 60 s (arrow 2) and ATP (10−4 M) (positive control) at 180 s (arrow 3) in non-transfected host CHO cells (A, grey line), in tpCHO cells (A, black line), in shGPR103-CHO cells (B), in thGPR103-CHO cells (C) and in tAhGPR103-CHO cells (D). Data are mean ± SEM of at least three independent experiments performed in triplicate.

Effect of Arg-modified h26RFa analogues on wild-type hGPR103

To further investigate the contribution of the side chain of the Arg moiety of h26RFa in the activation process of hGPR103, we have synthesized five analogues of the C-terminal heptapeptide substituted at position 25 and tested their effect on [Ca2+]i in shGPR103-CHO cells (Table 2). Replacement of the Arg25 residue by a lysine, an ornithine or a citrulline moiety led to analogues (LV-2108, LV-2109 and LV-2174, respectively) that were totally devoid of agonistic and antagonistic activity in the calcium mobilization assay (Table 3). Similarly, substitution of the Arg25 residue by a symmetrical dimethyl arginine generated the analogue [SDMA25]26RFa(20–26) (LV-2199) that did not exhibit any agonistic or antagonistic activity, whereas introduction of an asymmetrical dimethyl arginine led to [ADMA25] 26RFa(20–26) (LV-2185) that reduced by 67.5% h26RFa-evoked [Ca2+]i increase with an IC50 of 5.1 ± 1.1 μM (Table 3, Figure 6).

Table 3.

Effect of Arg25-substituted 26RFa(20–26) analogues on basal [Ca2+]i and h26RFa-induced [Ca2+]i increase in shGPR103-CHO cells

Compounds Amino acid formula of residue 25 Agonist Antagonist
EC50 (nM)a pEC50 IC50 (nM)a pIC50 Max effect (%)b
h26RFa, LV-2002 graphic file with name bph0171-4425-g1.jpg 10.4 ± 1.5 8.10 ± 0.04 nd
26RFa(20–26), LV-2021 739 ± 149*** 6.40 ± 0.09 nd
[Lys25]26RFa(20–26) graphic file with name bph0171-4425-g2.jpg >105 >105
LV-2108
[Orn25]26RFa(20–26) graphic file with name bph0171-4425-g3.jpg >105 >105
LV-2109
[Cit25]26RFa(20–26) graphic file with name bph0171-4425-g4.jpg >105 >105
LV-2174
[SDMA25]26RFa(20–26) graphic file with name bph0171-4425-g5.jpg >105 >105
LV-2199
[ADMA25]26RFa(20–26) graphic file with name bph0171-4425-g6.jpg >105 5140 ± 1100 5.30 ± 0.11 67.5
LV-2185
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aData are the mean ± SEM of at least three distinct experiments performed in triplicate. bThe maximal effect, at a concentration of 10−4.5 M, is expressed as a percentage of the mean calcium response inhibition induced by 10−6 M h26RFa. ***P < 0.001 versus control as assessed by Mann and Whitney test. Cit, citrulline; nd, not determined; Orn, ornithine.

Figure 6.

Figure 6

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Effect of graded concentration of [ADMA25]26RFa(20–26) (LV-2185) on the h26RFa-evoked intracellular calcium increase in shGPR103-CHO cells. Data are mean ± SEM of at least three independent determinations. The values are expressed as percentages of the response induced by 10−6 M 26RFa. The pIC50 value calculated from the data was 5.30 ± 0.11 for [ADMA25]26RFa(20–26) (LV-2185).

Discussion and conclusion

There is now compelling evidence that the neuropeptide 26RFa (QRFP) and its cognate receptor GPR103 are implicated in the regulation of several physiological functions including energy homeostasis and bone mineralization (see Chartrel et al., 2011; Leprince et al., 2013). To further characterize this peptidergic system, identification of receptor residues that are involved in ligand binding and receptor activation is a fundamental step for understanding the principles of ligand-receptor recognition.

Taking advantage of the high-resolution structure of the human β2-adrenoceptor that shares 26% sequence identity with hGPR103, we have developed a homology model for the h26RFa receptor that was subsequently used for ligand docking studies. These comparative models of GPCRs in combination with side-directed mutagenesis are commonly used to provide a molecular template for both ligand binding and functional studies (Chakraborty et al., 2012; Heifetz et al., 2012). In very much the same way as the human β2-adrenoceptor, our GPR103 model exhibited a relatively narrow and deep binding pocket for accommodating its cognate ligand. Among the solved GPCR structures available, the main structural differences are located in the ECLs. Notably, ECL2, which links the TM4 and TM5 domains, adopts different conformations. In contrast to the buried β-sheet characterized in rhodopsin, the ECL2 of the hGPR103 model was strongly exposed to the solvent and contained an extra helical segment (from Ile188 to Lys196) named EH that is also present in the β2-adrenoceptor (Figure 1 and 2A). While the length, sequence and secondary structure of ECL2 vary, a conserved disulfide bridge connecting ECL2 with the top of TM3 is present in all solved GPCR structures. Numerous studies have shown that this disulfide bond is critical not only for GPCR folding and surface localization, but also for ligand binding (de Graaf et al., 2008). Therefore, sequence alignment of ECL2 in our model was carried out in order to maintain the disulfide bond between Cys118 (TM3 domain) and Cys201 (ECL2). The hGPR103 model exhibited a second non-TM helix in the intracellular extremity between residues Glu337 and Val347, named IH. In other GPCRs, it has been shown that IH is involved in receptor signalling. In particular, the IH of rhodopsin, adrenoceptors and angiotensin II receptors is implicated in G-protein coupling and activation (Munch et al., 1991; Sano et al., 1997; Ernst et al., 2000; Lu et al., 2002; Katragadda et al., 2004). IH is also important for phosphorylation of the C-terminal tail of class A GPCRs by GPCR kinases as well as for arrestin recruitment (Gehret et al., 2010; Kirchberg et al., 2011).

We have previously shown that the α-helix of h26RFa (Pro4-Arg17) cannot activate hGPR103, while the C-terminal region of the peptide is essential for biological activity (Le Marec et al., 2011). Preliminary results showed that the α-helix of h26RFa, characterized in methanol (Thuau et al., 2005), extends over two additional residues, in its C-terminal side, up to the Lys19 residue in diphosphocholine micelles (A. Marotte, unpubl. data). To get insight as to how the peptide helix stands in hGPR103, we have used the (19–26) fragment of h26RFa (26RFa(19–26)) for docking simulations into the homology model of hGPR103. These docking experiments revealed that 26RFa(19–26) bound in two different orientations in the ligand-binding pocket within the TM bundle. Both of these binding modes displayed excellent structural complementarities between the ligand and the receptor with numerous intermolecular interactions. According to the docking results, the C-terminal part of 26RFa(19–26) sank in depth into the cavity while the Lys19 residue remained at the receptor surface. In the first mode, the side chain of the Phe26 residue of 26RFa(19–26) faced the Gln125 moiety of hGPR103 while the Arg25 residue established an interaction with the Ser126 moiety through its Nε atom. In the second mode, the Phe26 residue faced a hydrophobic area (Ile218, Leu222, Val129 and Val130) and the Arg25 residue strongly interacted with the Gln125 side chain via its two Nζ atoms. The existence of multiple active conformations has been previously described for other GPCR systems, where agonists, that favour one signalling pathway over another, have been characterized (Galandrin et al., 2007; Rajagopal et al., 2010). We therefore hypothesize that hGPR103 exists in different active conformations depending on h26RFa fitting, leading to the activation of different signalling pathways. In support of this hypothesis, it has been reported that 26RFa/43RFa stimulated [Ca2+]i and inhibited cAMP formation via Gi/0 and/or Gq protein (Fukusumi et al., 2003; Jiang et al., 2003). Moreover, we have recently shown that h26RFa displaces 125I-h26RFa binding with an IC50 of 1.11 nM (Neveu et al., 2012). In the light of the two docking solutions, the data were refitted using an equation for displacement of radioligand by competitors from two binding sites. Interestingly, the two-site curve-fitting model fitted the data significantly better than the one-site model, as determined by an F-test at a significance level of P < 0.05 (P = 0.015), suggesting the existence of a heterogeneous population of binding sites. Taken together, these findings suggest that h26RFa may act as a biased agonist of hGPR103, that is an agonist that stabilizes distinct receptor conformations that differ in their signalling partner preference, leading to different biological responses as recently shown for the βbeta2-adrenoceptor (Liu et al., 2012), the cholecystokinin CCK-2 receptor (Magnan et al., 2011) and the somatostatin sst-2 receptor (Cescato et al., 2010). However, it should be noted that the difference of affinity between the two h26RFa binding sites (IC50 −1 = 0.3 ± 0.2 nM; IC50 −2 = 7.9 ± 0.3 nM) is relatively low. Thus, the existence of high- and low-affinity binding sites for h26RFa deserves further investigation.

As mentioned earlier, the modelling of ECL2 was more complex, because of its length and its low-sequence homology with its counterpart in the human β2-adrenoceptor. Consistent with the selected 26RFa(19–26) position in the hGPR103 active site, if the whole h26RFa sequence is superimposed on the 26RFa(19–26) docked part, the N-terminal amphipathic helix will be situated outside the binding cavity of the receptor, close to ECL2. Taking into account the amphipathy of both the h26RFa and ECL2 α-helices, it is possible that interactions occur between these helices at the surface of hGPR103 (Figure 7).

Figure 7.

Figure 7

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Top view of the proposed position of the amphipathic helix of h26RFa on the extracellular surface of hGPR103. The EH and the N-terminal helix of h26RFa are represented by ribbons. Hydrophobic residues are coloured in grey and charged residues in orange and red for hGPR103 and h26RFa helices respectively. The figure was drawn with PYMOL, version 1.1eval (DeLano Scientific, 2002).

In the selected solution, the strongest interaction between 26RFa(19–26) and the receptor inside the binding pocket involved the Gln125 residue located close to the N-terminal region of the TM3 helix. Interestingly, sequence alignment of human RFamide peptide receptors shows that this Gln residue is fully conserved in all receptors of this superfamily, indicating that all these receptors may present common recognition or activation processes (Figure 8). As the Gln125 residue was paired with the Arg25 moiety, and as the latter is part of the RFamide motif that is common to all ligands for these receptors, we assumed that the Arg/Gln interaction plays a crucial role in receptor binding and/or activation. To test this hypothesis, we constructed the Q125A mutant of hGPR103 and evaluated the effect of h26RFa on its activation. The mutation and the addition of the C-terminal M2 flag did not affect the expression of the wild-type and mutant hGPR103 transcripts, but may modify the trafficking of the recombinant protein as shown by the subcellular distribution of wild-type and mutant tagged hGPR103 in transiently transfected CHO cells through confocal microscopy analysis. Of note, the C-terminal M2 flag did not affect the occurrence of hGPR103 at the plasma membrane (Chartrel et al., 2011). The recombinant wild-type and Q125A-substituted hGPR103 were similarly located at the plasma membrane although, in the absence of a ligand, both receptors were primarily trapped in the intracellular network, most probably in the endoplasmic reticulum. Similar observations have been recently reported for apelin receptor mutants transfected in the same host cells (Iturrioz et al., 2010). However, in the presence of cycloheximide (which by itself had no effect), h26RFa provoked an increase in fluorescence at the cellular membrane of wild-type hGPR103-transfected cells. These results suggest that activation of hGPR103 by h26RFa, initially present in small amounts at the cell surface, induces a signalling cascade leading to receptor recruitment from cytosolic stores to the plasma membrane. This agonist-induced regulation of the density of GPCRs at the cell surface has already been reported for many receptors such as the dopamine D1 receptor, opiate receptors and thrombin receptor (Brismar et al., 1998; Holtbäck et al., 1999; Cahill et al., 2007; Achour et al., 2008). Mutation of the Gln125 residue by an alanine in the TM3 domain of hGPR103 totally suppressed h26RFa-evoked recruitment of the receptor at the plasma membrane, confirming that Gln125 is a key amino acid for ligand recognition and/or receptor activation. It has been recently proposed that the positively charged arginine side chain of the C-terminal motif of RFamide peptides interacts with a Glu residue of their cognate receptor located at the extremity of TM6 domain close to EL3 (Findeisen et al., 2011a). As this amino acid is well conserved in RFamide peptide receptors, and as it is involved in NPY-receptor binding (Merten et al., 2007), it has been mutated in all RFamide peptide receptors (Findeisen et al., 2011a,b). Site-directed mutation of Glu297 of GPR103 with an alanine partly reduces the 26RFa effect (Findeisen et al., 2011a). The hGPR103 model and docking study described herein strongly suggest that the side chain of the Lys19 moiety of h26RFa rather than the Arg25 residue interacts with this glutamic acid located in the ECL3 of the receptor template (Figure 2E). In support of this hypothesis, the Cα of the Lys19 residue of 26RFa(19–26) was 8 Å apart from that of the Glu297 moiety of hGPR103, whereas the distance between the Cα of the Arg25 residue and the Cα of the Glu297 moiety was 14 Å. As the Lys19 and Glu297 side chains were oriented in opposite directions in the hGPR103 model, the amine and carboxylic groups were at a considerable distance from each other. However, the N-terminal part of h26RFa, that was missing in our simulations, may introduce a rearrangement of these side chains, leading to a favourable interacting distance between their chemical functions.

Figure 8.

Figure 8

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Sequence alignment of the different members of the human RFamide peptide receptor superfamily between residue 79 and residue 148 of hGPR103. hGPR103, h26RFa receptor; hGPR54, metastin/kisspeptin receptor; hGPR10, prolacting-releasing peptide receptor; hNPFF-1, neuropeptide FF/RFRP1 receptor; hNPFF-2, RFRP-3 receptor. The conserved Gln125 residue is highlighted by a star. Fully conserved amino acids are highlighted with red boxes and highly conserved amino acids with white boxes.

In order to determine if the main Arg25/Gln125 interaction is implicated in hGPR103 activation, we designed five Arg-modified 26RFa(20–26) analogues. Replacement of the Arg25 residue by a mono-functional and/or a short side chain moiety was deleterious for 26RFa(20–26) effect on wild-type hGPR103, suggesting that the peptide needs at least two anchoring points for efficient interactions with the receptor. More importantly, from the observation that [ADMA25]26RFa(20–26) (LV-2185) exhibited antagonistic activity, we can assume that the Arg25/Gln125 interaction is primarily involved in receptor activation. In addition, these data indicate that chemical modifications of the Arg25 moiety of h26RFa may yield selective and potent hGPR103 antagonists.

In conclusion, the present docking and site-directed mutagenesis studies reveal that the Gln125 residue in TM3 domain of hGPR103 is involved in the interaction of the receptor with the Arg25 residue of the C-terminal motif of h26RFa. This crucial Arg25/Gln125 interaction between h26RFa and its receptor can be exploited for the rational design of selective GPR103 agonists and antagonists.

Acknowledgments

This study was supported by INSERM (U982), the European Regional Development Fund (ERDF – PeReNE) and the Région Haute-Normandie. The authors thank the CRIHAN (Centre de Ressources Informatiques de Haute-Normandie) for the molecular modelling software. C. N. was the recipient of a fellowship from the Lille-Amiens-Caen-Rouen-Neuroscience Network.

Glossary

[Ca2+]i

intracellular calcium concentration

ADMA

asymmetrical dimethyl arginine

DCM

dichloromethane

DIEA

N,N-diisopropylethylamine

ECL

extracellular loop

EH

extracellular helix

HBTU

O-benzotriazol-1-yl-N,N,N’,N’-tetramethyluronium hexafluorophosphate

HOBt

1-hydroxy-benzotriazole

ICL

intracellular loop

IH

intracellular helix

NMP

N-methyl-2-pyrrolidone

PTX

Pertussis toxin

Q125AhGPR103

Gln125Ala human GPR103 mutant

SDMA

symmetrical dimethyl arginine

shGPR103-CHO

stably transfected human GPR103 CHO

tAhGPR103-CHO

transiently transfected Q125A human GPR103 CHO

TBME

tertbutylmethylether

TFA

trifluoroacetic acid

thGPR103-CHO

transiently transfected human GPR103 CHO

TIS

triisopropylsilane

TM

transmembrane

tpCHO

transiently pcDNA3.1-transfected CHO

Author contributions

J. L. and J. S-d. O. S conceived and designed the study. Data were acquired by C. N., F. D., B. L., L. Ga., C. C. and L. Gu. Data were analysed and interpreted by C. N., F. D., L. Ga., J. C. and I. S.-M. The paper was drafted by C. N., F. D., J. S.-d. O. S. and J. L. Critical revisions were contributed by R. B., S. R., J. A. B., D. V., H. V. and J. L.

Conflict of interest

The authors disclose no conflict of interest.

Supporting Information

Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: http://dx.doi.org/10.1111/bph.12808

Figure S1 Confocal images showing localization of the Flag M2-tagged hGPR103 and Q125AhGPR103 in stably and transiently transfected CHO cells in the presence of cycloheximide. Immunocytochemical labelling was performed with a primary antibody against Flag M2 and a secondary antibody labelled with green-fluorescence Alexafluor 488. Nuclei were stained with DAPI (blue). The Flag M2-tagged receptors were detected in shGPR103-CHO cells (A1), in thGPR103-CHO cells (A2) and in tAhGPR103-CHO cells (A3). Scale bars = 10 μm.

bph0171-4425-sd1.tif (1.1MB, tif)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 Confocal images showing localization of the Flag M2-tagged hGPR103 and Q125AhGPR103 in stably and transiently transfected CHO cells in the presence of cycloheximide. Immunocytochemical labelling was performed with a primary antibody against Flag M2 and a secondary antibody labelled with green-fluorescence Alexafluor 488. Nuclei were stained with DAPI (blue). The Flag M2-tagged receptors were detected in shGPR103-CHO cells (A1), in thGPR103-CHO cells (A2) and in tAhGPR103-CHO cells (A3). Scale bars = 10 μm.

bph0171-4425-sd1.tif (1.1MB, tif)

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