Nuclear localization of Formyl-Peptide Receptor 2 in human cancer cells

Accepted Manuscript Nuclear localization of formyl-peptide receptor 2 in human cancer cells Fabio Cattaneo, Melania Parisi, Tiziana Fioretti, Daniela Sarnataro, Gabriella Esposito, Rosario Ammendola PII:

S0003-9861(16)30153-9

DOI:

10.1016/j.abb.2016.05.006

Reference:

YABBI 7280

To appear in:

Archives of Biochemistry and Biophysics

Received Date: 7 April 2016 Revised Date:

6 May 2016

Accepted Date: 6 May 2016

Please cite this article as: F. Cattaneo, M. Parisi, T. Fioretti, D. Sarnataro, G. Esposito, R. Ammendola, Nuclear localization of formyl-peptide receptor 2 in human cancer cells, Archives of Biochemistry and Biophysics (2016), doi: 10.1016/j.abb.2016.05.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Nuclear Localization of Formyl-peptide Receptor 2 in human cancer cells

Fabio Cattaneo*1, Melania Parisi*1, Tiziana Fioretti1, Daniela Sarnataro1,2, Gabriella Esposito1,2,

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Rosario Ammendola§1

Department of Molecular Medicine and Medical Biotechnology, School of Medicine, University of

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Naples Federico II, Via S. Pansini 5, Naples 80131, Italy

CEINGE-Biotecnologie Avanzate s.c.a.r.l., Via G. Salvatore 486, Naples 80145, Italy

*

§

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These authors contributed equally to this work.

Corresponding author: Department of Molecular Medicine and Medical Biotechnology, School of

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Medicine, University of Naples Federico II, Via S. Pansini 5, Naples 80131, Italy. Tel. +39 081

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7463145; Fax: +39 081 7464359; e-mail: [email protected]

ACCEPTED MANUSCRIPT Abstract

Current models of G protein-coupled receptors (GPCRs) signaling describe binding of external agonists to cell surface receptors which, in turn, trigger several biological responses.

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paradigms indicate that GPCRs localize to and signal at the nucleus, thus regulating distinct

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signaling cascades. The formyl-peptide receptor FPR2 belongs to the GPCR super-family and is coupled to PTX-sensitive Gi proteins. We show by western blot analysis, immunofluorescence experiments and radioligand binding assays that FPR2 is expressed at nuclear level in CaLu-6 and AGS cells. Nuclear FPR2 is a functional receptor, since it participates in intra-nuclear signaling, as

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assessed by decreased G protein-FPR2 association and enhanced ERK2, c-Jun and c-Myc phosphorylation upon stimulation of intact nuclei with the FPR2 agonist, WKYMVm. We analyzed FPR2 sequence for the search of a nuclear localization sequence (NLS) and we found a stretch of

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basic aminoacids (227-KIHKK-231) in the third cytoplasmic loop of the receptor. We performed single (K230A) and multiple (H229A/K230A/K231A) mutagenesis of NLS. The constructs were individually overexpressed in HEK293 cells and immunofluorescence and western blot analysis showed that nuclear localization or translocation of FPR2 depends on the integrity of the H229 and

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K231 residues within the NLS.

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Keywords: FPR2; G protein-coupled receptors; Nuclear localization; Signal transduction.

ACCEPTED MANUSCRIPT Introduction

The G-protein-coupled receptors (GPCRs) comprise a large family of transmembrane proteins activated by a broad range of ligands and implicated in many patho-physiological processes [1]. In

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conventional GPCR signaling, receptors are localized to the plasma membrane and their stimulation with a specific agonist triggers the activation of G proteins [2] and, in turn, the activity of second messengers, ion channels or membrane-associated enzymes. However, a number of GPCRs, such as β-adrenergic, lysophosphatidic acid, gonadotropin releasing hormone type I, metabotropic

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glutamate, endothelin, platelet-activating factor, prostaglandin, angiotensin 2 type I, C-X-C 4 receptors, also localize to and signal at the nuclear membrane or within the nucleus [3-10] in several cell types [11-14]. Nuclear GPCRs represent distinctive signaling units that respond to specific

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intracellular agonists, by transducing nuclear transcription signals. They may be constitutively active or activated by endogenous produced non-secreted ligands, thus regulating a number of physiological processes, such as inflammatory responses, cell proliferation, DNA synthesis and transcription [8, 10, 15-17]. A GPCR may translocate from the plasma to nuclear membrane, constituting complexes that transduce distinctive signals in relation to the intracellular levels of

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their cognate agonists [13].

A full complement of downstream signal transduction components is present on the nuclear membranes and in the nucleoplasm including G proteins, enzyme effectors, ion channels, pumps and exchangers [14, 17-21]. Furthermore, several second messenger signaling cascades, such as

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ERKs, p38MAPK, PKB, PKA and PKC are activated upon binding of cognate agonists to nuclear GPCRs [4, 15, 22-25]. Gα and Gβγ subunits are key regulators of cellular signaling events and are

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associated, or co-purificate, or co-immunoprecipitate with nuclear GPCRs [26]. Gβγ have also a role in the assembly and trafficking of receptor-based complexes in endoplasmic reticulum and Golgi apparatus [27], as well as in the regulation of nuclear histone deacetylase 5 [28] and transcriptional repressor AEBP1 [29]. Targeting of proteins to the nucleus requires a nuclear localization signal (NLS) embedded in the protein, which consist of mono- or bi-partite basic aminoacids residues, usually lysines and arginines, or glycine-arginine repeats [30]. Nuclear translocation is generally regulated by importins, which bind to the NLS motif, and by beta-arrestin, which plays a key role in receptor internalization. A NLS motif has been identified in the eighth helix of adenosine, growth hormone, motilin, purine, angiotensin, bradikinin and endothelin receptors or in the third intracellular loop of apelin receptor [31], even though several heterogenous

ACCEPTED MANUSCRIPT sequences that do not resemble to classical basic NLS can promote nuclear import of several proteins [32]. The human Formyl-peptide Receptors 1, 2 and 3 (FPR1, FPR2 and FPR3) belong to GPCR family. They are all coupled to the Gi family of G proteins, as indicated by the total loss of cell response to their agonists upon exposure to pertussis toxin (PTX), and their activation by specific ligands

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triggers distinct signaling cascades in several cell types [33-35]. Many FPR2 agonists are peptides, with the exception of lipoxin A4 and of the synthetic small-molecular weight ligands isolated by compound library screens. Several FPR2 peptide ligands have been identified and purified from living organisms and a number of these peptides have been synthesized on the basis of the sequence

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of known proteins, but their physiological function and their presence in vivo has to be proven. FPR2 transduces the anti-inflammatory effects of lipoxin A4, but it can also mediate proinflammatory responses to serum amyloid A. WKYMVm, a modified peptide isolated by screening

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synthetic peptide libraries, binds FPR2 with high efficiency [36] and triggers several intracellular signaling pathways [33, 37]. In human fibroblasts, WKYMVm induces ERKs activation, p47phox translocation and NADPH-dependent superoxide generation [38], which requires the hexapeptidedependent activation of PKCα and PKCδ [39]. In human lung cancer CaLu-6 cells, stimulation with WKYMVm induces EGFR tyrosine phosphorylation, which provide docking sites for recruitment

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and triggering of the STAT3 pathway [40]. Similarly, in PNT1A cell line, WKYMVm induces HGF receptor transactivation and triggers some of the molecular responses elicited by c-Met/HGF binding [41]. These results indicate that, despite FPR2 lacks intrinsic tyrosine kinase activity, tyrosine phosphorylation of tyrosine kinase receptors occurs in response to the binding of an FPR2

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agonist and activates intracellular mitogenic cascades [42]. Herein, we show that FPR2 is expressed in nuclei isolated from human lung carcinoma CaLu-6 and

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human gastric adenocarcinoma AGS cell lines. We identified a NLS in the third intracellular loop of the receptor, whose identity was confirmed by site-directed mutagenesis, and we demonstrated that nuclear FPR2 is a functional receptor since its stimulation with WKYMVm triggers Gi activation, and ERK2, c-Jun and c-Myc phosphorylation.

Materials and Methods

Reagents and cell culture

The WKYMVm and WRWWWW (WRW4) peptides were synthesized and HPLC-purified by PRIMM (Milan, Italy). SDS–PAGE reagents were from Bio-Rad (Hercules, CA, USA). Protein

ACCEPTED MANUSCRIPT A/G Plus agarose, anti-ERKs, anti-active phosphorylated ERK1/2, anti-tubulin, anti-FPR2, antiphospho-c-Myc, anti-c-Myc, anti-YY1, anti-LAP2, anti-CD44, anti-rabbit, anti-goat and anti-mouse antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-Jun and anti-RAB7 were from Cell Signaling Technology (Danvers, MA, USA). CaLu-6 and AGS cell lines were grown in Dulbecco's modified Eagle's medium (DMEM)

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containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, 1% lglutamine, and 1% modified Eagle's medium. Cells were cultured until they reached 70% confluence and nuclei were isolated.

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Nuclei isolation

Nuclei were isolated as described [31]. Briefly, washed and pelleted cells were resuspended in lysis

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buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 5 µg/ml leupeptin, 10 µg/ml benzamidine, and 5 µg/ml soybean trypsin inhibitor), homogenized by several short bursts of lowforce Polytron disruption on ice, and then centrifuged at 700 × g for 10 min at 4 °C. The nuclear pellet was resuspended in lysis buffer, layered over a 4.5 ml discontinuous sucrose gradient of 2.0 M and 1.6 M sucrose containing 1 mM MgCl2, and centrifuged at 100,000 × g for 1 h at 4 °C.

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Protein levels were determined by the Bradford assay according to the manufacturer's instructions (Bio-Rad, Hercules, CA, USA).

Purified nuclei were stimulated with WKYMVm peptide at the final concentration of 10 µM for various times, as indicated in the figures. In other experiments, nuclei were preincubated with 10

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Mutagenesis

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µM WRWWWW for 15 min before stimulation.

K230A point mutation and H229A/K230A/K231A triple mutant within Myc-tagFPR2 fusion protein were generated by using Geneart site-directed mutagenesis system kit (Invitrogene, Carlsbad, CA, USA) according to the supplier’s instructions. The pCMV6-ENTRY vector (OriGene Technologies Inc., Rockville, MD, USA) containing wild type FPR2 sequence served as the template.

The

forward

and

reverse

primers

to

create

K230A

GCAGCCAAGATCCACGCAAAGGGCATGATTAA-3’

mutant

were

and

5’5’-

TTAATCATGCCCTTTGCGTGGATCTTGGCTGC-3’, respectively. The forward and reverse primers

to

produce

the

H229A/K230A/K231A

triple

ATTGCAGCCAAGATCGCCGCAGCGGGGCATGATTAAATC-3’

change and

were

5’5’-

ACCEPTED MANUSCRIPT GATTTAATCATGCCCGCTGCGGCGATCTTGGCTGCAAT-3’, respectively. Mutant FPR2 clones were selected with kanamycin and the resulting plasmids FPR2mut3 and FPR2mutBis were purified by maxi-prep (Qiagen, Hiden, Germany). The mutations were confirmed by DNA sequencing on an ABI 3130 xl Gene Analyzer Sequencer (CEINGE Biotecnologie Avanzate

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s.c.a.r.l., Naples, Italy).

Transfection

HEK293 cells (8x105/well) were seeded in a 6-multiwell and transfected with FPR2wt or FPR2mut3 or FPR2mutBis. Briefly, 30 µg of plasmids were diluted in 700 µl of DMEM without

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antibiotics and growth factors. 150 µl of this solution was incubated with 10 µl of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) diluted in 150 µl of DMEM without antibiotics and growth

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factors, for 5 min at room temperature (RT). DNA complexes were incubated with HEK293 cells for 24 h at 37 °C.

Immunofluorescence

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Immunofluorescence was performed as previously described [43]. Briefly, CaLu-6 cells, or HEK293 cells transfected with FPR2wt or FPR2mut3 or FPR2mutBis were washed in PBS containing Ca++ and Mg++, fixed in 4% paraformaldehyde (PFA) for 20 min at RT and quenched with 50 mM NH4Cl for 10 min at RT. After blocking with 1% BSA in PBS for 10 min, cells were

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permeabilized in 0.2% TritonX-100 (Sigma, Saint Louis, MO, USA) 1% BSA in PBS for 10 min at RT. Cells were then washed in 1% BSA in PBS at RT and incubated with anti-FPR2 (1:100;

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Thermoscientific, Waltham, MA, USA) or anti-LAP2 (1:100; Santa Cruz Biotechnology, CA, USA) antibodies, for 20 min. Cells were washed with 1% BSA in PBS and stained with goat antimouse Alexa Fluor 488 (1:200; Invitrogen, Carlsbad, CA, USA) or donkey anti-goat Alexa Fluor 680 (1:200; Invitrogen, Carlsbad, CA, USA) secondary antibodies, for 1 h at RT. Staining was performed by incubating cells with DAPI (1 µg/ml; Invitrogen, Carlsbad, CA, USA) in PBS for 10 min. Cells were then washed with 1% BSA in PBS and images were captured by using a Zeiss LSM 510 meta confocal microscope equipped with an oil immersion plan Apochromat 63×objective 1.4 NA.

Western blot and immunoprecipitation analysis

ACCEPTED MANUSCRIPT Cytosolic and membrane fractions were purified as previously described [39]. Briefly, CaLu-6 or AGS cells were resuspended in a cold hypotonic solution (0.25 M sucrose, 10 mM Tris, 5 mM MgCl2, 2 mM EGTA, 2 mM EDTA, pH 7.4) including protease inhibitors. The supernatant was ultracentrifugated at 126,000g for 1.5 h. Cytosolic and membrane fractions were obtained from the supernatant and pellet fractions, respectively, and analysed by western blotting. Nuclear protein

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extracts were prepared with a Qproteome nuclear protein kit (Qiagen, Hiden, Germany), according to the manufacturer's instructions. Protein concentration was determined using a Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Western blot analysis was performed as previously described [44]. Antigen–antibody complexes were detected with the ECL chemiluminescence reagent kit (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK). In immunoprecipitation

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experiments, nuclei were stimulated with 10 µM WKYMVm for 5 min. Equal amounts of nuclear proteins (400 µg) were incubated with 3 µg of anti-FPR2 for 12 h at 4 °C. Immunocomplexes were

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mixed with 30 µl of Protein A/G Plus agarose and rotated for 45 min at 4 °C. The immunoprecipitates were then washed three times with cold PBS, resuspended in 40 µl of Laemmli buffer, boiled for 5 min, pelleted by short centrifugation, and electrophoresed on 10% SDS–PAGE. Phosphorylated protein levels were quantitatively estimated by densitometry, using a Discover

Receptor binding assays

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Pharmacia scanner equipped with a Sun Spark Classic densitometric workstation.

Radioligand binding assays were performed as described [45] in a binding buffer containing 50 mM

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Tris-HCl (pH 7.4), 100 mM NaCl, 10 mM MgCl2, 0.1% BSA, 1 mM BSA, 1.0 mM phenylmethylsulfonyl fluoride. Nuclei were incubated with increasing concentrations of

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[125I]WKYMVm (PerkinElmer, Waltham, MA, USA) in binding buffer supplemented (non-specific binding) or not (specific binding) with a 1000-fold molar excess of unlabeled WKYMVm, for 90 min at 30 °C with gentle rocking. Competitive binding assays were performed on purified nuclei by using 0.5 nM [125I]WKYMVm in binding buffer, containing increasing concentrations of unlabeled WKYMVm for 90 min at 30 °C. Reactions were stopped by rapid filtration under reduced pressure, through Whatman glass microfiber filters (GF/C) (Sigma-Aldrich, Saint Louis, Mo, USA). Tubes and filters were rinsed three times with cold 25 mM Tris-HCl, pH 7.5. Radioactivity on the filters was quantified using a β-counter. Non-specific binding was subtracted from total binding to obtain the specific binding. Kd was calculated with the Origin program.

Statistical analysis

ACCEPTED MANUSCRIPT All the presented data are expressed as means ± S.D. and are representative of three or more independent experiments. Statistical analyses were assessed by Student’s t test for paired data. Results were considered significant at p<0.05.

FPR2 is expressed in nuclear fractions of CaLu-6 and AGS cells

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Results and Discussion

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The expression of several functional GPCRs is not exclusively restricted to the plasma membrane but is also extended to different compartments, including the nuclear membrane [28]. Many of the observed nuclear effects elicited by these receptors are not prevented by classical inhibitors, that

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exclusively target cell surface GPCRs, presumably because of their structures, lipophilic properties, or affinity for nuclear receptors [11]. Furthermore, these intracellular receptors may regulate signaling cascades that differ from those of their cellular membrane counterparts [46, 47]. We isolated membrane, cytosolic and nuclear fractions from human lung carcinoma CaLu-6 cells, which express membrane-bound FPR2 [40] and we examined FPRs subcellular distribution by

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western blot. The results show that an anti-FPR2 antibody, but not anti-FPR1 or anti-FPR3 antibodies (data not shown), detects the receptor in membrane and nuclear extracts (Fig. 1A). We also assessed colocalization of FPR2 with the nuclear lamina-associated polypeptide LAP2. Confocal microscopy revealed a fluorescent signal for FPR2 (green) distributed intracellularly (Fig.

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1B, Panel A) and, as expected, an intense fluorescent signal for LAP2 (Fig. 1B, Panel B) on the nuclear membrane (red). By using Imaris image analysis software, we demonstrated that FPR2 colocalizes with the nuclear LAP2 (yellow/orange) (Fig. 1B, Panel C). We isolated membrane,

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cytosolic and nuclear fractions also from human gastric adenocarcinoma AGS cells stably transfected with a short hairpin RNA (shRNA) targeting FPR2 (AGS shFPR2) or expressing a nontargeting shRNA (AGS shCTR) [48]. AGS shFPR2 cells express a reduced level of the receptor [48]. Western blot analysis showed that an anti-FPR2 antibody detects the receptor in membrane and nuclear extracts in AGS shCTR cells (Fig. 1C) and its significantly reduced expression in membrane fraction of AGS shFPR2 cells (Fig. 1D). We did not detect FPR2 in nuclear extracts from AGS shFPR2 cells (Fig. 1D). All the purified cellular components were validated by using anti-LAP2, anti-CD44 and anti-Tubulin antibodies, for nuclear, membrane, and cytosolic fractions, respectively. The nuclear fractions does not contain endosomes as demonstrated by western blot experiments performed with an anti-RAB7 antibody.

ACCEPTED MANUSCRIPT Following stimulation with the appropriate agonist, several GPCRs have been detected in the perinuclear or nuclear regions [49-51], whereas a perinuclear localization of the rat UT receptor has been observed without stimulation by its endogenous ligand [52]. The nuclear (or perinuclear) localization can be attributed to translocation of a GPCR from the cell surface or de novo synthesis

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of the receptor.

FPR2 aminoacidic sequence contains a NLS in the third intracellular loop

Generally, nuclear localization of proteins requires a NLS characterized by a short stretch of basic

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amino acid residues. This is recognized by members of the importin family, which act as carriers to transport the substrate protein across the nuclear pore structure. Nuclear GPCRs might be derived from the cell membrane and their transfer to the nucleus can be attributed to a nuclear localization

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motif. We analyzed FPR2 sequence for the search of NLS by using cNLS mapper program (http://nls-mapper.iab.keio.ac.jp) [53] and the results identified a stretch of basic aminoacids (Table I) in the third cytoplasmic loop of the receptor (Fig. 2). Apelin Receptor and Urotensin II receptor, which are members of the GPCR family, also show the presence of a NLS in the third cytoplasmic loop [31, 54]. NLS activity was ranked in 10 levels based on both Green Fluorescent Protein (GFP)-

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NLS localization phenotypes and the proportion of cells with the specific GFP phenotype among GFP-positive cells. The scoring was standardized as follows: score 10 (99% of cells with phenotype exclusively Nuclear [N]), 9 (99–90% of N), 8 (89–70% of N), 7 (69–40% of N, other partially nuclear [Nc]), 6 (Nc>N>nucleus and cytoplasm [NC] in a similar extend), 5 (N70%

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of NC, Nc>C), 3 (NC>C), 2 (NC
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localize to the nuclear membrane via de novo synthesis and retrograde transport [45]. Multiple alignment of the FPR sequences shows that K231 of FPR2, within the sequence KIHKK, is replaced by the polar aminacids Q in FPR1 and N in FPR3 (Table II). K230 of FPR2 is replaced by another basic aminoacid (R) in FPR3 (Table II). Additionally, a HomoloGene/NCBI database search for the NLS within FPR2 revealed that the KIHKK sequence has been conserved among species. K230 and K231 are replaced by the basic residues RR in mouse and rat (Table III). [125I]WKYMVm specifically binds FPR2 in isolated nuclei

ACCEPTED MANUSCRIPT To determine whether nuclear FPR2 immunoreactivity was representive of the presence of a functional ligand-binding receptor, radioligand binding assays were performed on nuclei purified from CaLu-6 cells, by using [125I]WKYMVm as labeled agonist. The results revealed a binding that was specifically displaced by increasing concentrations of unlabeled ligand in a concentrationdependent manner (Fig. 3A). The saturability of the binding sites for [125I]WKYMVm was tested by

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adding increasing concentrations of the radioligand, and receptor binding analysis performed with the Origin program predicted a Kd of 245 pM (Fig. 3B). The same program predicted a Kd of 182,5 pM for the receptor expressed on plasma membrane (data not shown).

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Nuclear-associated FPR2 is a functional receptor

The physiological functions of nuclear GPCRs are poorly understood. They can regulate signaling

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pathways that differ from those of their counterparts at the cell surface [54] and the consequent biological responses could result from the integration of extracellular and intracellular signaling events. A number of GPCR cell surface interactors, including Gαs, Gαi and Gαq, are also associated with the nucleus or the nuclear membrane [18], suggesting a variety of downstream signaling pathways available for nuclear GPCRs. We wished to assess what role nuclear FPR2

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might play and determine what pathways might be activated downstream of nuclear receptor activation. Upon ligand stimulation and activation, GPCRs dissociates from a trimer of G proteins (Gα and Gβγ) which trigger intracellular signaling pathways. Thus, a reducion in the Gαi associated with FPR2 represents an active state of a receptor [10]. Therefore, to evaluate whether

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nuclear-associated FPR2 was active, we co-immunoprecipated FPR2 and Gαi from purified nuclei stimulated or not stimulated with WKYMVm and we determined Gαi expression levels by western

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blot analysis. In untreated cells, we observed a basal level in Gαi expression, which decreased upon stimulation with WKYMVm (Fig. 4A). The same results were obtained with nuclei purified from AGS shCTR cells (Fig. 4B). The α-FPR2 antibody was not able to co-immunoprecipitate FPR2 and Gαi from purified nuclei of AGS shFPR2 cells (data not shown). These data suggest that nuclearassociated FPR2 is a functional receptor and can respond to a specific agonist. Effectors associated with post G protein GPCR signaling, including mitogen-activated protein kinase (MAPK) pathway, is also present in the nucleus [8, 17, 55, 56]. Therefore, we tested the functionality of nuclear-associated FPR2 by assessing the ability of the receptor to activate ERKs [32]. p42MAPK and p44MAPK are nuclear and cytoplasmic protein kinases and in both compartments MAPK signal transduction system is regulated by serine/threonine phosphorylation [29, 55, 56]. Isolated intact CaLu-6 or AGS shCTR nuclei were stimulated with WKYMVm for different times

ACCEPTED MANUSCRIPT and nuclear lysated were incubated with an α-pERK antibody. The results showed a time-dependent phosphorylation of nuclear p42MAPK, but not of p44MAPK, in both type of cells (Fig. 5A and 5B) which was prevented by preincubation with the FPR2 antagonist peptide WRWWWW (WRW4) (Fig. 5C and 5D). Activated MAPK phosphorylate and activate several transcription factors, such as c-Jun and c-Myc [55, 57], resulting in the modulation of genes associated with proliferation.

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Furthermore, FPR2 activation by WKYMVm effectively phosphorylates c-Jun N-terminal kinase (JNK) [58]. In order to activate gene expression, transcription factors have to localize to the nucleus. Nuclear abundance of c-Jun correlates with target gene activity and excessive c-Jun activation can drive cells towards apoptosis or changes in differentiation. On the other hand,

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decreased c-Jun function can reduce proliferation [59]. c-Myc is located predominantly in the nucleus where it is found in a dispersed granular nucleoplasmic pattern [60] and can be directly phosphorylated by ERKs [61]. We analysed c-Jun and c-Myc activation in nuclei purified from

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CaLu-6 and AGS shCTR cells and stimulated with WKYMVm. We observed a time-dependent phosphorylation of the two transcriptional factors in both type of cells (Fig. 6A and 6B), which was prevented by the preincubation with the FPR2 antagonist (Fig. 6C and 6D). We did not observe ERKs phosphorylation, and c-Myc and c-Jun activation in nuclei isolated from AGS shFPR2 cells (data not shown). Interestingly, the nuclear signaling triggered by FPR2 is not prevented by PTX

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(data not shown), presumably because of its affinity for nuclear receptors, lipophilic properties or specific structures assumed by nuclear receptors [11]. These results indicate that nuclear (or perinuclear) FPR2 might play a more direct role in regulating gene expression and our in progress studies are aimed to identify which genes are regulated by

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nuclear FPR2. The nuclear expression of the receptor is relevant to cell physiology, but it remains to be demonstrated whether it has a defined role in the context of intact cells, rather than isolated

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nuclei. The development of specific ligands that can discriminate between surface and nuclear FPR2 will be of great utility in dissecting membrane and nuclear signaling.

Analysis by mutagenesis of the NLS of FPR2

We next examined the putative NLS located in the third intracellular loop of FPR2 and its involvement in the nuclear localization of the receptor. We performed single mutagenesis of the 227-KIHKK-231 sequence in a Myc-tagFPR2 construct, substituting the basic residue K230 into Alanine (FPR2mut3). This construct served as template for multiple mutagenesis in which we substituted H229 and K231 into non-polar aminoacid Alanine (FPR2mutBis) (Fig. 7A). FPR2wt and the two mutated constructs were individually overexpressed in HEK293 cells, which do not express

ACCEPTED MANUSCRIPT FPR2 [34], and analysed by immunofluorescence. The results show that fluorescence was partially localized in the nucleus of cells transiently transfected with Myc-tagFPR2wt or Myc-tagFPR2mut3 (K230A point mutation) (Fig. 7B, Panels B and C). In contrast, nuclear localization of FPR2 was abolished in cells transfected with Myc-tagFPR2mutBis (H229A/K230A/K231A triple mutant) (Fig. 7B, Panel D). Western blot analysis, performed on nuclear proteins purified from HEK293

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cells transfected with the three constructs, was in agreement with immunofluorescence results. Cells transfected with FPR2wt showed nuclear localization of the receptor (Fig. 7C, lane B), which was partially prevented when cells were transfected with FPR2mut3 (Fig. 7C, lane C). FPR2 nuclear localization was completely abolished in cells transfected with the triple mutant FPR2mutBis (Fig. 7C, lane D). We also analyzed FPR2 expression in total cellular lysates of HEK293 cells transfected

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with the three constructs and we observed that the α-Myc-tag antibody detects comparable levels of total receptor (Fig. 7D). Taken together these data indicate that H229 and K231 residues in the

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KIHKK sequence play a key role in the nuclear localization or translocation of FPR2. Accordingly, we did not observe nuclear expression of FPR1 and FPR3, where K231 is replaced by the polar aminoacids Q and N, respectively (Table II).

Our results indicate a nuclear localization for FPR2 but do not report on the possible orientation of the receptor at the nuclear membrane. EP1 receptor is located at both the inner and outer nuclear

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membranes [62] and in both locations, the receptor has easy access to its ligand since the prostanoid synthesis occurs at both the inner and outer nuclear membranes [63]. On the other hand, mGluR1a and 5a receptors are oriented with their ligand-binding domain within the nuclear envelope;

[46].

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Conclusions

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therefore, agonists must cross both the plasma and nuclear membranes to access binding domains

The results of this study provide evidence that: (i) FPR2 localizes in nuclear fractions of CaLu-6 and AGS cells, as demonstrated by western blot, immunofluorescence and radioligand binding experiments; (ii) FPR2 sequence contains a putative, functional NLS which excludes FPR2 from the nucleus when mutated; and (iii) nuclear FPR2 activation prompts a decreased Gαi-FPR2 association and triggers ERK2, c-Jun and c-Myc phosphorylation. The demonstration that FPR2 is expressed at the nuclear level raises questions about its function at this location. In general, nuclear-localized receptors may regulate distinct signaling pathways, suggesting that biological responses mediated by FPR2 are not only initiated at the cell surface but might result from the integration of extracellular and intracellular signaling pathways. Therefore, an

ACCEPTED MANUSCRIPT important focus is to target single pathways associated with a plasma membrane or nuclear GPCR. This pathway-selective strategy might be based on targeting the assembly or trafficking of signaling complexes to distinct subcellular destinations through biased ligands, thus providing a selective set of discriminatory modulators. Pharmaceutical retention of FPR2 at the plasma membrane could be a novel strategy for inhibiting nuclear activities of FPR2 in pathological processes that involve this

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receptor. The access of ligands to nuclear receptors is another open question. Unknown endogenous ligand(s) can somehow be routed to the nuclear membrane to cause an activation of FPR2 and thus trigger signaling pathways. Such ligands must be biosynthesized inside the cells or to cross the cellular membrane. FPR2 agonists that do not permeate cells have access only to receptors

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expressed on the plasma membrane. This observation provides a new concept for bioavailability of a drug, which should be defined also in terms of access of an agonist to intracellular domains of a

Acknowledgements

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receptor, and not only in the current terms of systemic absorption and tissue distribution.

The authors are grateful to Dr. N. Prevete for providing the human AGS shCTR and AGS shFPR2 cells. This work was supported by POR Campania FSE 2007-2013 Project CREME and Ministry of

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Health, Italy, RF-2011-02349269.

ACCEPTED MANUSCRIPT Legend to Figures

Fig. 1. Nuclear FPR2 expression. Membrane (M), cytosolic (C) and nuclear (N) fractions were purified from CaLu-6 (A), AGS shCTR (C) or AGS shFPR2 (D) cells. Protein extracts (30 µg) were resolved on 10% SDS-PAGE and immunoblots were probed with α-FPR2. The purified cellular

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components were validated by using α-LAP2, α-CD44, α-RΑΒ7 and α-Tubulin antibodies, for nuclear, membrane, endosome and cytosolic fractions, respectively. The blots are representative of three separate experiments of identical design (B) Confocal immunofluorescence microscopy of CaLu-6 cells. Localization of FPR2 was detected by incubating cells with an α-FPR2 antibody.

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Cells were then washed in 1% BSA in PBS and incubated with goat anti-mouse Alexa Fluor 488conjugated secondary antibody (Panel A; green). Nuclear protein LAP2 was detected by incubating

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cells with α-LAP2 antibody for 20 min and with secondary donkey anti-goat Alexa Fluor 680 antibody for 1 h (Panel B; red). Staining was performed by incubating cells with DAPI. Merged signals (Panel C; yellow/orange) show co-localization of FPR2 and LAP2 (arrows). Cells showed no staining with secondary antibody alone. Data are representative of three independent experiments.

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Fig. 2. Schematic representation of the human FPR2. The putative NLS in the third intracellular loop, containing basic aminoacids, is indicated.

Fig. 3. Specific binding of [125I]WKYMVm to nuclear FPR2. (A) Purified nuclei from CaLu-6

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cells were incubated for 90 min at 30 °C in binding buffer containing 0.5 nM [125I]WKYMVm and increasing concentrations (0.05, 0.25, 0.5, 0.75, 1 and 10 nM) of unlabeled peptide, as indicated.

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(B) Nuclei were incubated with increasing concentrations of the radioligand in binding buffer supplemented (non-specific binding) or not (specific binding) with a 1000-fold molar excess of unlabeled WKYMVm, as indicated, for 90 min at 30 °C. Specific binding was determined by the difference between total and non-specific binding. Radioactivity was quantified using a β-counter. The experiments were performed in triplicate.

Fig. 4. Nuclear FPR2 is a functional receptor. Nuclei were isolated from CaLu-6 (A) or AGS shCTR (B) cells and incubated in the absence (-) or in the presence (+) of 10 µM WKYMVm for 5 min. Nuclear lysates (400 µg) were immunoprecipitated with α-FPR2 antibody, electrophoresed by 10% SDS-PAGE and transferred to a nitrocellulose membrane. Immunocomplexes were probed with α-Gi or α-FPR2 antibodies. Antigen–antibody complexes were detected with an ECL

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Fig. 5. Nuclear FPR2 triggers ERKs activation. Nuclei were purified from serum-deprived CaLu6 (A) or AGS shCTR (B) cells and stimulated with 10µM WKYMVm for the indicated times, or (C

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and D) preincubated with the indicated concentration of WRWWWW (WRW4) before stimulation. Nuclear lysates (30 µg) were resolved on 10% SDS–PAGE. ERK phosphorylation was detected by western blot with an α-phospho-ERK antibody (α-pERK). The arrow indicates the phosphorylated form of p42MAPK (pERK2). An α-ERK antibody served as a control for protein loading. The arrows

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indicate the non-phosphorylated forms of ERK1 and ERK2. α-Tubulin and α-YY1 antibodies were used as markers for nuclear purity. All the blots are representative of three separate experiments of

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identical design. Protein phosphorylation levels were quantitatively estimated by densitometry using a Discover Pharmacia scanner equipped with a Sun Spark Classic densitometric workstation. *p<0.05 compared with unstimulated nuclei.

Fig. 6. Nuclear FPR2 elicits c-Jun and c-Myc phosphorylation. Nuclei were purified from serum-deprived CaLu-6 (A and C) or AGS shCTR (B and D) cells. Nuclei were incubated with

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10µM WKYMVm for the indicated times (A and B), or preincubated with the indicated concentration of the FPR2 antagonist (WRW4) before stimulation (C and D). Thirthy micrograms of nuclear lysates were resolved on 10% SDS–PAGE and c-Jun and c-Myc phosphorylation was detected with α-phospho-Jun (α-p-Jun) and α-phospho-Myc (α-p-Myc) antibodies, respectively.

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An α-YY1 was used as markers for nuclear purity and as loading control. All the blots are representative of three separate experiments of identical design. Protein phosphorylation levels

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were quantitatively estimated by densitometry using a Discover Pharmacia scanner equipped with a Sun Spark Classic densitometric workstation. *p<0.05 and °p<0.05 compared with unstimulated nuclei.

Fig. 7. Analysis by mutagenesis of the NLS in the third intracellular loop of FPR2. (A) The aminoacid sequences of the NLS of FPR2 and of the two constructs containing the mutated aminocids are boxed in red. The mutated aminoacids are shown in white with the name of the respective constructs. (B) HEK293 cells were transfected with empty vector (Panel A), or with FPR2wt (Panel

B),

or with FPR2mut3 (K230A) (Panel C),

or with FPR2mutBis

(H229A/K230A/K231A) (Panel D). Immunofluorescence was performed as described in Materials and Methods. Localization of FPR2 and LAP2 was detected by incubating cells with α-FPR2 and

ACCEPTED MANUSCRIPT α-LAP2 antibodies, respectively. Staining was performed by incubating cells with DAPI. Merge (yellow/orange) show co-localization of DAPI, FPR2 and LAP2 (arrows). Cells showed no staining with secondary antibody alone. Data are representative of three independent experiments. Nuclear (C) or total cellular lysates (D) were purified from HEK293 cells transfected with empty vector (lane A), or FPR2wt (lane B), or FPR2mut3 (lane C), or FPR2mutBis (lane D). Proteins (30 µg)

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were resolved on 10% SDS-PAGE and FPR2 was detected by western blot with an α-Myc-tag antibody. α-YY1 was used as a marker for nuclear purity. α-Tubulin was used as loading control. Relative expression was quantitatively estimated by densitometry using a Discover Pharmacia scanner equipped with a Sun Spark Classic densitometric workstation. *p<0.05 compared with cells

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transfected with the empty vector. The blots are representative of three separate experiments of

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identical design.

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273 (1998) 9886-9893.

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Table I. cNLS Mapper results of Nuclear Localization Sequence in FPR2 Gene FPR2

NLS KIHKK

Position 227-231

Score 5.8

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Higher scores (8-10) indicate that the protein is exclusively localized to the nucleus. A score of 6-7 indicates a partial localization to the nucleus, whereas a score of 3-5 a localization to both the nucleus and the cytoplasm. A score of 1 or 2 indicates a cytoplasmic localization (http://nlsmapper.iab.keio.ac.jp).

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FPR1 FPR2 FPR3

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Multiple Sequence Alignment of FPRs in the third intracellular loop of the three receptors.

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Table III Multiple sequence aligment

Human (NP001453.1) Chimpanzee (XP003953631.1) Monkey (XP002801420) Dog (XP0056162691) Mouse (NP0320651) Rat (XP0010735081)

201-RGIIRFVIGFSLPMSIVAICYGLIAAKIHKKGMIKSSRPLRVLTAVVASF-250 201-RGIIRFVIGFSLPMSIVAICYGLIAAKIHKKGMIKSSRPLRVLTAVVASF-250 201-RGIIRFVIGFSMPMSIVAICYGLIAAKIHKKGMIKSSRPLRVLTAVVASF-250 201-RGIIRFIIGFSMPMSIVAICYGLIAAKIHKKGMIKSSRPLRVLTAVVASF-250 201-RGIIRFLIGFSMPMSIVAVCYGLIAVKINRRNLVNSSRPLRVLTAVVASF-250 201-RGTIRFVIGFTMPMSIVAICYGLIAVKIHRRALVNSSRPLRVLTAVVVSF-250

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Multiple Sequence Alignment of the FPR2 NLS in eukaryotic genomes. (http://www.ncbi.nlm.nih.gov/homologene).

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FPR2 is expressed at nuclear/perinuclear level in CaLu-6 and AGS cells;

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FPR2 sequence analysis reveals a nuclear localization signal in the third intracellular loop; [125I]WKYMVm specifically binds nuclear-associated FPR2;

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Stimulation of nuclear FPR2 triggers G proteins activation and enhanced

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