Palmitoylation of the P2X7 receptor, an ATP-gated channel, controls ...

The FASEB Journal article fj.08-114637. Published online October 29, 2008.

The FASEB Journal • Research Communication

Palmitoylation of the P2X7 receptor, an ATP-gated channel, controls its expression and association with lipid rafts P. Gonnord,* C. Delarasse,* R. Auger,* K. Benihoud,* M. Prigent,† M. H. Cuif,† C. Lamaze,‡,1,2 and J. M. Kanellopoulos*,1,2 *Institut de Biochimie et Biophysique Mole´culaire et Cellulaire, CNRS UMR 8619, and †Institut de Ge´ne´tique et Microbiologie, CNRS UMR 8621, Universite´ Paris Sud, Paris, France; and ‡Institut Curie, Centre de Recherche, Laboratoire Trafic, Signalisation, et Ciblage Intracellulaires, CNRS UMR 144, Paris, France The P2X7 receptor (P2X7R) is an ATPgated cationic channel expressed by hematopoietic, epithelial, and neuronal cells. Prolonged ATP exposure leads to the formation of a nonselective pore, which can result in cell death. We show that P2X7R is associated with detergent-resistant membranes (DRMs) in both transfected human embryonic kidney (HEK) cells and primary macrophages independently from ATP binding. The DRM association requires the posttranslational modification of P2X7R by palmitic acid. Treatment of cells with the palmitic acid analog 2-bromopalmitate as well as mutations of cysteine to alanine residues abolished P2X7R palmitoylation. Substitution of the 17 intracellular cysteines of P2X7R revealed that 4 regions of the carboxyl terminus domain are involved in palmitoylation. Palmitoylation-defective P2X7R mutants showed a dramatic decrease in cell surface expression because of their retention in the endoplasmic reticulum and proteolytic degradation. Taken together, our data demonstrate that P2X7R palmitoylation plays a critical role in its association with the lipid microdomains of the plasma membrane and in the regulation of its half-life.—Gonnord, P., Delarasse, C., Auger, R., Benihoud, K., Prigent, M., Cuif, M. H., Lamaze, C., Kanellopoulos, J. M. Palmitoylation of the P2X7 receptor, an ATP-gated channel, controls its expression and association with lipid rafts. FASEB J. 23, 000 – 000 (2009) ABSTRACT

Key Words: ATP 䡠 degradation 䡠 membrane microdomains 䡠 posttranslational modification

The p2X7 receptor (P2X7R) belongs to the P2X receptor family of ATP-gated cation channels. Seven members of the P2X receptor family share the same predicted structure composed of a large extracellular loop of ⬃300 amino acids that binds ATP, 2 transmembrane domains, and 2 intracellular N and C termini. The P2X7R differs from the other P2X receptors by its C terminus, which is 200 amino acids longer (1). Brief activation of P2X7R with extracellular ATP in its tetra0892-6638/09/0023-0001 © FASEB

anionic form, ATP4⫺, or the more potent agonist 2⬘,3⬘-O-(benzoyl-4-benzoyl)-ATP (Bz-ATP) opens cation-specific ion channels. Prolonged ligation of P2X7R results in the formation of nonselective membrane pores, permeable to molecules of molecular mass up to 900 Da, as shown experimentally by the uptake of fluorescent dyes. Recently, Pelegrin et al. (2) have shown that pannexin-1 is involved in the early phase of dye uptake, suggesting that this hemichannel can be activated in a P2X7R-dependent way. Prolonged ATP ligation of P2X7R can lead to membrane blebbing (3) and cell death by apoptosis (4) or lysis/necrosis (5), depending on the cell type. Recently, the role of P2X7R in the activation of the cryopyrin inflammasome has also been demonstrated (6, 7). Thus, macrophages stimulated with LPS express proinflammatory cytokines such as pro-IL-1␤ and pro-IL-18, but do not secrete the processed cytokines. Triggering of P2X7R leads to K⫹ efflux, caspase-1 activation, and proteolytic cleavage of pro-IL-1␤ and pro-IL-18 followed by the secretion of the mature cytokines (reviewed in ref. 8). Recently, 2 groups have reported that in rat submandibular glands and mouse lung alveolar epithelial cells a fraction of the P2X7R present at the plasma membrane could associate with detergent-resistant membranes (DRMs; refs. 9, 10). It has been assumed that the asymmetric assembly of cholesterol and glycosphingolipids in the lateral plane of biological membranes can lead to the formation of highly dynamic lipid microdomains that can be further stabilized. These membrane domains have been referred to as DRMs or lipid rafts. The names reflect the biochemical properties of these 1

These authors contributed equally to this work. Correspondence: J.M.K., Institut de Biochimie et Biophysique Mole´culaire et Cellulaire, CNRS UMR 8619, Universite´ Paris Sud, F-91405 Orsay cedex, France. E-mail: jean. [email protected]; C.L., Institut Curie, Centre de Recherche, Laboratoire Trafic, Signalisation, et Ciblage Intracellulaires, CNRS UMR 144, 26 rue d’Ulm, F-75248 Paris Cedex 05, France. E-mail: [email protected] doi: 10.1096/fj.08-114637 2

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structures: their resistance to solubilization by nonionic detergents and their buoyancy in sucrose density gradients (11). The signals targeting transmembrane proteins to microdomains are ill defined. However, several posttranslational modifications, such as addition of glycosylphosphatidylinositol anchors, myristoylation, or palmitoylation, favor the insertion of membrane-spanning proteins into a liquid-ordered phase enriched in cholesterol and sphingolipids. The acyl chains are packed more efficiently in the liquid-ordered phase than in the cholesterol-poor liquid-disordered phase (12). Palmitoylation is a posttranslational modification in which a 16-carbon fatty acid palmitate is covalently linked to a protein, most of the time via a thioester bond. Palmitate bound to cysteine side chains (Spalmitoylation) increases the overall hydrophobicity of the protein domain. Thus, palmitoylation can play a role in the correct targeting of the modified proteins to membranes. Moreover, contrary to other fatty acid modifications, the palmitate linkage is reversible. Thus, palmitoylation can serve as a reversible membrane localization signal (reviewed in ref. 13). Transmembrane proteins such as G protein-coupled receptor, immunoreceptors, and neurotransmitter receptors are frequently modified on juxtamembranous cysteines (13). Palmitoylation of ion channels such as the ␤2a subunit of L-type Ca2⫹ channels, the GluR6 kainate receptor, and the Kv1.1 channel regulates their function (14 –16). Palmitoylation can also influence the stability of modified proteins (17), receptor trafficking (18, 19), and cell signaling (20). In the present work we examined whether P2X7R was palmitoylated and the role of this posttranslational modification in P2X7R trafficking and association with membrane microdomains.

MATERIALS AND METHODS Reagents and antibodies ATP, 2⬘,3⬘-O-(4-benzoylbenzoyl)adenosine 5⬘-triphosphate (Bz-ATP), 2-bromopalmitate (BrP), hydroxylamine hydrochloride (NH2OH), N-ethyl-maleimide (NEM), horseradish peroxidase (HRP) -conjugated cholera toxin subunit B is from Sigma (Sigma-Aldrich, St. Louis, MO, USA). Cell culture media are from Invitrogen (Carlsbad, CA, USA). For Western blot, immunofluorescence, and flow cytometry analyzes, the following primary antibodies (Abs) were used: mouse monoclonal anti-FLAG M2 (Sigma), rabbit polyclonal anti-caveolin (BD Biosciences, Franklin Lakes, NJ, USA), mouse monoclonal anti-calnexin (BD Biosciences), mouse monoclonal anti-Lamp1 (BD Biosciences), rabbit polyclonal anti-Rab 6 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and mouse monoclonal anti-actin (Sigma). The following affinity-purified anti-immunoglobulin G (IgG) Abs were used as secondary Abs: peroxidase-coupled Goat antirabbit IgG Abs (Rockland Immunochemicals, Gilbertsville, PA, USA), peroxidase-coupled goat anti-mouse IgG Abs (Sigma), phycoerythrin-coupled goat anti-mouse IgG Abs (Invitrogen), rhodamine-coupled goat anti-rabbit IgG Abs (DAKO, Ely, Cam2

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bridgeshire, UK), and Alexa 488-coupled goat anti-mouse IgG Abs (Invitrogen). Two different rabbit polyclonal anti-P2X7R Abs were used in this work: 1) the affinity-purified rabbit polyclonal anti-P2X7R Ab-recognizing residues 576 –595 of P2X7R (Alomone Labs, Jerusalem, Israel), and 2) rabbit polyclonal anti-P2X7R antisera we generated by genetic immunization with pcDNA3.1 containing mouse P2X7R cDNA. The specificity of these antisera was demonstrated by comparing immunostaining of human embryonic kidney (HEK) cells and HEK cells transfected with P2X7R and T lymphocytes from P2X7R⫹/⫹ and P2X7R⫺/⫺ mice. For immunoprecipitation, the IgGs were purified on protein-G agarose beads and covalently coupled to protein-G agarose according to the manufacturer’s instructions (Seize X protein G immunoprecipitation kit, Pierce, Rockford, IL, USA). Cells, plasmids, and transfections The cDNA encoding the P2X7R was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) from splenocytes of BALB/c mice and introduced into pcDNA3.1. The FLAG epitope (DYKDDDDK) was introduced in the P2X7R sequence between H85 and S86 by overlapping PCR as described previously (21). Cysteine to alanine mutant constructs were generated from the P2X7R-FLAG cDNA using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). HEK 293 cells were transfected using Gene Juice (Novagen, Darmstadt, Germany) according to the manufacturer’s instructions. Cells were selected in G418 medium or used after transient transfection. After transfection with the wild-type (wt) P2X7R construct, a stable cell line was established, and a clone was selected for. HEK 293 cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS). Macrophages were obtained from BALB/c mice, 5 days after intraperitoneal injection of thioglycolate. Metabolic labeling HEK 293 cells were transiently transfected with wt or mutant constructs. Two days later, cells were incubated with 0.15 mCi/ml 3H-palmitic acid ([9,10-3H] palmitic acid; specific activity 60 Ci/mmol; Isobio, Fleurus, Belgium) in DMEM supplemented with 0.2% of delipidated bovine serum albumin (BSA) (PAA Laboratories, Pasching, Austria) at 37°C for 5 h. For pulse-chase experiments, cells were incubated with 0.15 ␮Ci/ml 35S-methionine/cysteine mixture (Easytag express protein labeling mix [35S]; specific activity 1175 Ci/ mmol; Perkin Elmer, Waltham, MA, USA) in methionine/ cysteine-free DMEM containing 0.2% of BSA at 37°C for 2 h. For chase, cells were washed twice and cultured for the indicated times in DMEM supplemented with 10% FCS and 1 mM methionine/cysteine. After 3 washes, cells were lysed in Tris buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% Triton X-100; and protease inhibitors) containing or not 50 mM NEM. Lysates were immunoprecipitated with antiFLAG M2 beads (Sigma). After overnight incubation at 4°C, beads were washed 3 times and eluted with nonreducing sample buffer. Immunoprecipitates were analyzed by SDSPAGE and transferred to nitrocellulose membranes. Membranes were exposed to a tritium screen (Kodak, Geneva, Switzerland) or phosphor screen for several days and revealed with a Storm phosphorimager (Molecular Dynamics, Amersham Biosciences Corp., Piscataway, NJ, USA). Blots were then immunostained with anti-P2X7R Abs (Alomone Labs) at 4°C overnight and probed with HRP-conjugated secondary

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Abs. Specific bands were visualized by enhanced chemiluminescence (Perkin Elmer).

RESULTS

Drug treatments

P2X7R is associated with DRMs independently from ATP ligation

Palmitoylation was inhibited by pretreating cells with 100 ␮M 2-bromopalmitate at 37°C for 1 h and during the biosynthetic labeling with 3H-palmitic acid. Chemical removal of S-palmitoylation was performed by treating cell lysates at room temperature with 1 M NH2OH hydrochloride, pH 7.2, for 1 h. DRM preparation Cells were harvested after 1 h culture without FCS and submitted to surface biotinylation using sulfo-NHS-LC-Biotin (Pierce) at 1 mg/ml at 4°C for 30 min. Five million cells were resuspended in Tris-buffered saline (TBS) (50 mM Tris, 150 mM NaCl, and 1 mM EDTA, pH 7.4) containing either 0.5 or 0.05% Triton X-100 and protease inhibitors (Roche, Basel, Switzerland) and passed 6 times through a 26 G3/8 needle. Lysates were adjusted to 45% sucrose and placed at the bottom of a SW41 centrifuge tube. A sucrose step gradient was performed by layering 6 ml of 36% and 3.5 ml of 5% (w/v) sucrose in TBS. Sucrose percentages were assessed by refractometry. After centrifugation at 38,000 rpm at 4°C for 15 h, 2 ml aliquots of the gradients were collected from the top and submitted to precipitation with neutravidin-agarose beads (Pierce) at 4°C overnight. Neutravidin-bound proteins were analyzed by SDS-PAGE. Immunofluorescence HEK-P2X7R cells were seeded on polylysine-coated glass coverslips. Cells were fixed with 4% paraformaldehyde for 30 min. Nonspecific sites were blocked using PBS containing 0.5% BSA (blocking buffer). If required, permeabilization was carried out using 0.1% saponin in blocking buffer for 10 min, and saponin was included in washing buffers. Abs were diluted to their final concentration in blocking buffer and applied for 1 h at room temperature (RT). Cells were then washed 3 times, and secondary Abs applied for 1 h at RT. Dapi at a final concentration of 1 ␮g/ml was added in the secondary Abs mix. Finally, cells were washed and mounted onto slides with Vectashield (Vector Laboratories, Burlingame, CA, USA) as a mounting medium. Cells were observed on a DMIRE2 microscope (Leica, Wetzlar, Germany), and images were captured by a CoolSNAPHQ CCD camera (Roper Scientific, Tucson, AZ, USA). MetaMorph software (Universal Imaging Corp., Marlow, Buckinghamshire, UK) was used to deconvolute Z-series and treat the images.

We examined whether P2X7R is found in DRMs as reported for other transmembrane signaling receptors. The mouse P2X7R was tagged with a FLAG epitope inserted in the extracellular domain and was stably transfected in HEK 293 cells (HEK-P2X7R cells). This P2X7R is fully functional as assessed by measuring channel activity, nonselective pore formation, and cell death after stimulation with ATP or Bz-ATP (Supplemental Fig. 1). DRM fractions were prepared from HEK-P2X7R cells as described (22) and analyzed for the presence of P2X7R (Fig. 1A). The vast majority of P2X7R was solubilized with 0.5% Triton X-100 and appeared in the high-density fractions together with the transferrin receptor (TfR), a receptor classically excluded from DRMs. When the Triton X-100 concentration was reduced to 0.05%, ⬃50% of the P2X7R was resistant to detergent solubilization and could be recovered in the lighter fractions containing both caveolin and the ganglioside GM1, 2 markers of lipid microdomains (Fig. 1B). In primary peritoneal macrophages, we confirmed that P2X7R was equally distributed in DRMs and non-DRM fractions (Fig. 1C), ruling out an unusual distribution of the receptors due to their overexpression in HEKP2X7R cells. For several transmembrane receptors, ligand binding can result in the clustering of receptor-ligand complexes within lipid rafts, which is necessary for initiation or termination of signal transduction (reviewed in refs. 23, 24). To investigate whether P2X7R association with DRMs could be influenced by ATP binding, we treated HEK-P2X7R cells or macrophages with 3 mM ATP under conditions where cell lysis could not occur, and we isolated DRM fractions. As shown in Fig. 1 (C, D), no significant variations in the distribution of P2X7R between low- and high-density fractions could be observed in ATP-stimulated HEK-P2X7R cells or in macrophages in comparison with untreated cells. Thus, ATP does not trigger P2X7R recruitement into DRMs. P2X7R is palmitoylated

Flow cytometry Two days after transfection, cells were harvested, washed once with PBS supplemented with 1% BSA and 0.01% sodium azide (wash buffer), and incubated with primary Abs diluted to final concentration in wash buffer at 4°C for 30 min. Cells were washed and probed with secondary Abs at 4°C for 30 min. Cells were then washed and analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA). Statistical analysis Data are expressed as means ⫾ sd. Data were analyzed by using Student’s t test, and P ⬍ 0.05 (at least) was considered statistically significant. P2X7 RECEPTOR PALMITOYLATION

Posttranslational modifications such as palmitoylation are involved in the distribution and/or clustering of various surface receptors through lipid raft association (13). We determined whether P2X7R is palmitoylated by incubating HEK-P2X7R cells with 3 H-palmitic acid. The receptors were immunoprecipitated with anti-FLAG Ab and subjected to Western blot analysis. As shown in Fig. 2A, a single tritiated band of apparent molecular mass 70 kDa was revealed that corresponds to P2X7R as confirmed by immunostaining with anti-P2X7R-specific Abs (we often observed a proteolytic product of P2X7R at an apparent molecular mass of 60 kDa). We show that 3

Figure 1. P2X7R is associated with DRMs of the plasma membrane. A, B)After cell surface labeling with sulfo-NHS-LC-biotin, HEK-P2X7R cells were extracted by 0.5% (A) or 0.05% (B) Triton X-100 at 4°C and fractionated on a 36%/5% sucrose density gradient. Neutravidin-bound proteins were resolved on SDS-PAGE and analyzed by Western blot to detect P2X7R, caveolin, and TfR. To detect ganglioside GM1, 20 ␮l of each fraction was dot blotted on nitrocellulose membranes and revealed with HRP-conjugated cholera toxin (0.5 mg/ml). Low-density fraction (fraction 2) contains DRMs, as confirmed by the presence of GM1 ganglioside. High-density fractions (fractions 4 –5) contained the bulk of solubilized membrane proteins. C) HEK-P2X7R cells and primary mouse peritoneal macrophages labeled with sulfo-NHS-LC-biotin were incubated with or without 3 mM ATP at 37°C for 15 min, lysed in 0.05% Triton X-100 before fractionation on a 36%/5% sucrose density gradient. Neutravidin-bound proteins were resolved on SDS-PAGE and analyzed by Western blot to detect P2X7R. D) Western blots were quantified using ImageQuant software (GE Healthcare, Little Chalfont, UK). Percentages of P2X7R in DRMs represent the ratio of the amount of P2X7R in DRMs (fraction 2) vs. the total amount of P2X7R. Error bars correspond to sd. Differences between control and ATP-treated cells are not statistically significant: HEK-P2X7R cells (n⫽7), P ⫽ 0.2; peritoneal macrophages (n⫽3), P ⫽ 0.6.

P2X7R is specifically palmitoylated because treatment with the nonmetabolizable palmitate analog BrP (25) prevented the incorporation of 3H-palmitic acid into the receptor (Fig. 2A). In addition, we found that palmitoylation occurs on cysteine residues via a thioester bond because NH2OH, which cleaves the thioester bond, removed the 3H-palmitic acid incorporated into P2X7R (Fig. 2A). The palmitoylation status of P2X7R was also tested after ATP stimulation (Fig. 2A). No significant changes in the amount of 3H-palmitic acid incorporated into the receptor were observed after ATP treatment. Importantly, we confirmed that P2X7R is also palmitoylated in primary peritoneal macrophages (Fig. 2B). Palmitoylated P2X7R is associated with DRMs We next analyzed whether palmitoylation controls P2X7R association with DRMs. HEK-P2X7R cells were labeled with 3H-palmitic acid and lysed in 0.05% Triton X-100 before fractionation on sucrose density gradient. 4

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As shown in Fig. 3A, the pool of palmitoylated P2X7R was almost entirely recovered from DRM fractions. In addition, ATP did not alter the association of palmitoylated P2X7R with DRMs. To determine whether P2X7R palmitoylation is required for its association with DRMs, cells were pretreated prior to fractionation with 100 ␮M BrP. As shown in Fig. 3B, the balance between the 2 pools of P2X7R was strongly modified. Thus, the amount of P2X7R recovered from DRM fractions was highly reduced, and the fraction of nonpalmitoylated P2X7R was more abundant in high-density fractions. To show that the organization of DRMs was not altered on BrP treatment, we followed the distribution of caveolin, which is localized in lipid rafts independently of its palmitoylation status (26). As shown in Fig. 3B, BrP treatment had no effect on caveolin localization, while the amount of P2X7R present in DRMs decreased. Taken together, these data demonstrate that palmitoylation is required for the association of surface P2X7R with DRMs.

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Figure 2. P2X7R is palmitoylated. A) Cells were labeled with 3H-palmitic acid as described in Materials and Methods. P2X7R was immunoprecipitated from lysates of HEK-P2X7R cells with anti-FLAG M2 agarose beads. Immunoprecipitated proteins were separated on SDS-PAGE and transferred to nitrocellulose membrane. Top panel: 5 day exposure to tritium screen (3H-palmitate). Control, untreated cells; ATP, cells stimulated by 3 mM ATP at 37°C for 15 min; BrP, cells incubated with 100 ␮M BrP; NH2OH, immunoprecipitates treated with 1 M NH2OH (pH 7.4) at RT for 1 h to cleave the thioester bond and remove the palmitate. Bottom panel: Western blot performed with anti-P2X7R Ab. Tritium incorporation was measured by analysis with phosphorimager, and Western blots were quantified using ImageQuant software. Percentage of palmitoylation is expressed as the ratio of 3H-palmitic acid quantity vs. P2X7R quantity. Results are normalized to the control value. Error bars correspond to sd (n⫽3); **P ⬍ 0.01. B) Thioglycolate-elicited macrophages were labeled with 3H-palmitic acid as described in Materials and Methods. P2X7R was immunoprecipitated from lysates of macrophages with rabbit polyclonal anti-P2X7R IgG covalently bound to protein G-agarose beads. Immunoprecipitated proteins were separated on SDS-PAGE and transferred to nitrocellulose membrane. 3H-palmitate, 30 day exposure to tritium screen; anti-P2X7R, Western blot probed with anti-P2X7R Ab (Alomone Labs).

Carboxyterminal cysteines of P2X7R are required for its palmitoylation To analyze precisely the role of palmitoylation in the cellular localization of P2X7R, we attempted to block its palmitoylation through mutagenesis of intracellular cysteine residues. The mouse P2X7R contains 17 cysteines in its cytoplasmic tails, among which 16 are conserved between human, rat, and mouse species. It

was previously shown that mutation of palmitoylation sites in multiple cysteine motifs can lead to palmitoylation of the remaining cysteines, which otherwise are not palmitoylated (27, 28). Thus, the 16 potential sites for S-palmitoylation were mutated by groups of 2, 3, or 4 neighboring cysteines as summarized in Fig. 4A. To test the palmitoylation status of the different P2X7R mutants, HEK cells were transiently transfected with the various constructs and labeled with 3H-palmitic acid. As

Figure 3. Palmitoylated P2X7R is associated with DRMs. A) Five million HEK-P2X7R cells were labeled for 5 h with 3H-palmitic acid at 37°C and then incubated with or without 3 mM ATP for 15 min. HEK-P2X7R cells were extracted by 0.05% Triton X-100 at 4°C and fractionated on a 36%/5% sucrose density gradient. Fractions were submitted to immunoprecipitation using anti-FLAG M2 agarose beads overnight at 4°C. Immunoprecipitates were resolved on SDS-PAGE and transferred to nitrocellulose membranes. Membranes were exposed 5 days to a tritium screen and immunostained for the P2X7R. B) HEK-P2X7R cells were incubated with or without 100 ␮M BrP for 16 h at 37°C. After surface protein labeling with sulfo-NHS-LC-biotin, 5 million cells were extracted, and neutravidinbound membrane proteins were resolved on SDS-PAGE and analyzed by Western blot to detect P2X7R and caveolin. Western blots were quantified using ImageQuant software. Percentages of P2X7R in DRMs represent the ratio of the amount of P2X7R in DRMs (fraction 2) vs. the total amount of P2X7R. Error bars correspond to sd (n⫽3); *P ⬍ 0.05. P2X7 RECEPTOR PALMITOYLATION

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Figure 4. C-terminal cysteines of P2X7R are involved in palmitoylation. A) Position of the conserved mutated cysteines is indicated in gray on the receptor schematic structure and in the amino acid sequence. A nonconserved cysteine is labeled with a star. The 2 transmembrane domain sequences are in bold and italics, and the extracellular domain sequence is underlined. Cysteines were mutated by groups numbered from 1 to 7 (indicated by black bar above mutated residues). P2X7R mutants that encompass several groups (2– 4, 5–7, and 2–7) were also generated. B) Two days after transfection with the different mutant constructs, HEK-transfected cells were incubated with 3H-palmitic acid for 5 h before immunoprecipitation with anti-FLAG Abs. Immunoprecipitates were run on SDS-PAGE and transferred to nitrocellulose membranes. These membranes were exposed 5 days to a tritium screen (top panel) and immunoblotted with anti-P2X7R Abs (bottom panel). Tritium incorporation was measured by analysis with a phosphorimager, and Western blots were quantified using ImageQuant software. Percentage of palmitoylation is expressed as the ratio of 3H-palmitic acid quantity vs. P2X7R quantity. Results were normalized to the values of wt P2X7R. Error bars correspond to sd (n⫽3); *P ⬍ 0.05, **P ⬍ 0.01.

shown in Fig. 4B, P2X7R mutants from groups 1, 2, and 4 were palmitoylated as efficiently as the wt P2X7R. In contrast, 2 different profiles of altered palmitoylation were found with mutants of groups 3, 5, 6, and 7. The group 3 mutant showed a 50% decrease of palmitoylation, compared to wt P2X7R, which was further confirmed with the mutant group 2– 4, indicating that the cysteine residues from group 3 are involved in P2X7R palmitoylation. In contrast, palmitoylation was abolished in P2X7R mutants from groups 5–7 (Fig. 4B). The lack of palmitoylation was also observed with mutants 5–7 and 2–7. Because groups 5 and 6 contained several nonjuxtaposed cysteines, we generated single C to A mutants to determine which cysteines are involved in P2X7R palmitoylation. With the single cysteine mutants, palmitoylation of the P2X7R was abrogated, as observed in mutants of groups 5 and 6, suggesting a contribution of each cysteine to the bulk of P2X7R palmitoylation (Supplemental Fig. 2). Thus, 6

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our results indicate that each cysteine of groups 5 and 6 is critical for palmitoylation of P2X7R. Palmitoylation-deficient P2X7Rs are not expressed at the plasma membrane We then investigated the effect of palmitoylation on P2X7R cell surface expression. HEK cells transiently transfected with the different P2X7R mutants were analyzed by flow cytometry for the expression of the FLAG-tagged P2X7R. As shown in Fig. 5A, the expression profile of the palmitoylated P2X7R mutants (groups 1, 2, 4) was similar to that of wt P2X7R. In contrast, the palmitoylationdeficient P2X7R mutants (groups 5–7) were not expressed at the plasma membrane. Likewise, the cell surface expression was also strongly reduced in P2X7R mutants of group 3, which showed a 50% decrease of palmitoylation (Fig. 5A, panel 3). Figure 5B shows that the

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Figure 5. Palmitoylation-deficient P2X7R mutants are not expressed at the plasma membrane. A) Forty-eight hours after transfection with the wt or P2X7R mutants, HEK-transfected cells and nontransfected cells were harvested and labeled at 4°C with anti-FLAG M2 Ab for 30 min and then with PE-conjugated anti-mouse IgG for 30 min. Expression patterns of HEK-P2X7R cells (thick black line) are superimposed with those of HEK-nontransfected cells (gray shading). B) HEK transiently transfected cells were lysed. Lysates were analyzed by SDS-PAGE, and P2X7R and actin were detected by Western blotting.

P2X7R mutants of groups 5–7, which were not present at the cell surface, were still expressed within the cell. We then studied the subcellular localization of the wt and the mutants P2X7R. Immunofluorescence microscopy showed that wt P2X7R was localized predominantly at the plasma membrane (Fig. 6). In contrast, the palmitoylation-deficient mutants of group 6 showed no expression at the cell surface and displayed a punctuate intracellular staining. The P2X7R mutant was partially colocalized with calnexin, a marker of the endoplasmic reticulum (ER; Fig. 6A), and with Lamp1, a marker of lysosomes (Fig. 6B). In addition, the P2X7R mutant was absent from the Golgi apparatus, as shown with Rab6 staining (Fig. 6C). All P2X7R mutants [i.e., groups 3, 5–7, and one lacking all cysteines (Cys⫺)] were analyzed for their intracellular distribution (Sup-

plemental Figs. 3–5). We find that the colocalization patterns observed with these mutants are similar to those obtained with mutant 6. However, it is worth noting that in all P2X7R mutants the labeling with anti-P2X7R Abs was much weaker than with the wt P2X7R: to obtain similar staining intensities, the exposure time was at least 1 s, whereas for the wt receptor it was only 100 –500 ms. Thus, these observations suggest that the P2X7R mutants are degraded. To determine whether different P2X7R proteolytic fragments are generated during the processing of the various mutants, we used 3 different anti-P2X7R Abs recognizing distinct regions of the receptor (Supplemental Fig. 6). No major differences in staining intensities with the 3 anti-P2X7R Abs were observed for all P2X7R mutants at the same exposure time, indicating that there is no

Figure 6. Palmitoylation-deficient P2X7R mutants are retained in the ER and colocalized with Lamp1. Permeabilized HEK-P2X7R cells were stained with rabbit polyclonal anti-P2X7R antiserum raised by genetic immunization (A, C) or with mouse monoclonal anti-FLAG M2 Ab (B) and with anticalnexin (A), anti-Rab6 (B), and anti-Lamp1 Abs (C) and observed with a wide-field 3-dimensional microscope. Images are single sections of a deconvoluted Z series. Scale bars ⫽ 10 ␮m. P2X7 RECEPTOR PALMITOYLATION

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preferential accumulation of a proteolytic fragment of P2X7R (Supplemental Figs. 3–5). Taken together, these data show that the lack of P2X7R palmitoylation leads to the retention of receptors in the ER and their increased targeting to lysosomes. Palmitoylation-deficient P2X7Rs have a reduced half-life To understand the differential expression of palmitoylation-deficient vs. wt P2X7R, we measured the life span of the different P2X7Rs. HEK-P2X7R cells were labeled with 35S-methionine/cysteine and chased at 37°C for different times. Data shown in Fig. 7A reveal 2 kinetic components of P2X7R degradation in both wt and mutant receptors. In the first 4 h, the percentage of 35 S-labeled P2X7R decreased to 48, 33, and 16% for the wt and mutants 3 and 6, respectively. In the second phase of the degradation process, the half-life was also reduced from 54 h for the wt to 25 and 21 h for mutants 3 and 6, respectively. In addition, the amount of 35 S-labeled P2X7R from group 6 at t ⫽ 0 was reduced to ⬃60% of the levels obtained for wt P2X7R. These

results strongly suggest that palmitoylation-deficient P2X7Rs of groups 3 and 6 are more rapidly degraded. However, mutant 3, in which palmitoylation deficiency is partial, is more stable than mutant 6, which is not palmitoylated. Thus, there is a correlation between lack of palmitoylation of P2X7R and its stability. We next used 3 different pharmacological inhibitors to test the involvement of the lysosomal and proteasomal pathways in proteolysis of P2X7R (Fig. 7B). Inhibitors of lysosome acidification such as chloroquine and NH4Cl, enhanced the stability of mutant 6, in agreement with the colocalization of this mutant with Lamp1 (Fig. 6C). Lactacystin, a selective inhibitor of the proteasomes, strongly increased the amount of the mutant 6 and wt P2X7R. Thus, the absence of palmitoylation leads to the increased degradation of the P2X7R, preferentially through proteasomes.

DISCUSSION In this study, we show that P2X7R is palmitoylated and that this posttranslational modification is responsible for its association with lipid rafts at the plasma mem-

Figure 7. Palmitoylation-defective P2X7R mutants have a reduced half-life. A) HEK cells were transfected with P2X7R wt, mutant 3, or mutant 6, and transfectants were metabolically labeled with 35S-methionine/cysteine for 2 h and chased for 4, 24, 48, and 72 h. Immunoprecipitates of P2X7R were run on SDS-PAGE, transferred to nitrocellulose membranes, and exposed to a phosphor screen for 48 h. 35S incorporation was measured with phosphorimager and quantified using ImageQuant software. Quantity of 35S-labeled P2X7R is normalized to the value of the wt P2X7R at t ⫽ 0. B) HEK cells transfected with wt and mutant 6 P2X7R were treated with chloroquine (100 ␮M), NH4Cl (50 mM), and lactacystin (10 ␮M) overnight. Western blot of cell lysates was revealed with anti-P2X7R and anti-actin Abs and quantified using ImageQuant software. Results are normalized to the quantity of P2X7R in untreated cells (percentage of control). Error bars correspond to sd (n⫽3); *P ⬍ 0.01, **P ⬍ 0.001. 8

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brane. Mutations of specific intracellular cysteines prevented P2X7R palmitoylation and resulted in strong defects in trafficking and expression of the receptor at the cell surface. We found that 50% of the total pool of P2X7R at the plasma membrane was associated with DRMs isolated from HEK-P2X7R cells and macrophages. These data agree with the recent findings that P2X7R was associated with lipid rafts in rat submandibular glands (9) and mouse lung alveolar epithelial cells (10). In both studies P2X7R was equally distributed between low- and high-density sucrose fractions. We also found that P2X7R was almost completely solubilized by 0.5% Triton X-100, in agreement with Garcia-Marcos et al. (29), indicating that the receptor is only partially resistant to Triton X-100 solubilization. Similar detergent solubility has also been observed for the P2X1 and P2X3 purinergic receptors (30, 31) and for different immunoreceptors, such as Fc␥RIIa and FcεR (32, 33). The fact that caveolin remained associated with DRMs at 0.5% Triton X-100 indicates that P2X7R was only weakly associated with caveolin. Accordingly, we found that P2X7R and caveolin partially colocalized at the cell surface of HEK-P2X7R cells in agreement with results obtained with alveolar epithelial cells (Supplemental Fig. 7) (10). Ligand-induced aggregation of cell surface immunoreceptors such as FcεR, B, and T cell receptors leads to their recruitements into lipid rafts, where they associate with signaling partners (34 –36). Coaggregation of raftassociated molecules consecutive to receptor ligation is thought to represent a general mechanism for initiating and increasing cell signaling. However, in contrast to immunoligands, we found that ATP does not drive the P2X7R into DRMs, suggesting that additional recruitment of these receptors into lipid rafts is not required to amplify P2X7R signaling. Our experiments also establish that ATP does not stimulate the basal level of P2X7R palmitoylation in HEK-P2X7R-transfected cells and macrophages, in contrast to what has been described for the ␤2 adrenergic and vasopressin receptors (37, 38). Palmitoylation has been shown to promote DRM association of some transmembrane receptors (13, 39), such as Fas (40), Fc␥RIIa (33), and the mouse 5-hydroxytryptamine1A receptor (41). Our biochemical experiments show that palmitoylation is also required for the association of P2X7R with DRMs. However, palmitoylation is not always a strict requirement for DRM localization, because the palmitoylation of the anthrax toxin receptors prevents their partition into DRMs (42), whereas the transferrin receptor is palmitoylated but does not associate with lipid rafts (43). Finally, although caveolin-1 is palmitoylated, this posttranslational modification is not required for DRM association (26). Because the lack of P2X7R palmitoylation can lead to its degradation, we have tested whether the loss of P2X7R association with rafts after BrP treatment (Fig. 3B) may be due to its proteolysis. We have performed P2X7 RECEPTOR PALMITOYLATION

raft fractionation of HEK-P2X7R cells incubated with lactacystin and BrP or control. As shown in Supplemental Fig. 8, the raft association of the full-length P2X7R still decreases in the presence of BrP and lactacystin (Supplemental Fig. 8D). In the absence of palmitoylation, the P2X7R does not associate with rafts, and its degradation by the proteasome is not the principal mechanism involved in this loss of raft association. It is worth noticing that BrP treatment does not abolish surface expression of nonpalmitoylated P2X7R (Fig. 3B). This apparent discrepancy could be due to the length of BrP treatment, which is shorter than the half-life of P2X7R. Longer treatments with BrP were tried but lead to significant cell death. To study the functional role of P2X7R palmitoylation, we therefore generated several mutants in which 16 conserved cysteines were substituted with alanine. Our results show that the cysteines from group 1 (C4, C5), 2 (C363), and 4 (C388) are not modified by palmitoylation. In contrast, 2 separate regions of the carboxyterminal domain are important for P2X7R palmitoylation. The juxtamembrane cysteines from group 3 (C371, 373, 374) are clearly implicated in palmitoylation because their replacement by alanines leads to a 50% decrease in palmitate incorporation, in agreement with several reports showing that juxtamembrane cysteines of transmembrane proteins are often palmitoylated (reviewed in ref. 44). In addition, we found that the cysteines of mutants from group 5 (C477, 479, 482), 6 (498, 499, 506), and 7 (572, 573) are essential for palmitoylation, because palmitoylation was abrogated in all mutants. A similar observation has been reported for the ␤2a subunit of L-type Ca2⫹ channels where all the cysteines are essential for palmitoylation (14). In addition, the level of P2X7R expression at the plasma membrane correlates with its ability to be palmitoylated, as previously reported for the CCR5 chemokine receptor by Percherancier et al. (28). Interestingly, Smart et al. (45) performed a glycinescanning mutagenesis of C548 and C572 from rat P2X7R. They found that the C572G mutant was not expressed at the cell surface, whereas the mutation at C548, which is not a conserved residue between species, has no deleterious effects. These authors have identified in the carboxyterminal region of P2X7R a nonselective pore-enabling sequence (551–582) containing 2 cysteines (group 7 C572, C573), which we found to be involved in palmitoylation. We propose that this region interacts with the cytoplasmic leaflet of the plasma membrane through increased hydrophobicity due to the palmitoylated cysteine(s) and contributes to P2X7R stability and its capacity to recruit signaling partners. The sequestration of nonpalmitoylated P2X7R in the ER suggests that P2X7R palmitoylation is required for its maturation and exit from the ER. These observations indicate that this posttranslational modification may play a role in the macromolecular organization of the P2X7R. Indeed, several works have shown that the role of palmitoylation is not limited to a mere increase in hydrophobicity of the acylated protein but can mediate 9

specific protein-protein interactions. Thus, it has been previously demonstrated that palmitoylation of 3 cysteines of the cytoplasmic tail of influenza virus hemagglutinin was required for its oligomerization and for virus assembly (46). Moreover, Escherichia coli hemolysin is palmitoylated on 2 lysine residues, and this acylation is essential for its ability to form a pore in the host membrane (47– 49). Removal of one acyl group results in an almost complete loss of cytolytic activity of the hemolysin. The palmitoylation of the P2X7R could be a prerequisite for its trimerization in the ER and to allow its correct folding. The palmitoylation-mediated oligomerization of the receptor could mask an ER retention motif in the cytoplasmic tail of P2X7R. Indeed, several receptors possess such an arginine-based ER retention motif (LRSR for ␥-aminobutyric acid receptor subunit B, LRKR for Kir6.2, NRKR for sulfonylurea receptor 1), which is hidden after multimeric assembly of the receptors and allows their correct maturation and transport to the plasma membrane (reviewed in ref. 50). The P2X7R presents an “LRHR” motif in its carboxyterminal tail localized between the cysteines of mutants 6 and 7. This motif has already been described as an inner nuclear membrane retention motif for the glycoprotein B of human cytomegalovirus (50). This motif may thus be involved in the ER sequestration of palmitoylation-deficient P2X7R. We further observed that the palmitoylation-deficient P2X7Rs are more concentrated into lysosomes and that degradation of P2X7R mutants involves lysosomes and proteasomes. Because proteasomes are involved in the degradation of misfolded proteins sequestred in the ER (51, 52), we hypothesize that the lack of P2X7R palmitoylation prevents their correct folding, leading to the early degradation of ER-sequestered P2X7R by proteasomes. Interestingly, in all P2X7R mutants, the lack of palmitoylation in different regions of the C-terminal portion of the receptor does not generate different proteolytic fragments (as assessed with the 3 anti-P2X7R Abs used), suggesting that the misfolded receptors share similar cleavage sites. Altogether, these results are in agreement with other recent studies linking palmitoylation and degradation. Thus, palmitoylation of the anthrax toxin receptors increases their life span by preventing their internalization and targeting to lysosomes (42). For the yeast SNARE Tlg1, palmitoylation inhibits ubiquitinylation, which, in turn, prevents the degradation of Tlg1 (53). Finally, the palmitoylation-deficient mutant of human adenosine A1 receptors exhibit an enhanced proteolysis (17). In conclusion, our studies show that palmitoylation of the carboxyterminal region of P2X7R plays a critical role at several stages along the biosynthetic pathway of the receptor. It allows the correct maturation of P2X7R and the bona fide intracellular trafficking and insertion into the plasma membrane. It is also required for the dynamic distribution of P2X7R in plasma membrane microdomains, potentially promoting its interaction with signaling partners. 10

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We are grateful to Keith Mitchell for his advice and help during DRM preparation. We warmly thank Dr. David Holowka and Dr. Yann Percherancier for critical review of our manuscript. This work was supported by CNRS and Gefluc. P.G. was supported by a grant from the Fondation pour la Recherche Me´dicale. Work in C.L.’s laboratory was supported by grants from the Association pour la Recherche sur le Cancer (ARC no. 3143) and Agence Nationale de la Recherche (no. BLAN0211).

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