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The Journal of Immunology, 2001, 167: 1871-1876.
Copyright © 2001 by The American Association of Immunologists

Cutting Edge

Cutting Edge: The Nucleotide Receptor P2X7 Contains Multiple Protein- and Lipid-Interaction Motifs Including a Potential Binding Site for Bacterial Lipopolysaccharide1

Loren C. Denlinger2,*,{dagger}, Philip L. Fisette2,*,§, Julie A. Sommer*, Jyoti J. Watters*, Usha Prabhu*, George R. Dubyak, Richard A. Proctor{dagger},{ddagger} and Paul J. Bertics3,*

Departments of * Biomolecular Chemistry, {dagger} Medicine, and {ddagger} Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison, WI 53706; § Department of Medicine, Boston University Medical Center, Boston, MA 02118; and Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44106.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
The nucleotide receptor P2X7 has been shown to modulate LPS-induced macrophage production of numerous inflammatory mediators. Although the C-terminal portion of P2X7 is thought to be essential for multiple receptor functions, little is known regarding the structural motifs that lie within this region. We show here that the P2X7 C-terminal domain contains several apparent protein-protein and protein-lipid interaction motifs with potential importance to macrophage signaling and LPS action. Surprisingly, P2X7 also contains a conserved LPS-binding domain. In this report, we demonstrate that peptides derived from this P2X7 sequence bind LPS in vitro. Moreover, these peptides neutralize the ability of LPS to activate the extracellular signal-regulated kinases (ERK1, ERK2) and to promote the degradation of the inhibitor of {kappa}B-{alpha} isoform (I{kappa}B-{alpha}) in RAW 264.7 macrophages. Collectively, these data suggest that the C-terminal domain of P2X7 may directly coordinate several signal transduction events related to macrophage function and LPS action.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
During the last decade, numerous studies have shown that extracellular adenine nucleotides can modulate the actions of LPS (endotoxin) on macrophage function (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11), and may also have a role in the killing of intracellular pathogens (12). Whereas the role for a specific nucleotide receptor in the clearance of Mycobacterium tuberculosis is less clear (12, 13), the nucleotide receptor P2X7 has been reported to potentiate the LPS-induced macrophage production of inflammatory mediators that are important to the pathophysiology of septic shock (14, 15). Our laboratory and others have shown that treatment of macrophages with P2X7 agonists or antagonists can modulate LPS-stimulated production of IL-1{beta} and NO (3, 4, 6, 8). Additionally, LPS-stimulated macrophages from P2X7-knockout mice produce less IL-1{beta} and IL-6 after coadministration of the nucleotide ATP (11). Moreover, cotreatment with a P2X7 ligand, 2-methylthio-ATP, protects mice from a lethal challenge with LPS and promotes a reduction in LPS-induced serum levels of TNF-{alpha} and IL-1{alpha} (2). Collectively, these data provide pharmacological and molecular evidence suggesting that P2X7 modulates the response of macrophages to LPS.

The P2X7 C-terminal region that is distal to the second putative transmembrane domain is thought to control several functions of this ATP-gated ionotropic receptor (16). For example, prolonged stimulation of P2X7 results in the formation of a pore that permits the passage of small molecules (<=900 Da), an activity that may depend upon the P2X7 C-terminal domain (17). This pore has been linked to P2X7-stimulated apoptosis, and disruption of this pore by a spontaneous amino acid substitution (Glu496 to Ala) is associated with resistance to ATP-induced cell death (18). Additionally, other P2X7-dependent signaling events may be distinct from pore formation, including the activation of phospholipase D (PLD)4 and several members of the mitogen-activated protein kinase family, including extracellular signal-regulated kinase (ERK)1 and ERK2 (8, 19, 20). Because these signaling events are dependent on interactions with adaptor and/or effector proteins in other systems (21), it is likely that the P2X7 C-terminal domain binds to intracellular signaling components.

Despite great interest in the C-terminal region of P2X7, there is little information regarding structural motifs that are involved in receptor localization and signal transduction. However, in the present report we have identified several motifs in P2X7 that are homologous to those known to be involved in protein-protein interactions and LPS binding. The data presented here suggest that the C-terminal region of P2X7 may directly associate with proteins and/or lipids that are important for regulating macrophage function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Chemicals and peptides

Phenol-extracted LPS (Escherichia coli O111:B4) and FITC-LPS were purchased from Sigma (St. Louis, MO). A 10 mg/ml LPS stock in 20 mM HEPES (pH 7.4) was sonicated on maximum power in a bath sonicator for 3 min before use. The peptides used for LPS neutralization experiments were synthesized at Quality Controlled Biochemicals (Hopkinton, MA) and solubilized in PBS (137 mM NaCl, 8 mM Na2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4, pH 7.4). Amino acid sequences for two P2X7 peptides and a control peptide from LPS binding protein (LBP) are as follows:

LBP-RK: 89-GRWKVRKSFFKLQGSFD-amide

P2X7-RK: 573-CRWRIRKEFPKSEGQYS-amide

P2X7-EE: 573-CRWRIEEEFPKSEGQYS-amide

A second set of N-terminal biotinylated peptides used in the FITC-LPS binding experiments contained the same sequences but were designed with a spacer arm (two alanine residues) between the biotinyl group and the N terminus of the peptides designated above (SynPep, Dublin, CA). These peptides were also solubilized in PBS.

Cell culture

Murine RAW 264.7 cells were grown to 50–75% confluence and routinely passaged in RPMI 1640 medium (Mediatech, Herndon, VA) containing 5% cosmic calf serum (Mediatech) and 100 U/ml penicillin/streptomycin (Life Technologies, Gaithersburg, MD). Before experimentation, the cells were plated overnight in Falcon 24-well plates (8.5 x 104 cells/well). The following day, the cells were treated with LPS and/or synthetic peptides at the concentrations indicated in the figures.

LPS neutralization

LPS was premixed with synthetic peptides for 30 min in serum-free RPMI 1640 medium. These mixtures were then used to stimulate RAW macrophages for 15 min, followed by cell lysis in SDS-PAGE sample buffer. Equal amounts of protein (25 µg as measured by the MicroBCA Protein Assay, Pierce, Rockford, IL) were loaded per lane and separated by 10% SDS-PAGE. The proteins were transferred to Immobilon polyvinylidene difluoride membrane (Millipore, Bedford, MA), and the membrane was blocked in 5% milk/TBST (10 mM Tris pH 8.0, 150 mM NaCl, 0.1% Tween 20). Immunoblotting for active ERK1/2 and inhibitor of {kappa}B-{alpha} isoform (I{kappa}B-{alpha}) was performed as previously described (8). Anti-active ERK1/2 Abs (Promega, Madison, WI) were used at a final concentration of 1/5000, whereas anti-I{kappa}B-{alpha} (Santa Cruz Biotechnology, Santa Cruz, CA) Abs were diluted 1/1000. The immunoreactive bands were visualized using secondary Abs conjugated to HRP (Santa Cruz Biotechnology) and Lumi-Glo chemiluminescent detection methods (Kirkegaard & Perry Laboratories, Gaithersburg, MD). For sequential analysis of the same blot, the membranes were stripped using 62.5 mM Tris-HCl, pH 6.7, 2% SDS, and 100 mM DTT at 70°C for 30 min followed by blocking in 5% milk/TBST. To confirm equal protein loading, immunoblotting was performed with pan-reactive anti-ERK1/2 Abs (1/5000 dilution; Upstate Biotechnology, Lake Placid, NY). These Abs recognize the total ERK1/2 protein pool, both active and inactive.

FITC-LPS binding assays

N-terminal biotinylated peptides were immobilized on Reacti-Bind NeutrAvidin-coated 96-well black polystyrene plates (Pierce) for 1 h at 37°C, followed by extensive washing with PBS. The plates were blocked with 0.1% Tween 20/PBS for 30 min at 37°C, followed by incubation with FITC-LPS solutions containing 0.01% Tween 20 for 1 h at 37°C. After repeated washing to remove unbound FITC-LPS, the bound fraction was liberated from the plate with 10 mM NaOH/0.1% SDS in PBS and quantified using a plate fluorometer ({lambda}excitation = 492 nm and {lambda}emission = 535 nm). These readings were compared with a FITC-LPS solution standard curve.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Protein interaction motifs

Because multiple lines of evidence suggest that the C-terminal domain of P2X7 (residues 352–595) is critical for signaling (8, 17, 18, 19), we first performed a sequence analysis that revealed multiple potential protein- and lipid-interaction motifs, i.e., these domains were similar (>=40% amino acid conservation) to known functional motifs. As shown in Table IGo, a PXXP motif is present within residues 441–460, suggesting that the P2X7 C-terminal region is able to bind proteins containing Src homology 3 (SH3) domains. Whereas this motif in P2X7 is not completely characteristic of either type I or type II binding sequences (RXXPXXP and PXXPXR, respectively), there are conserved arginine and glutamine residues on either side (Table IGo), with evidence in the literature to support binding site laxity (21, 22). In addition, residues 436–531 of P2X7 are similar to a region of TNFR1 that overlaps its death domain (Table IGo), suggesting a potential mechanism for P2X7-induced caspase activity and apoptosis (20, 23, 24, 25). Thus, the P2X7 C-terminal region may have similar interactions with adaptor or effector proteins to facilitate pore-independent signal transduction.


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  		Table I. Alignments of predicted P2X7 amino acid sequences in comparison with domains of other proteins1

 
The P2X7 C-terminal domain may also bind to cytoskeletal proteins or other components important for receptor trafficking. With respect to potential interactions with cytoskeletal proteins, P2X7 contains regions homologous to domains within high molecular weight protein 3 (HMW3) from Mycoplasma genitalium and the hypothetical Caenorhabditis elegans protein C18H2.1 (Table IGo, residues 389–405 and 494–508, respectively). Although domain analysis has not yet been reported for these proteins, HMW3 is involved in the localization of adhesin proteins to the attachment organelle of Mycoplasma (26). Additionally, C18H2.1 also contains two ankyrin repeats, domains that are involved in cytoskeletal organization in other systems (27). Of note, the motif shared with C18H2.1 includes a residue (Glu496) essential to P2X7-dependent pore formation (18), an activity that likely involves the recruitment of other P2X7 monomers and/or accessory proteins. Thus, the C terminus of P2X7 may interact with the cytoskeleton to facilitate localization of the receptor.

LPS-binding motif

Because P2X7 has been shown to modulate LPS-induced macrophage activation and inflammatory mediator production (8, 11), it is noteworthy that residues 573–590 of P2X7 share strong amino acid homology with the entire LPS binding site of LBP (Table IGo). This region is at the distal end of the P2X7 C-terminal domain and does not overlap with residues important for nucleotide binding (Fig. 1GoA). Sequence alignment of P2X7 with all known species of LBP and bactericidal/permeability increasing protein (BPI), a protein found in neutrophil granules that shares the same LPS binding motif, shows 83% conservation (Fig. 1GoB). Computer modeling using the SYBYL program and the BPI crystal coordinates (28) suggests that this region of the P2X7 C terminus is structurally similar to the LPS binding sites of both BPI and LBP (Fig. 1GoC). Specific features that are retained include the hairpin loop, the distribution of positively charged amino acid side chain projections, and the hydrophobic troughs found in known LPS binding sites (Fig. 1GoC). The conserved basic residues may be important for binding to the negatively charged phosphates of LPS, which have been shown to be important for biotoxicity (29). Additionally, all three proteins share nonvariant phenylalanine, tryptophan, and glycine residues in this region, suggesting that they are also critical for the structure and/or function of these proteins.



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  		FIGURE 1. Modeling of the P2X7 C-terminal domain based on amino acid sequence similarity with the LPS binding motif in LBP and BPI. A, Schematic model of P2X7. The blackened M1 and M2 regions refer to the transmembrane domains, and the C1 and C2 shaded segments refer to cysteine-rich folding domains (16 17 ). Residues K64, K66, R294, and K311 are conserved in all P2X family members, and mutagenesis experiments on the P2X1 and P2X2 receptors suggest that these amino acids are important for nucleotide binding (39 40 ). The LPS binding motif within the distal C-terminal region is designated by the hatched box labeled LPS. B, Aligned sequences of the putative LPS-binding regions of P2X7, LBP, and BPI from various species. A 90% consensus sequence is listed below using the same designations for conserved residues as in Table IGo. Accession numbers used include XP 012121, NP 062129, NP 035157, XP 012965, I56246, NP 032515, B35843, AAB31144, CAA36797, and Q28739. C, Three-dimensional structures of the putative LPS-binding site of BPI, LBP, and P2X7. The structures were produced from the atomic coordinates of the mature form of human BPI using the SYBYL structure alignment program. Key conserved residues are indicated.

 
P2X7-LPS interactions

Of all the motifs identified within the P2X7 C-terminal region (Table IGo), the putative LPS binding motif has the highest conservation (~90%) to a domain with known function. Therefore, using an approach that has been used to confirm the LPS binding site of LBP (30, 31), we evaluated whether synthetic peptides corresponding to the putative LPS-binding region could neutralize the ability of LPS to initiate signal transduction in macrophages. Indeed, premixing LPS with synthetic peptides corresponding to aa 573–590 of the human P2X7 sequence (P2X7-RK) dose dependently blocks the ability of LPS to induce RAW cell activation of ERK1 and ERK2. Similar effects were seen with the control LBP-RK peptides (Fig. 2Go). In addition to the prevention of protein kinase cascade activation, coadministration of P2X7-RK or LPB-RK peptides also neutralized the ability of LPS to mobilize the transcription factor NF-{kappa}B in RAW cells, as determined by monitoring the degradation of its inhibitor I{kappa}B-{alpha} in RAW cells (Fig. 2Go). Similar to published studies with LBP (30, 31), substitution of two residues in the P2X7 peptide (R578K579 -> EE) prevents this peptide from neutralizing LPS-induced macrophage activation of ERK1/ERK2 and degradation of I{kappa}B-{alpha} (Fig. 2Go). Moreover, the addition of serum prevented the ability of the peptides to neutralize LPS-induced signaling, suggesting a competition between the peptides and LBP (and/or other serum proteins) for free LPS (data not shown).



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  		FIGURE 2. Activation of ERK1/2 and degradation of I{kappa}B-{alpha} in RAW cells treated with LPS in the presence and absence of synthetic peptides. RAW 264.7 macrophages were plated the night before experimentation, and then cotreated with the indicated concentrations of premixed LPS and peptide solutions in serum-free medium for 15 min at 37°C. Sequential immunoblotting for active ERK1/2, I{kappa}B-{alpha}, and total ERK1/2 was performed as described in Materials and Methods. Similar results were obtained from at least three separate experiments.

 
To further address potential nonspecific interactions between the synthetic peptides and RAW cells that could be responsible for the observed alterations in LPS-induced signal transduction, we developed a fluorescent, solid-phase, LPS binding assay. A second set of peptides was synthesized with an N-terminal biotinyl group to facilitate immobilization to Neutravidin plates. These peptide-coated plates were then treated with FITC-LPS for fluorescent detection of LPS binding after multiple washes (Fig. 3Go). Similar to the results of the neutralization experiments, the biotinyl-AA-P2X7-RK peptide bound FITC-LPS, an observation that is dependent upon the doses of both the peptides and FITC-LPS (Fig. 3Go). This effect was not seen with the biotinyl-AA-P2X7-EE peptide. Because the binding of FITC-LPS to the biotinyl-AA-LBP-RK peptide was <2-fold that observed with the biotinyl-AA-P2X7-RK peptide, it is likely that the dissociation constants of these peptides for LPS are comparable within an order of magnitude. Collectively, these data suggest that aa 573–590 of the P2X7 C terminus form a domain that has the potential to bind LPS in a manner similar to that observed with the LPS binding domain of LBP.



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  		FIGURE 3. Binding of FITC-LPS to immobilized biotinylated peptides. N-terminal biotinylated peptides were immobilized on Reacti-Bind NeutrAvidin-coated 96-well black polystyrene plates and incubated with FITC-LPS as descibed in Materials and Methods. The binding of FITC-LPS to biotinyl-AA-LBP-RK is included for comparison at a peptide-coating concentration of 10 µM followed by the addition of 100 µg/ml FITC-LPS. The data shown are the means and ranges of duplicate samples. Similar results were observed in two experiments using 1–30 µg/ml FITC-LPS and in five experiments using 0 and 100 µg/ml FITC LPS.

 
Because hydropathy plots predict that the P2X7 C-terminal domain is located inside the cell (16, 17), LPS would have to be internalized before binding to P2X7. In this regard, internalized LPS can be found in multiple cellular compartments, including the cytoplasm of macrophages (32, 33), and can activate NF-{kappa}B via its binding to an intracellular protein Nod1 (34), suggesting that LPS interactions with an intracellular domain of P2X7 are possible. Additionally, this motif may represent a lipid/phospholipid binding domain shared by multiple proteins. This concept is supported by observations that LBP binds to phospholipids and acts as a transfer protein to shuttle them to high-density lipoprotein particles (35). Therefore, an interesting possibility is that phospholipid and/or LPS interactions with the P2X7 C-terminal domain control the trafficking of this receptor, similar to the ability of ceramide to alter phosphoinositide binding and protein localization via interactions with pleckstrin homology domains of multiple proteins (36). Moreover, as macrophages more readily internalize phospholipids and/or LPS relative to this ability in lymphocytes, enhanced P2X7 monomer recruitment to the cell surface in macrophages might explain the formation of larger pores (37, 38). Thus, LPS treatment of macrophages may directly influence P2X7 action.

Several domain-specific functions can be predicted from these data. First, the concept that the C-terminal domain of P2X7 directly regulates endpoints distinct from receptor-dependent pore activity was first introduced by el-Moatassim and Dubyak (19), who found that P2Z-stimulated PLD activity could be dissociated from the pore, and that the PLD activity was dependent on GTP. As the family of SH3 domain binding proteins contains many GTPase activating proteins and guanine nucleotide exchange factors, it is plausible that the putative SH3 domain binding motif of P2X7 (Table IGo) may be involved in regulating receptor-dependent PLD activity via interaction with Rho or other small m.w. G proteins. Second, the organization of other P2X7 monomers or cytosolic proteins, which may be required for pore formation, likely involves interaction with cytoskeletal proteins, possibly via the motifs shared with HMW3 and C18H2.1 (Table IGo). Consistent with this is the observation that spontaneous mutation of Glu496 results in a reduction of P2X7-dependent pore activity (18). Finally, cell surface localization of the receptor is likely controlled via interactions with the P2X7 C-terminal domain (17, 38), possibly via interactions with phospholipids that bind the conserved motif shared with the LPS binding site of LBP and BPI. In summary, this study provides further support for a modulatory role of P2X7 in macrophage function and LPS action.


   Footnotes
 
1 This work was supported by National Institutes of Health/National Heart, Lung, and Blood Institute Grant 1 T32 HL07899 (to L.C.D.) and National Institutes of Health Grants HL56396 and AI34891 (to P.J.B.). Back

2 L.C.D. and P.L.F. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Paul J. Bertics, Department of Biomolecular Chemistry, University of Wisconsin Medical School, 1300 University Avenue, Room 571, Madison, WI 53706. E-mail address: pbertics{at}facstaff.wisc.edu Back

4 Abbreviations used in this paper: PLD, phospholipase D; ERK, extracellular signal-regulated kinase; I{kappa}B-{alpha}, inhibitor of {kappa}B-{alpha} isoform; LBP, LPS binding protein; SH, Src homology; HMW3, high molecular weight protein 3; BPI, bactericidal/permeability increasing protein. Back

Received for publication May 17, 2001. Accepted for publication June 28, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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