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Published as doi: 10.1096/fj.07-9294com.
 (The FASEB Journal. 2008;22:861-869.)
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(The FASEB Journal. 2008;22:861-869.)
© 2008 FASEB

ADP-ribosylation at R125 gates the P2X7 ion channel by presenting a covalent ligand to its nucleotide binding site

Sahil Adriouch*,{ddagger}, Peter Bannas*, Nicole Schwarz*, Ralf Fliegert{dagger}, Andreas H. Guse{dagger}, Michel Seman{ddagger}, Friedrich Haag* and Friedrich Koch-Nolte*,1

* Institute of Immunology and

{dagger} Institute of Biochemistry and Molecular Biology I, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; and

{ddagger} INSERM, U519, Faculté de Médicine et Pharmacie, Université de Rouen, Rouen, France

1Correspondence: Institute of Immunology, Diagnostic Center, University Medical Center Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany. E-mail: nolte{at}uke.uni-hamburg.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ADP-ribosylation is a post-translational modification regulating protein function in which amino acid-specific ADP-ribosyltransferases (ARTs) transfer ADP-ribose from NAD onto specific target proteins. Attachment of the bulky ADP-ribose usually inactivates the target by sterically blocking its interaction with other proteins. P2X7, an ATP-gated ion channel with important roles in inflammation and cell death, in contrast, is activated by ADP-ribosylation. Here, we report the structural basis for this gating and present the first molecular model for the activation of a target protein by ADP-ribosylation. We demonstrate that the ecto-enzyme ART2.2 ADP-ribosylates P2X7 at arginine 125 in a prominent, cysteine-rich region at the interface of 2 receptor subunits. ADP-ribose shares an adenine-ribonculeotide moiety with ATP. Our results indicate that ADP-ribosylation of R125 positions this common chemical framework to fit into the nucleotide-binding site of P2X7 and thereby gates the channel.—Adriouch, S., Bannas, P., Schwarz, N., Fliegert, R., Guse, A. H., Seman, M., Haag, F., Koch-Nolte, F. ADP-ribosylation at R125 gates the P2X7 ion channel by presenting a covalent ligand to its nucleotide binding site.


Key Words: post-translational modification • purinoceptors • ADP-ribosyltransferases • leukocyte ecto-enzymes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ADP-RIBOSYLATION IS AN enzyme-catalyzed post-translational protein modification in which ADP-ribosyltransferases (ARTs) transfer the large, negatively charged ADP-ribose moiety from NAD onto a specific amino acid residue in a target protein (1 2 3) . ADP-ribosylation is best known for its role in bacterial pathogenesis wherein ADP-ribosylating bacterial toxins act as virulence factors which inactivate the function of key regulatory proteins in host cells by ADP-ribosylation (4 , 5) . For example, the toxin-catalyzed ADP-ribosylation of actin at arginine 177 prevents the polymerization of cytoskeletal actin (6 7 8) , and the toxin-catalyzed ADP-ribosylation of elongation factor 2 at diphthamide residue 699 effectively shuts off the elongation cycle of protein synthesis (9 10 11) . ADP-ribosylation does not induce a conformational alteration of actin or of elongation factor 2 (8 , 11) but rather seems to inactivate these proteins by sterically blocking their interaction with other proteins.

Mammalian cells express endogenous toxin-related ecto-ARTs, such as the promiscuous arginine-specific mouse T-cell surface enzyme ART2.2 (12 13 14 15 16) . On exposure to extracelluar NAD, ART2.2 ADP-ribosylates several membrane proteins, including the ATP-gated P2X7 ion channel (16 17 18) . Interestingly, ART2.2-catalyzed ADP-ribosylation leads to activation rather than inactivation of P2X7, resulting in influx of calcium, followed by exposure of phosphatidyl-serine on the outer leaflet of the plasma membrane, the formation of a nonselective pore that allows the passage of DNA-staining dyes such as YO-PRO-1, mitochondrial dysfunctioning, DNA fragmentation, and, ultimately, cell death (18 , 19) . The half-life of extracellular NAD is restricted by CD38, which catabolizes NAD to ADP-ribose and cyclic ADP-ribose, each of which can act as a second messenger (20 21 22 23) . ADP-ribose can react nonenzymatically with free cysteine residues in secretory proteins in a process resembling ADP-ribosylation (24) . Soluble ADP-ribose, however, is neither a substrate for cell surface ARTs, nor does it function as a ligand for P2X7 (13 , 18) .

P2X7 is widely expressed on immune cells and has sparked interest because of its crucial roles in the release of the leaderless proinflammatory cytokine interleukin-1, the killing of intracellular bacteria, and in cell death (18 , 19 , 25 26 27 28 29 30 31) . Like other ligand-gated P2X purinoceptors, P2X7 is thought to form trimers (32) , with each subunit having cytosolic N- and C-termini and two membrane-spanning domains (33 34 35 36 37) . Gating of P2X7 requires very high concentrations of ATP (33 , 37) . The extracellular ATP-binding domain contains ~280 amino acid residues (aa 47–329 in P2X7), including 10 conserved cysteine residues that probably are engaged in intrachain disulfide bonding (38 , 39) (Fig. 1 ). The short (27 aa) N-terminal and long (250 aa) C-terminal cytosolic tails of P2X7 mediate the interaction with other proteins, including the pore-forming hemichannel pannexin 1 (40 , 41) .


Figure 1
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  		Figure 1. Schematic diagram of P2X7 illustrating conserved arginine residues (yellow diamonds), cysteine residues (red circles), potential glycosylation sites (green triangles), and predicted β-strands (blue arrows). The connectivity of cysteine residues corresponds to that proposed for P2X1 and P2X2 (35) .

The purpose of this study was to identify the arginine residues on P2X7 that serve as targets for ADP-ribosylation and to determine how P2X7 is activated by ADP-ribosylation. To this end, we analyzed the functional effects of substituting the 11 conserved arginines in the extracellular domain of P2X7 with lysine. Our results show that ADP-ribosylation of residue R125 is necessary and sufficient to gate P2X7, providing the basis for the first molecular model for the activation of a target protein by ADP-ribosylation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Expression vectors and cell transfections
Expression vectors for wild-type P2X7 (42) and wild-type ART2.2 (43) were cloned as described previously. Mutants were generated by site-directed PCR-mutagenesis using the QuickChange system (Stratagene, La Jolla, CA, USA). The expression construct for monomeric red fluorescent protein (mRFP) was from Clontech Laboratories Inc. (Palo Alto, CA, USA). Expression constructs (5 µg per 106 cells) were transfected into HEK cells with the jetPEI transfection reagent (Q-Biogen, Illkirch, France). Anti-P2X7 antibodies were generated by genetic immunization, as described previously (43 , 44) . Antibodies were conjugated to Alexa 488 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

FACS analyses
Cells were harvested by trypsinization 20 h post-transfection, followed by staining with Alexa 488-conjugated antibodies (1 µg/2x105 cells/100 µl). Separate aliquots of cells were washed and then incubated in the absence or presence of the indicated concentrations of ATP in 10 mM Hepes pH 7.5, 140 mM NaCl, 5 mM KCl, and 10 mM glucose for 60 min at 37°C in the presence of 1 µM YO-PRO-1 (Molecular Probes Inc., Eugene, OR, USA). Cells were washed and stained with APC-conjugated Annexin V for 20 min at 4°C according to the manufacturer’s instructions (Molecular Probes). All measurements were performed on a FACS-Calibur and analyzed with the Cellquest-Pro software (BD, Franklin Lakes, NJ, USA).

ADP-ribosylation, immunoprecipitation, and immunoblot analyses
Cells were harvested by trypsinization 20 h post-transfection. For ADP-ribosylation of cell surface proteins, cells were incubated for 15 min at 37°C with 1µM 32P-labeled NAD (1µCi/106 cells/100 µl PBS). Washed cells were lysed in PBS, 1% Triton-X100, 1 mM AEBSF (Sigma-Aldrich Corp., St. Louis, MO, USA) for 20 min at 4°C. Insoluble material was pelleted by high-speed centrifugation (15 min, 13,000 g). P2X7 was immunoprecipitated with K1G antibody conjugated to protein G-sepharose beads (1 µg Ab/20 µl beads/106 cells). The K1G antibody was used for immunoprecipitation because protein G binds rabbit antibodies more efficiently than rat antibodies. Immunoprecipitated proteins and soluble proteins in total cell lysates (1x105 cell equivalents/lane) were size fractionated on precast SDS-PAGE gels (Invitrogen) and blotted onto PVDF membranes. P2X7 was detected with rabbit anti-P2X7 C-terminal peptide antibody (1:1000) (Alomone, Jerusalem, Israel) and PO-conjugated anti-rabbit IgG (1:5000) using the ECL system (Amersham GE-Healthcare, Munich, Germany).

Calcium imaging by fluorescence microscopy
For ratiometric calcium imaging (45) , HEK cells were cotransfected with expression vectors for P2X7, ART2.2, and mRFP and plated at low density on glass bottom culture dishes (35 mm, MatTek, Ashland, MA, USA). At 20 h post-transfection, the culture medium was removed gently and replaced with prewarmed (37°C) buffer (15 mM HEPES, pH 7.4, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.35 mM CaCl2, 10 mM glucose, 0.1% BSA) containing 4 µM Fura-2/AM (Calbiochem, EMD Biosciences Inc., San Diego, CA, USA) for 30 min at 37°C. Imaging of cells was performed with a Leica DM-IRBE fluorescence microscope equipped with perfusion system (Warner Instruments, Hamden, CT, USA) and an x40 objective (1.3 numerical aperture). Cells were perfused continuously with prewarmed (37°C) buffer containing the indicated concentrations of ATP or NAD. Alternating excitation at 340 and 380 nm was achieved using a monochromator system (polychromator II; TILL Photonics, Graefelfing, Germany). Two images were acquired every 5 s with a grayscale CCD camera (type C4742–95-12NRB; Hamamatsu, Enfield, UK). Raw data images were stored on hard disk, and ratio images (340/380 nm) were calculated using Openlab software (v3.09; Improvision, Tübingen, Germany).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
R151 at the tip of the cysteine-rich region of P2X7 is part of the paratope recognized by antibodies K1G and Hano 44
The extracellular domain of P2X7 contains 11 arginine residues that are strictly conserved in mouse, rat, and human P2X7 (Fig. 1) . Residues R307 and R316 are located in a predicted β-stranded region upstream of Tm2 (Supplemental Fig. 1). Residues R125 and R151 lie on the side and at the tip of a conspicuous "finger" that is connected by 3 closely spaced disulfide bridges. The other conserved arginine residues lie outside of well-defined secondary structure units, consistent with a location in loops on the surface of the protein, as would be expected for charged amino acid residues (Supplemental Fig. 1). These 11 arginine residues were substituted with lysine in mouse P2X7, and each mutant was transfected into HEK cells. Cell surface expression levels were assessed 20 h post-transfection by flow cytometry using three different fluorochrome-conjugated antibodies that recognize P2X7 in native conformation (44) (Fig. 2 ). All mutants were expressed at wild-type levels on the cell surface except mutant R277K, which was expressed at a slightly lower level, and mutant R307K, which was barely detectable (Fig. 2A, B ). The three antibodies used for expression analyses, rat monoclonal antibodies Hano 43 and Hano 44 as well as rabbit antiserum K1G, yielded comparable results for each of the mutants, except in the case of mutant R151K, which was detectable with Hano 43 but not with Hano 44 or K1G (Fig. 2C ). The lack of staining of R151 mutants by antibodies K1G and Hano 44 indicates that R151 is part of the paratope recognized by these antibodies. This result is consistent with the notion that the cysteine-rich region containing R151 forms a finger-like structure accessible to antibodies.


Figure 2
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  		Figure 2. Cell surface and overall expression levels of P2X7 mutants in transfected HEK cells. A) FACS analyses of native P2X7 on the surface of HEK cells 20 h after transfection with expression constructs for wild-type P2X7 (shaded histograms), mutant P2X7 (open histograms), or irrelevant control vector (dashed lines). Cells were stained with fluorochrome-conjugated monoclonal antibody Hano 43 recognizing a native epitope in the extracellular domain of P2X7. B) Western blot analyses of total expression levels of P2X7 mutants in transfected HEK cells (control for A). HEK cells were lysed 20 h post-transfection in 1% Triton-X 100. Proteins in cell lysates were size fractionated by SDS-PAGE and subjected to immunoblot analyses with an antibody directed against the C-terminal peptide of P2X7. Residue numbers are indicated on the bottom (m=mock transfectants). C) FACS-analyses of native P2X7 on the surface of HEK cells 20 h after transfection with expression constructs for wild-type P2X7 (shaded histograms), P2X7 mutant R151K (open histograms), or irrelevant control vector (dashed lines). Cells were stained with fluorochrome-conjugated monoclonal antibodies Hano 43, Hano 44, or with polyclonal rabbit antibody K1G.

Lysine substitutions of R294 or R307 result in loss of P2X7 function; lysine substitutions of R206, R276, or R277 result in gain of P2X7 function
On exposure to ATP, P2X7-transfected HEK cells showed characteristic apoptotic responses (i.e., exposure of phosphatidylserine and uptake of YO-PRO-1) (Fig. 3 ). The ATP sensitivities were similar for wild-type P2X7 and for most R–K mutants (Fig. 3 , panel 2), except mutants R294K and R307K, which lacked detectable responses (panel 1) and mutants R206K, R276K, and R277K, which showed 10- to 20-fold enhanced sensitivities to ATP (panel 3). Lack of ATP-responses by mutant R294K despite its good expression on the cell surface implies that residue R294 forms part of the ligand binding site, consistent with a previous study (44) . Lack of reactivity of mutant R307K likely reflects the poor expression of this mutant on the cell surface. The enhanced ATP-responses of mutants R206K, R276K, and R277K imply that these residues play a role in binding and/or gating of the receptor, analogous to the previously reported gain of function mutants A127K and H155Y (46 , 47) .


Figure 3
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  		Figure 3. ATP-sensitivity of P2X7 mutants in transfected HEK cells. At 20 h post-transfection, cells were harvested by trypsinization and were incubated for 60 min in the presence of the DNA-staining dye YO-PRO-1 (1 µM) and the indicated concentrations of ATP. Cells were counterstained with Annexin V-APC before FACS analyses. Dose response curves illustrate the percentage of HEK cells stained with YO-PRO-1 and AnnexinV as a function of the concentration of ATP.

R125 and R133 in the cysteine-rich region of P2X7 are the targets for ART2.2
To identify the arginine residues in P2X7 modified by ART2.2, we assayed for ADP-ribosylation of P2X7 mutants in ART2.2-cotransfected HEK cells using radioactive [32P]-NAD as substrate (Fig. 4 ). Autoradiography (AR) of cell lysates showed ART2.2-dependent radiolabeling of numerous cell surface proteins (Fig. 4A , lane 12), and, additionally, a strong radiolabeled band for each mutant except R125K, R277K, and R307K (Fig. 4A , lanes 2, 8, 10). The reduced radiolabeling of R277K and R307K reflects the reduced expression levels of these mutants on the cell surface rather than poor ADP-ribosylation. The reduced radiolabeling of R151K in P2X7-immunoprecipitates (Fig. 4B , lane 3) likely results from the poor recognition of mutant R151K by the K1G antibody (see Fig. 2C , panel 3), which was used here for immunoprecipitation.


Figure 4
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  		Figure 4. R125 and R133 serve as the targets for ART2.2-catalyzed ADP-ribosylation. A) At 20 h post cotransfection with expression constructs for ART2.2 and wild-type or mutant P2X7, cells were harvested and incubated for 15 min in the presence of radiolabeled NAD. Washed cells were lysed in 1% Triton-X 100, and P2X7 was immunoprecipitated with antibody K1G immobilized on protein G sepharose. Top panel: proteins in cell lysates. Bottom panel: proteins in immunoprecipitates. Both were size fractionated by SDS-PAGE, and radiolabeled proteins were detected by autoradiography (AR). Residue numbers of R–K mutants are indicated on the bottom (c=control cells transfected with ART2.2 alone). B) The arginine doublets R124/R125 and R133/R134 in the cysteine-rich finger were mutated to lysine individually or in combination as indicated by the residue numbers. (To save space, the quadruple mutant is labeled "24 25 33 34" instead of "124 125 133 134", c=control cells transfected with ART2.2 alone). At 20 h post cotransfection, HEK cells were radiolabeled as above. P2X7 was immunoprecipitated and subjected to SDS-PAGE autoradiography and immunoblot (IB) analyses. In the schematic diagram of the cysteine-rich finger, shaded and empty diamonds indicate conserved and nonconserved arginines, respectively; crescents, the attached ADP-ribose moieties. C) Cell surface expression levels of P2X7 mutants were assessed by FACS as in Fig. 2 . Shaded histograms indicate wild-type P2X7; open histograms indicate mutant P2X7; dashed lines indicate irrelevant control vector.

The reduced radiolabeling of R125K (Fig. 4A , lane 2) despite its strong expression (Fig. 2A ) suggested that R125 is a target of ART2.2, albeit not the only one. Two conspicuous arginine doublets, R124/R125 containing the conserved R125 and the nearby nonconserved doublet R133/R134, were investigated further. The schematic diagram in Fig. 4B illustrates the location of these residues within the cysteine-rich finger. We tested whether mutation of any of these arginine residues alone or in combination with R125K would abolish ADP-ribosylation of P2X7. Immunoblotting (Fig. 4B , bottom row) and FACS analyses (Fig. 4C ) confirmed similar cell surface expression levels of these mutants. The results of the radiolabeling analyses reveal that comutation of residues R125 and R133 abolished ADP-ribosylation of P2X7 (Fig. 4B , top row, lanes 7, 9), whereas other single or double mutants were still radiolabeled (lanes 2–6, 8). These results demonstrate that R125 and R133 on P2X7 are both ADP-ribosylated by ART2.2.

ADP-ribosylation of R125 is necessary and sufficient to activate P2X7
We next assayed whether ADP-ribosylation at R125 or R133 could activate P2X7. To this end, we analyzed calcium fluxes in response to NAD by live cell imaging of HEK cells attached to cover slips 20 h after cotransfection with ART2.2 and the highly responsive R276K mutant, which was further mutated at the other arginine residues in the cysteine-rich finger, including the ADP-ribosylation target sites R125 and R133 (Fig. 5 ). The results reveal that replacement of R125 abolishes calcium responses to NAD (Fig. 5A , panel 4). In contrast, substitution of other arginine residues, including the second target site for ART2.2-catalyzed ADP-ribosylation, R133, did not abolish the responses to NAD (Fig. 5A , panels 3, 5, 6). These results pinpoint R125 as the essential target residue for the activation of P2X7 by ADP-ribosylation. Importantly, substitution of residue R125, either alone or in combination with R133, abolished responses to NAD but did not alter the sensitivity to ATP (Fig. 5B, C ), indicating that the ATP-binding site itself is not affected by these substitutions (Supplemental Movies 1 and 2). The double mutant was used here because this mutant cannot be ADP-ribosylated at all, while R125K can still be ADP-ribosylated on R133 (Fig. 4B , lanes 7, 3). The transient responses to high concentrations of NAD (250 µM) observed in mock transfected cells (Fig. 5A , panel 1) likely are mediated by endogenously expressed P2Y11 receptor (48) .


Figure 5
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  		Figure 5. ADP-ribosylation of R125 is necessary and sufficient to activate P2X7. HEK cells were cotransfected with expression constructs for mRFP, ART2.2, and wild-type or mutant P2X7. At 20 h post-transfection, cells were loaded with Fura-2 and subjected to single-cell imaging. At the indicated times, the perfusion buffer (37°C) was changed to subject cells to increasing doses of NAD (A, panels 1–6; and B, panel 1), or ATP (B, panel 2). Gray lines show single cell tracings; red lines, the calculated mean. C) Images from Supplemental Movies 1 and 2. Top panel: Images from Supplemental Movie 1 illustrate the potent calcium responses and blebbing of ART2.2/R276K cotransfected HEK cells exposed to a drop of NAD added at recording time 0:30. Bottom panel: Images from Supplemental Movie 2 illustrate the lack of responses to a drop of NAD added at recording time 0:30, contrasting with retained vigorous responses to a drop of ATP added at recording time 3:30 of HEK cells cotransfected with ART2.2 and a non-ADP-ribosylatable P2X7 mutant.

NAD released from cells on mild mechanical stimulation suffices to induce ART2-dependent activation of P2X7
When analyzing apoptotic responses of cells harvested by trypsinization 20 h after transfection, we consistently observed strong spontaneous responses, even in the absence of exogenously added NAD or ATP, in cells cotransfected with ART2.2 and R276K (Fig. 6 ) but not in cells transfected with R276K alone (Fig. 3) . Mutation of R125, the critical residue for activation of P2X7 by ADP-ribosylation, abolished spontaneous PS-exposure and YO-PRO-1 uptake (Fig. 6A , panel 3); however, mutation of R133, the second target site for ADP-ribosylation, did not (Fig. 6A , panel 4). The spontaneous apoptotic responses of ART2.2/R276K cotransfected cells were dependent on the enzymatic function of ART2.2 as they were not observed in cells cotransfected with a catalytically inactive mutant of ART2.2 (not shown). These results suggested that cells release NAD during harvesting in sufficient quantity to activate P2X7 by ART2.2-catalyzed ADP-ribosylation. To test this hypothesis further, we performed experiments designed to prevent the activation of P2X7 by ADP-ribosylation during cell harvesting. We previously had shown that etheno-NAD (an analog with a modified adenine group) (Fig. 7 A) is an efficient substrate for ART2.2 but that etheno-ADP-ribosylation does not activate P2X7 and, further, that etheno-ADP-ribosylation prevents the subsequent ADP-ribosylation and activation of P2X7 in response to NAD (18) . Indeed, pretreatment of ART2.2/R276K cotransfected cells with etheno-NAD for 10 min prior to harvesting markedly inhibited spontaneous apoptotic responses (Fig. 6B , panel 2), as did inclusion of the P2X7 antagonist KN62 during cell harvesting (not shown).


Figure 6
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  		Figure 6. NAD released during cell harvesting causes R125-dependent activation of P2X7. HEK cells were cotransfected with expression constructs for ART2.2 as well as wild-type or mutant P2X7. At 20 h post-transfection, cells were harvested by trypsinization either without (A) or following (B) a 10 min preincubation with 10 µM etheno-NAD. Cells then were incubated further for 60 min in the presence of 1 µM YO-PRO-1. Cells were counterstained with Annexin V-APC before FACS analyses. Numbers indicate the percentages of cells in the respective quadrants.


Figure 7
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  		Figure 7. Model for the activation of P2X7 by ADP-ribosylation at R125. A) The soluble ligand ATP and ADP-ribose in covalent linkage to R125 share a common adenine-ribonucleotide chemical framework, which is modified in etheno-ADP-ribose. Residues K64 and K311 may coordinate the negatively charged phosphate groups; residues F293 and R294 may coordinate the adenine-ribose moiety (35 , 37) . The modified etheno-adenine ribonucleotide moiety does not fit into the binding site. B) P2X7 in closed conformation as viewed from above. Dashed crescents indicate unoccupied ligand binding sites. Circles indicate the two transmembrane regions of each subunit. Lines in the finger-like cysteine-rich region mark potential intrachain disulfide bridges. Residue R151 lies in an exposed position accessible to binding by antibodies K1G and Hano 44. C) ADP-ribosylation at R125 places the attached adenine-ribonucleotide in the ligand-binding site, inducing the open conformation that allows passage of calcium ions. ADP-ribosylation at residue R133 places the chemical framework out of reach of the binding site.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A functional role for the cysteine-rich region
Our results indicate that the cysteine-rich region of P2X7 plays a functional role and may form an exposed finger-like structure. This region is accessible to ART2.2 (i.e., can be ADP-ribosylated at R125 and R133) (Fig. 4B ) as well as to antibodies, with residue R151, at the tip of the finger, evidently forming part of the paratope recognized by antibodies K1G and Hano 44 (Fig. 2C) . In line with the notion of a functional role of the cysteine-rich region are recent findings showing gain of P2X7 function following mutation of other residues in this region (A127K and H155Y) (46 , 47) . The two target arginines in the cysteine-rich region of P2X7 possibly conform to a more general ADP-ribosylation target motif. Both R125 and R133 exist as a part of an arginine doublet flanked by intrachain disulfide bonds (Fig. 4B ). Interestingly, a similar motif has been described in human defensin 1 as a target site for ART1-catalyzed ADP-ribosylation (49) .

Activation of P2X7 by a soluble vs. a covalently bound ligand
When comparing the responses of P2X7 to soluble ATP vs. immobilized ADP-ribose (generated by ART2.2 on exposure of cells to NAD), several points are worth noting. First, 40- to 50-fold lower concentrations of NAD than ATP were required to activate R276K. Similar results were observed with the other two gain-of-function mutants R206K and R277K (not shown). A similar difference in the sensitivity of P2X7 to activation by ATP vs. NAD has been noted previously for primary T cells (18) . The higher sensitivity of P2X7 to NAD than to ATP likely reflects a higher stability of the covalently bound ADP-ribose vs. the soluble ligand ATP. Second, and in line with this notion, calcium signals subside more slowly during washout of saturating concentrations of NAD than of ATP. Similarly, PS-exposure by primary T cells induced by short pulse treatments with ATP was readily reversed, but PS-exposure induced by pulse treatments with NAD was not (18) . It is of interest to note that extracelluar NAD and ATP can be metabolized by extracellular nucleotide-hydrolizing enzymes like CD38 and CD39 to ADP-ribose and ADP, respectively (50) . Importantly, neither ADP-ribose nor ADP can activate P2X7, even at high mM concentrations (18 , 37) . Notwithstanding, the covalent attachment of ADP-ribose evidently provides an efficient means of gating P2X7.

Activation of P2X7 by NAD released from cells on mild mechanical stress
Interestingly, mild mechanical stress, (i.e., trypsinization, pipetting, and centrifugation during cell harvesting) induced cells to release NAD but not ATP in sufficient quantity to induce sustained activation of P2X7 (Fig. 6) . These results indicate that ADP-ribosylation could be a very sensitive pathway for the activation of P2X7 in physiological contexts. Note that the HEK cells used in this study do not express CD38. In physiological contexts, this potent NAD-hydrolase would restrict the availability of extracellular NAD as substrate for ART2.2 when expressed either on the same cell surface as ART2.2 or on neighboring cells (20) .

A model for the activation of P2X7 by ADP-ribosylation at R125
To date, two ART target proteins have been crystallized in the post-translationally modified form (8 , 11) . The ADP-ribose group attached to dipthamide 699 in elongation factor 2 (EF2) is visible in the 3D-structure as a prominent bulky extension on the tip of the protein (11) . The ADP-ribose moiety attached to arginine 177 in monomeric actin is not visible in the electron density map, indicating a high flexibility of this group in the crystal (8) . In both cases, the crystal structures of the unmodified and of the modified proteins are almost identical. Thus, ADP-ribosylation seems to inactivate EF2 and actin, not by inducing a conformational shift but rather by sterically blocking their interaction with other proteins. It is not unlikely that this holds also for the inactivation of other ADP-ribosylation targets in which the ADP-ribose moiety is attached to a region on the protein surface known or suspected to be involved in protein-protein interaction (e.g., Asn41 in rho, Cys 391 in Gi, Arg14 in Rap, and Arg129 in Gβ) (1 2 3 4 5) . Here we provide the first molecular model for the activation of a target protein by ADP-ribosylation.

ADP-ribose shares an adenine-ribonculeotide moiety with ATP. On the basis of our results, we propose that ADP-ribosylation of R125 positions this common chemical framework to fit into the nucleotide-binding site of P2X7 and thereby gates the channel (Fig. 7) . Although no crystal structure yet has been elucidated for any of the P2X purinoceptors, important structural insights have been provided by studies with receptor antagonists, crosslinking agents, and mutant receptors (35 , 37) . The transmembrane-spanning domains Tm1 and Tm2 of the three subunits of the receptor complex likely are arranged in an alternating ring-like fashion. Two conserved lysine residues at the outer ends of Tm1 and Tm2 (K64 and K311 in P2X7) have been implicated to coordinate the phosphate residues of ATP, and a triplet of consecutive residues (N292, F293, R294 in P2X7) to coordinate the adenosine and ribose moieties (35 , 37) (Fig. 7A ). In analogy to glutamate and nicotinic receptors, it has been proposed that the ligand-binding sites are located at the interfaces of two adjacent receptor subunits (34 , 35) (Fig. 7B ). Our model for the gating of P2X7 by ADP-ribosylation is in line with this notion of an intersubunit binding site and is supported by the following findings. First, the residue corresponding to R125 in P2X2, H120, recently has been shown to be located in a region on the protein surface facing the adjacent receptor subunit in the trimeric receptor complex (51) . A disulfide bond was formed between adjacent P2X2 subunits when this residue and residue H213 (which corresponds to S215 in P2X7) were mutated to cysteines. Hence, it is likely that R125 similarly lies in a region facing the adjacent P2X7 subunit in the region around S215 and that the ADP-ribose moiety attached to R125 is located at the interface of two receptor subunits. Second, mutations of residues R206, R276, and R277 enhance the sensitivity of P2X7 to activation by both soluble ATP (Fig. 3) and ADP-ribosylation (not shown), suggesting that ATP and the ADP-ribose attached to R125 fit into the same ligand-binding site (Fig. 7A, C ). Third, P2X7 is resistant to activation by covalently linked analogues of ADP-ribose, such as etheno-ADP-ribose, which would not be expected to fit into the ligand-binding site because of their modified adenosine moiety (Fig. 7A ). Fourth, our model accounts for the finding that ADP-ribosylation at the second target site, R133, does not activate P2X7 (Fig. 5) because the ADP-ribose unit attached to R133 would be out of reach of the ligand-binding site.

CONCLUSIONS
We here provide the first molecular model for the activation of a target protein by ADP-ribosylation. Conceivably, the model for the activation of P2X7 by ADP-ribosylation pertains also to other nucleotide-gated receptors. Moreover, it will be of interest to determine whether ADP-ribosylating toxins, such as the promiscuous arginine-specific exoenzyme S from Pseudomonas aeruginosa (52 , 53) , can hijack this mechanism to induce cell death by chronic activation of P2X7. Finally, our finding that minimal changes in the environment can induce cells to release NAD in sufficient quantity to activate P2X7 by ART2.2-catalyzed ADP-ribosylation (Fig. 6) provides a plausible scenario for the activation of P2X7 by ADP-ribosylation in physiological contexts.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft to F.K.N. and F.H. (No310/6), to F.K.N. and S.A. (418FRA112), and to A.H.G. (GU 360/7 and 360/9); by a stipend to S.A. from the Fondation pour la Recherche Medical; and by a stipend to P.B. from the Werner Otto Stiftung. We thank F. Braasch, G. Dubberke, F. Seyfried, and V. Schumacher for excellent technical assistance. We thank Drs. B. Fleischer, O. Boyer, and S. Rothenburg for critical reading of the manuscript. F.K.N., F.H., M.S., and A.H.G. designed and supervised this study. S.A. and P.B. cloned the P2X7 mutants. S.A. performed the experiments shown in Figs. 2 , 3 , and 4A, B ; P.B., Figs. 4C, D , and 6 ; and N.S. and R.F., Fig. 5 and Supplemental Movies 1 and 2. F.K.N. wrote the paper.

Received for publication June 21, 2007. Accepted for publication September 6, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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