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Epithelial Membrane Proteins Induce Membrane Blebbing and Interact with the P2X7 Receptor C Terminus

2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês

10.1074/jbc.m205120200

1083-351X

Heather L. Wilson, Stuart A. Wilson, Annmarie Surprenant, R. Alan North,

Mast cells and histamine

The binding of extracellular ATP to the P2X7 receptor opens an integral cation-permeable channel; it also leads to membrane blebbing and, in certain immune cells, interleukin-1β secretion and eventual death. The latter three effects are unique to the P2X7 receptor; also unique among P2X receptors is the long intracellular C terminus of the protein. We have shown that the C-terminal domain of the P2X7receptor is responsible for the cell blebbing phenotype. A screen for proteins that associate with the C-terminal domain of the P2X7 receptor and might mediate the blebbing phenotype, identified epithelial membrane protein 2 (EMP-2). The interaction between EMP-2 and P2X7 was confirmed biochemically by co-immunoprecipitation, co-purification, and glutathioneS-transferase pull-down assays, and this interaction was entirely dependent on the C-terminal domain of P2X7. The P2X7 receptor also interacted with the other members of the epithelial membrane protein family (EMP-1, EMP-3, and PMP-22). All four EMPs were found to be expressed in HEK-293 cells and in THP-1 monocytes, which express P2X7 receptors. Interestingly, the constitutive overexpression of any of the EMPs in HEK-293 cells led to cell blebbing, annexin V binding, and cell death, by a caspase-dependent pathway. These findings suggest that the P2X7 C-terminal domain associates with EMPs, and this interaction may mediate some aspects of the downstream signaling following P2X7 receptor activation. The binding of extracellular ATP to the P2X7 receptor opens an integral cation-permeable channel; it also leads to membrane blebbing and, in certain immune cells, interleukin-1β secretion and eventual death. The latter three effects are unique to the P2X7 receptor; also unique among P2X receptors is the long intracellular C terminus of the protein. We have shown that the C-terminal domain of the P2X7receptor is responsible for the cell blebbing phenotype. A screen for proteins that associate with the C-terminal domain of the P2X7 receptor and might mediate the blebbing phenotype, identified epithelial membrane protein 2 (EMP-2). The interaction between EMP-2 and P2X7 was confirmed biochemically by co-immunoprecipitation, co-purification, and glutathioneS-transferase pull-down assays, and this interaction was entirely dependent on the C-terminal domain of P2X7. The P2X7 receptor also interacted with the other members of the epithelial membrane protein family (EMP-1, EMP-3, and PMP-22). All four EMPs were found to be expressed in HEK-293 cells and in THP-1 monocytes, which express P2X7 receptors. Interestingly, the constitutive overexpression of any of the EMPs in HEK-293 cells led to cell blebbing, annexin V binding, and cell death, by a caspase-dependent pathway. These findings suggest that the P2X7 C-terminal domain associates with EMPs, and this interaction may mediate some aspects of the downstream signaling following P2X7 receptor activation. epithelial membrane protein glutathione S-transferase phosphate-buffered saline wild type lactose dehydrogenase reverse transcriptase benzyloxycarbonyl P2X7 receptors belong to a family of ion channels gated by extracellular ATP (1North R.A. Barnard E.A. Curr. Opin. Neurobiol. 1997; 7: 346-357Crossref PubMed Scopus (426) Google Scholar), all with the same predicted topology of two transmembrane domains and intracellular N and C termini (1North R.A. Barnard E.A. Curr. Opin. Neurobiol. 1997; 7: 346-357Crossref PubMed Scopus (426) Google Scholar). The P2X7 receptor shares 40–45% amino acid identity with the other P2X proteins, but it is structurally distinct at the C terminus, extending for an additional 100–200 amino acids (2Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1487) Google Scholar). P2X1–6 receptors are widely distributed in both neuronal and non-neuronal cells, whereas P2X7 receptors are most highly expressed in immune and epithelial cells (3Collo G. North R.A. Kawashima E. Merlo-Pich E. Neidhart S. Surprenant A. Buell G. J. Neurosci. 1996; 16: 2495-2507Crossref PubMed Google Scholar, 4Collo G. Neidhart S. Kawashima E. Kosco-Vilbois M. North R.A. Buell G. Neuropharmacology. 1997; 36: 1277-1283Crossref PubMed Scopus (438) Google Scholar). Brief stimulation (10–30 s) of the P2X7 receptor leads to the formation of a channel permeable to large cations, as is also seen for some of the other P2X receptors (2Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1487) Google Scholar, 5Virginio C. MacKenzie A.B. North R.A. Surprenant A. J. Physiol. 1999; 519: 335-346Crossref PubMed Scopus (323) Google Scholar). However, more prolonged activation of the P2X7 receptor results in extensive membrane blebbing from within seconds to minutes (5Virginio C. MacKenzie A.B. North R.A. Surprenant A. J. Physiol. 1999; 519: 335-346Crossref PubMed Scopus (323) Google Scholar), and eventual cell death (5Virginio C. MacKenzie A.B. North R.A. Surprenant A. J. Physiol. 1999; 519: 335-346Crossref PubMed Scopus (323) Google Scholar, 6Di Virgilio F. Chiozzi P. Falzoni S. Ferrari D. Sanz J.M. Venketaraman V. Baricordi O.R. Cell Death Differ. 1998; 5: 191-199Crossref PubMed Scopus (225) Google Scholar, 7Dubyak G.R. J. Auton. Nerv. Syst. 2000; 81: 64-68Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), although such responses are not observed with other P2X receptors. We hypothesized that the C terminus of the P2X7receptor might engage other cellular proteins to mediate its distinct responses of membrane blebbing and cell death. We tested this hypothesis by using a construct containing the soluble C-terminal domain (Asn356–Tyr595) of the rat P2X7 protein as bait in a yeast two-hybrid screen. Because HEK-293 cells expressing P2X7 receptors exhibit pronounced blebbing and eventual cell death when stimulated with ATP (5Virginio C. MacKenzie A.B. North R.A. Surprenant A. J. Physiol. 1999; 519: 335-346Crossref PubMed Scopus (323) Google Scholar),we sought interacting proteins using a HEK-293 cell library.RESULTSPrevious studies on the P2X7 receptor have shown that truncation of the receptor by removal of most of the intracellular C terminus reduces YOPRO-1 dye uptake and channel dilatation (2Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1487) Google Scholar). We have now verified that the C terminus is required for the P2X7receptor-mediated cell blebbing. In HEK-293 cells expressing the wild-type (WT) rP2X7 receptor, cells underwent membrane disruption within 1 min of activation with 100 μm Bz-ATP, whereas cells transfected with the truncated (Δ418) rP2X7receptor did not show any significant membrane disruption over a 4-min stimulation with 100 μm Bz-ATP (Fig.1, A–C). Both the WT rP2X7 receptor and the truncated receptor were expressed on the cell surface as determined by immunocytochemistry (Fig.1A). In agreement with previous electrophysiological recordings (2Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1487) Google Scholar), the Δ418 truncated rP2X7 receptor was shown to form a functional channel, because cells loaded with the calcium indicator Fluo-4AM displayed a prolonged Ca2+ rise after stimulation with 100 μm Bz-ATP (Fig.1B). Therefore, the P2X7 receptor C terminus is required for ATP-mediated cell blebbing. For this reason we used this intracellular domain to probe for interacting proteins, which might mediate this response.EMP-2 was identified as a strong positive hit in the yeast two-hybrid screen of a HEK-293 cell cDNA library with the P2X7receptor C terminus (P2X7CT). The interaction was confirmed by isolation of the EMP-2 Gal-4 activation domain fusion plasmid, retransformation into Y190 cells and assaying for growth on His− plates, and β-galactosidase activity. The interaction between full-length P2X7 receptor and EMP-2 was further tested using biochemical approaches. EMP-2myc/His was cotransfected with the rP2X7 receptor into HEK-293 cells; P2X7 receptor was copurified from cell extracts with EMP-2 attached to a cobalt affinity resin (Fig.2A, lane 5). In the absence of EMP-2myc/His, only a low background level of rP2X7 bound the cobalt affinity resin (Fig. 2A,lane 4). A complementary experiment was performed whereby EMP-2myc/His was co-immunoprecipitated with P2X7EE using an EE monoclonal antibody (Fig.2B). No EMP-2 was immunoprecipitated in the absence of P2X7EE (Fig. 2B, lane 2), nor did EMP-2 co-immunoprecipitate with an EE-tagged mRNA export factor, Ref 2–1EE (Fig. 2B, lane 3).Figure 2EMP-2 copurifies and co-immunoprecipitates with P2X7 receptor.A, cotransfection of EMP-2myc/His with P2X7 in HEK-293 cells followed by purification of EMP-2 using a cobalt-affinity resin resulted in the copurification of P2X7 (lane 5). Cells expressing only P2X7 receptors bound negligibly to the resin (lane 4). B, immunopreciptation of cell extracts expressing P2X7EE or Ref 2–1EE with EMP-2myc/His with anti-EE results in coprecipitation of EMP-2 with P2X7 (lane 4) but not with Ref 2–1EE (lane 3). EMP-2 was detected by immunoblotting with the c-Myc antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)EMP-2 is a member of a small family of epithelial membrane proteins, which also includes PMP-22, EMP-1, and EMP-3. RT-PCR using primers specific for each family member on extracts of HEK-293 cells indicated the presence of all four mRNAs (Fig.3A). Contamination by genomic DNA is unlikely because primers to β-actin (that span a short intron) gave a product of the expected size of the cDNA, and all EMP primers were chosen to span introns in the genomic sequences. RT-PCR products were also confirmed by sequencing. The RT-PCR was repeated on extracts from THP-1 monocytes, and mRNAs encoding all four EMP members were detected (Fig. 3B). Each of the family members (Myc/His-tagged) were co-expressed in HEK-293 cells with the P2X7 receptor. The P2X7 receptor copurified with each of the His-tagged proteins on the cobalt affinity resin (Fig.3C).Figure 3EMPs are expressed by HEK-293 cells and THP-1 monocytes and interact with P2X7 receptor.A, message for PMP-22, EMP-1, EMP-2, and EMP-3 was detected by RT-PCR of HEK-293 mRNA. The product size (241 bp) with actin primers is predicted from mRNA; amplification of genomic DNA would give a product of 400 bp. Other product sizes were as expected. B, message for PMP-22, EMP-1, EMP-2, and EMP-3 was detected by RT-PCR of THP-1 mRNA, using the same methods as for A. C, overexpression of four EMPs (with myc/His tags) with P2X7 in HEK-293 cells was followed by purification using a cobalt-affinity resin to bind the His tag. This resulted in the copurification of P2X7 with EMP-2, EMP-1, EMP-3, and PMP-22.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We also sought to confirm directly an interaction between the C terminus of the P2X7 receptor and the EMPs. Extracts were prepared from HEK-293 cells transfected with PMP-22, EMP-1, EMP-2, EMP-3, and Ref-2–1 (each Myc/His-tagged). Expression of the proteins was confirmed by direct analysis of samples of the extracts by immunoblotting with the Myc antibody (Fig.4A). Several bands were observed for the Myc-tagged EMPs because they exhibit different glycosylation states (12Ryan M.C. Notterpek L. Tobler A.R. Liu N. Shooter E.M. J. Neurochem. 2000; 75: 1465-1474Crossref PubMed Scopus (26) Google Scholar). Extracts were incubated either with GST (control) or GST-P2X7CT. The PMP-22, EMP-1, EMP-2, and EMP-3 proteins were pulled down by GST-P2X7CT although not by GST alone (Fig. 4B). No pull-down of any Myc-labeled products in the presence of GST-P2X7CT was observed using control extracts of HEK-293 mock-transfected or Ref 2–1myc-transfected cells. A deletion mutant of the P2X7 receptor lacking the C terminus (to residue N356, ΔCT-P2X7EE) was not capable of co-immunoprecipitating EMP-2 (Fig. 4C). The truncated receptor was expressed at the cell surface in a similar pattern to the WT P2X7 receptor (Fig. 4D). This indicates that EMPs specifically interact with the C terminus of the P2X7 receptor.Figure 4EMPs interact with the P2X7receptor C terminus.A, expression of EMPs or Ref 2–1myc in crude HEK-293 cell extracts before GST pull-downs was verified by immunoblotting with anti-Myc. B, these cell extracts were then reacted with GST or GST-P2X7CT bound to glutathione–Sepharose. Specific pull-down of PMP-22 and EMP 1,2, and 3 with GST–P2X7CT, compared with GST alone, was detected by c-Myc immunoblotting, whereas no pull-down was obtained for Ref 2–1myc. C, immunopreciptation of cell extracts expressing P2X7EE, or truncated P2X7EE (truncated to residue 356: ΔCTP2X7EE), or Ref 2–1EE with EMP-2myc/His, using anti-EE, results in coprecipitation of EMP-2 with P2X7 (lane 2) but not with the truncated receptor ΔCTP2X7EE (lane 3) nor with Ref 2–1EE (lane 4). EMP-2 was detected by immunoblotting with the c-Myc antibody. D, the truncated receptor, ΔCTP2X7EE, was expressed in a similar pattern to the WT receptor (P2X7EE), as assessed by immunocytochemistry using anti-EE.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Overexpression of PMP-22 and EMP-2 in NIH-3T3 cells has previously been shown to cause cell rounding, blebbing, and a reduction in survival rate (13Fabbretti E. Edomi P. Brancolini C. Schneider C. Genes Dev. 1995; 9: 1846-1856Crossref PubMed Scopus (157) Google Scholar, 14Brancolini C. Marzinotto S. Edomi P. Agostoni E. Fiorentini C. Muller H.W. Schneider C. Mol. Biol. Cell. 1999; 10: 2441-2459Crossref PubMed Scopus (66) Google Scholar, 15Wang C.X. Wadehra M. Fisk B.C. Goodglick L. Braun J. Blood. 2001; 97: 3890-3895Crossref PubMed Scopus (45) Google Scholar). We therefore sought to determine whether overexpression of all four members of the family mediate similar effects in HEK-293 cells. Overexpression of each protein led to a significant increase in cell death. A significantly higher release of LDH occurred in EMP-transfected cells after 36 h of treatment in low serum (Fig.5A). In addition, an increase in trypan blue uptake was observed as determined by cell counting (Fig.5B), compared with control mock-transfected cells also maintained in low serum. Fluorescence-activated cell sorter analysis of HEK-293 cells 48 h after transfection showed that each member of the EMP family resulted in a significant proportion of annexin-positive cells (Fig. 5, C and D). Mock-transfected cells and cells transfected with P2X2receptor cDNAs showed no significant annexin binding. The high percentage of propidium-positive cells in the mock-transfected control (Fig. 5C) is likely caused by the addition of transfection reagent. The LDH release and trypan blue uptake in EMP-overexpressing cells was significantly inhibited by the addition of the caspase inhibitor, z-VAD (Fig. 5, A and B).Figure 5Overexpression of epithelial membrane proteins induces cell death. Plasmids encoding each epithelial membrane protein were separately transfected into HEK-293 cells. At 12 h after transfection, cells were incubated in 1% serum supplemented with either 0.5% Me2SO or 30 μmz-VAD in 0.5% Me2SO. A, after 36 h, a sample of supernatant was withdrawn and assayed for LDH content (Sigma kit). An equivalent HEK-293 cell sample was treated with 1% Triton X-100 and assayed for LDH content, which was taken as the 100% maximal release. B, at 48 h after treatment, cells were harvested and viable vs. nonviable cells were assayed by trypan blue exclusion. C, HEK-293 cells overexpressing each EMP family member were tested for annexin V binding by flow cytometry, using propidium iodide (PI) as a cell-dead stain. For each sample, a cell count of 2,500 cells was taken, and cells were gated into four quadrants where Q1 is the proportion of nonviable (PI-positive), non-annexin V-bound cells; Q2 is PI-positive non-viable annexin V-positive cells; Q3 is PI-negative (viable) annexin V-negative cells; and Q4 represents annexin V-positive viable (PI-negative) cells. Typical flow cytometry traces are shown for mock-transfected, staurosporine-treated, and EMP-2 overexpressing HEK-293 cells. D, annexin V binding measured in the Q4 gating. Each column shows the percentage of annexin-positive/propidium-negative cells. In each experiment, 100% was assigned to the number of annexin-positive/propidium-negative cells observed with staurosporin (1 μm) treatment (n = 4 in each case).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Immunocytochemistry of HEK-293 cells transfected with each of the EMP cDNAs (Myc/His tagged) showed predominant staining at the cell surface (Fig. 6). All cells transfected with PMP-22, EMP-1, EMP-2, or EMP-3 exhibited several large membrane blebs on their surfaces (Fig. 6).Figure 6Overexpression of EMPs induces cell blebbing. Blebbing of the membrane was observed 48 h after HEK-293 cells were transfected with each of the EMPs. Immunocytochemistry was performed using the anti-Myc antibody to detect Myc-tagged EMPs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONThe P2X7 receptor is functionally distinct compared with the other members of the ATP-gated ion channel family because its activation leads to membrane blebbing (5Virginio C. MacKenzie A.B. North R.A. Surprenant A. J. Physiol. 1999; 519: 335-346Crossref PubMed Scopus (323) Google Scholar). Here we have demonstrated that the unique intracellular C terminus of the P2X7receptor is required for this response. The removal of the C-terminal domain of P2X7 also greatly reduces the uptake of dyes such as YOPRO-1, but it does not grossly alter the ATP-gated cation channel function (2Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1487) Google Scholar).The search for proteins that interact with the C-terminal domain of P2X7, and may be involved in the membrane-blebbing phenotype, led to the identification of EMP-2. EMP-2 consists of 167 amino acids containing four predicted transmembrane domains (16Taylor V. Suter U. Gene. 1996; 175: 115-120Crossref PubMed Scopus (87) Google Scholar) and exhibits 40% amino acid identity to PMP-22. In addition, two other related proteins have been identified by random sequencing of a mouse intestine library (EMP-1 (17Lobsiger C.S. Magyar J.P. Taylor V. Wulf P. Welcher A.A. Program A.E. Suter U. Genomics. 1996; 36: 379-387Crossref PubMed Scopus (39) Google Scholar)) and from related expressed sequence tag (EST) sequences (EMP-3 (16Taylor V. Suter U. Gene. 1996; 175: 115-120Crossref PubMed Scopus (87) Google Scholar)). Direct interaction of EMP-2 and the C terminus of the P2X7 receptor was verified by using biochemical approaches—copurification, coimmunoprecipitation, and GST pull-down assays. In addition, the related proteins EMP-1, EMP-3, and PMP-22 were also found to interact with the P2X7CT using these same techniques. Notably these interactions did not depend on activation of the P2X7receptor. Consistent with a role in the membrane-blebbing phenotype of P2X7 and not its channel functions, overexpression of EMP-2 in HEK-293 cells had no significant effect on P2X7-activated cation currents, YOPRO-1 uptake, or a significant shift in the EC50 value for Bz-ATP (data not shown).P2X7 has previously been found to associate with a multiprotein complex including cytoskeletal proteins, heat shock proteins, and two integral membrane proteins, receptor-phosphotyrosine phosphatase-β (RPTPβ) and integrin β2, although the domains of the P2X7 receptor responsible for most of these interactions have not yet been determined (18Kim M. Jiang L.H. Wilson H.L. North R.A. Surprenant A. EMBO J. 2001; 20: 6347-6358Crossref PubMed Scopus (334) Google Scholar). The C terminus of the P2X7 receptor is generally thought to be entirely intracellular, whereas EMPs have a predominantly intramembrane disposition with only a small part of the protein likely to be intracellular (amino acids 86–94 in EMP-2), although this domain may be sufficient for the observed interactions. Alternatively, there is a significantly hydrophobic segment in the P2X7 receptor C terminus (amino acids 512–536), raising the possibility that the main interaction occurs between hydrophobic regions within the membrane.The EMPs share 22–25% similarity to the “stargazin” or voltage-gated channel ã subunits, with common exon–intron boundaries and alignment of the four transmembrane domains (Fig.7). This is of interest, as the γ subunits (γ1–γ8) have been shown to interact with neuronal voltage-dependent calcium channels (19Kang M.-G. Chen C.-C. Felix R. Letts V.A. Frankel W.N. Mori Y. Campbell K.P. J. Biol. Chem. 2001; 276: 32917-32924Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 20Moss F.J. Viard P. Davies A. Bertaso F. Page K.M. Graham A. Canti C. Plumpton M. Plumpton C. Clare J.J. Dolphin A.C. EMBO J. 2002; 21: 1514-1523Crossref PubMed Scopus (69) Google Scholar), indicating that the P2X7 receptor–EMP interaction is not unique, and a number of ion channels may form complexes with tetra-span membrane proteins.Figure 7EMPs share homology to the γ subunits of voltage-activated Ca2+ channels. Alignment of the human five exon γ5 and γ7 to human PMP-22, EMP-1, EMP-2, and EMP-3 using the ClustalW algorithm. Bars above the sequence represent predicted transmembrane spanning domains; solid triangles (above sequence) show exon-intron boundaries for PMP-22, EMP-1, EMP-2, and EMP-3; open triangles(below sequence) show exon–intron boundaries for γ5 and γ7 subunits. The lower line provides the consensus sequence for the alignment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The normal physiological role of EMPs is not well understood. Genetic studies and the generation of PMP-22-deficient mice have established that it is responsible for a set of inherited peripheral neuropathies in mice and humans. One type of Charcot-Marie Tooth disease (CMT1A) results from a mutation in PMP-22 (21Valentijn L.J. Baas F. Wolterman R.A. Hoogendijk J.E. van den Bosch N.H. Zorn I. Gabreels-Festen A.W. de Visser M. Bolhuis P.A. Nat. Genet. 1992; 2: 288-291Crossref PubMed Scopus (403) Google Scholar), and Dejerine-Sottas syndrome, which also involves impaired nerve conductions, is a product of a deletion within the PMP-22 gene (22Chance P.F. Alderson M.K. Leppig K.A. Lensch M.W. Matsunami N. Smith B. Swanson P.D. Odelberg S.J. Disteche C.M. Bird T.D. Cell. 1993; 72: 143-151Abstract Full Text PDF PubMed Scopus (716) Google Scholar). In rodents, point mutations of PMP-22 are associated with the “Trembler” phenotype due to impaired conduction in peripheral nerves (23Suter U. Welcher A.A. Ozcelik T. Snipes G.J. Kosaras B. Francke U. Billings Gagliardi S. Sidman R.L. Shooter E.M. Nature. 1992; 356: 241-244Crossref PubMed Scopus (387) Google Scholar). However, the wide tissue distribution of PMP-22 suggests that it may play a more general role than in myelin formation. PMP-22 was originally isolated from NIH-3T3 cells and was first named gas-3 for its growth arrest-dependent expression (24Schneider C. King R.M. Philipson L. Cell. 1988; 54: 787-793Abstract Full Text PDF PubMed Scopus (800) Google Scholar). Northern analysis has shown that each member of the EMP family, including PMP-22, is widely expressed (16Taylor V. Suter U. Gene. 1996; 175: 115-120Crossref PubMed Scopus (87) Google Scholar, 24Schneider C. King R.M. Philipson L. Cell. 1988; 54: 787-793Abstract Full Text PDF PubMed Scopus (800) Google Scholar), and there is considerable overlap with the tissue distribution of P2X7 receptors. For example, many epithelial cells express both EMPs and P2X7 receptors (4Collo G. Neidhart S. Kawashima E. Kosco-Vilbois M. North R.A. Buell G. Neuropharmacology. 1997; 36: 1277-1283Crossref PubMed Scopus (438) Google Scholar). PMP-22 is highly expressed in Schwann cells (25Spreyer P. Kuhn G. Hanemann C.O. Gillen C. Schaal H. Kuhn R. Lemke G. Muller H.W. EMBO J. 1991; 10: 3661-3668Crossref PubMed Scopus (167) Google Scholar, 26Baechner D. Liehr T. Hameister H. Altenberger H. Grehl H. Suter U. Rautenstrauss B. J. Neurosci. Res. 1995; 42: 733-741Crossref PubMed Scopus (102) Google Scholar), where there is also good functional evidence for P2X7 receptors (27Grafe P. Mayer C. Takigawa T. Kamleiter M. Sanchez-Brandelik R. J. Physiol. 1999; 515: 377-383Crossref PubMed Scopus (44) Google Scholar,28Colomar A. Amedee T. Eur. J. Neurosci. 2001; 14: 927-936Crossref PubMed Google Scholar).The RT-PCR analysis confirmed that mRNAs encoding all members of the EMP family are present in both HEK-293 cells and in THP-1 monocytes. P2X7 receptors are strongly expressed in THP-1 monocytes, and their activation is an important trigger for interleukin-1β release (29MacKenzie A. Wilson H.L. Kiss-Toth E. Dower S.K. North R.A. Surprenant A. Immunity. 2001; 15: 825-835Abstract Full Text Full Text PDF PubMed Scopus (700) Google Scholar). After prolonged stimulation, P2X7 receptors mediate ATP-induced killing in human macrophages (30Kusner D.J. Adams J. J. Immunol. 2000; 164: 379-388Crossref PubMed Scopus (147) Google Scholar, 31Humphreys B.D. Rice J. Kertesy S.B. Dubyak G.R. J. Biol. Chem. 2000; 275: 26792-26798Abstract Full Text Full Text PDF PubMed Google Scholar). In NIH-3T3 cells, overexpression of PMP-22 also causes cell rounding, blebbing, and a reduction in the survival rate (13Fabbretti E. Edomi P. Brancolini C. Schneider C. Genes Dev. 1995; 9: 1846-1856Crossref PubMed Scopus (157) Google Scholar, 14Brancolini C. Marzinotto S. Edomi P. Agostoni E. Fiorentini C. Muller H.W. Schneider C. Mol. Biol. Cell. 1999; 10: 2441-2459Crossref PubMed Scopus (66) Google Scholar), and recently bleb formation has been reported following overexpression of EMP-2 (15Wang C.X. Wadehra M. Fisk B.C. Goodglick L. Braun J. Blood. 2001; 97: 3890-3895Crossref PubMed Scopus (45) Google Scholar). We have now extended these observations, and shown that membrane blebbing is induced and cell survival is decreased when any one of the EMPs is overexpressed in HEK-293 cells, as evidenced by increased LDH release, trypan blue uptake, annexin V binding, and immunofluorescence. The cell death could be inhibited by the addition of the caspase inhibitor, z-VAD, indicating that overexpression of EMP-1, EMP-2, EMP-3, or PMP-22 results in a caspase-dependent apoptotic-like phenotype.Although overexpression of EMPs leads to constitutive cell blebbing, the normal cellular trigger for an EMP to manifest this response is currently unknown. This study indicates that EMPs can associate with other integral membrane proteins such as the P2X7 receptor, which under the appropriate conditions (i.e. ATP stimulation), can provide that trigger and promote blebbing, although the mechanism by which the P2X7–EMP complex could promote cell blebbing is not understood at present. Because EMPs are widely expressed, even in cells lacking P2X7, this would suggest they may have additional integral membrane protein partners that link EMP-like proteins to apoptosis pathways. Identification of such proteins will be an important goal for the future together with understanding the mechanisms involved in the constitutive cell blebbing observed when EMP proteins are overexpressed. P2X7 receptors belong to a family of ion channels gated by extracellular ATP (1North R.A. Barnard E.A. Curr. Opin. Neurobiol. 1997; 7: 346-357Crossref PubMed Scopus (426) Google Scholar), all with the same predicted topology of two transmembrane domains and intracellular N and C termini (1North R.A. Barnard E.A. Curr. Opin. Neurobiol. 1997; 7: 346-357Crossref PubMed Scopus (426) Google Scholar). The P2X7 receptor shares 40–45% amino acid identity with the other P2X proteins, but it is structurally distinct at the C terminus, extending for an additional 100–200 amino acids (2Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1487) Google Scholar). P2X1–6 receptors are widely distributed in both neuronal and non-neuronal cells, whereas P2X7 receptors are most highly expressed in immune and epithelial cells (3Collo G. North R.A. Kawashima E. Merlo-Pich E. Neidhart S. Surprenant A. Buell G. J. Neurosci. 1996; 16: 2495-2507Crossref PubMed Google Scholar, 4Collo G. Neidhart S. Kawashima E. Kosco-Vilbois M. North R.A. Buell G. Neuropharmacology. 1997; 36: 1277-1283Crossref PubMed Scopus (438) Google Scholar). Brief stimulation (10–30 s) of the P2X7 receptor leads to the formation of a channel permeable to large cations, as is also seen for some of the other P2X receptors (2Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1487) Google Scholar, 5Virginio C. MacKenzie A.B. North R.A. Surprenant A. J. Physiol. 1999; 519: