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

Abstract

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 North R.A. Surprenant A. J. PubMed Scopus Google Scholar). of the P2X7 receptor in membrane blebbing to C. MacKenzie North R.A. Surprenant A. J. PubMed Scopus Google Scholar), and eventual cell C. MacKenzie North R.A. Surprenant A. J. PubMed Scopus Google Scholar, F. S. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar), are with other P2X receptors. We that the C terminus of the P2X7receptor might other proteins to mediate distinct of membrane blebbing and cell death. We this by a the C-terminal domain of the P2X7 protein as in a HEK-293 cells P2X7 receptors blebbing and eventual cell with ATP C. MacKenzie North R.A. Surprenant A. J. PubMed Scopus Google proteins a HEK-293 cell on the P2X7 receptor have shown that of the receptor by of most of the intracellular C terminus and channel (2Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1487) Google Scholar). We have that the C terminus is for the cell HEK-293 cells the cells membrane of with whereas cells with the any membrane a stimulation with the receptor and the receptor were expressed on the cell as by with (2Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1487) Google Scholar), the receptor was shown to a cells with the a stimulation with the P2X7 receptor C terminus is for cell this this intracellular domain to for proteins, which might mediate this was identified as a in the screen of a HEK-293 cell with the P2X7receptor C terminus The interaction was confirmed by of the EMP-2 domain cells and for on and The interaction between P2X7 receptor and EMP-2 was was with the receptor HEK-293 P2X7 receptor was cell with EMP-2 to a the of a of the A was was with an EMP-2 was in the of EMP-2 with an and with P2X7 of with P2X7 in HEK-293 cells by of EMP-2 a in the of P2X7 P2X7 receptors to the of cell with with in of EMP-2 with P2X7 but with EMP-2 was by with the is a of a family of epithelial membrane proteins, which also and for family on of HEK-293 cells the of all four by is to a a of the of the and all were to in the were also confirmed by The was on THP-1 monocytes, and all four members were of the family members were in HEK-293 cells with the P2X7 The P2X7 receptor with of the proteins on the are expressed by HEK-293 cells and THP-1 and with P2X7 for and was by of HEK-293 The with is predicted of a of were as for and was by of THP-1 the same as for A. overexpression of four EMPs with P2X7 in HEK-293 cells was by a to the in the of P2X7 with EMP-3, and also to an interaction between the C terminus of the P2X7 receptor and the were HEK-293 cells with EMP-3, and of the proteins was confirmed by of of the by with the were for the EMPs J. PubMed Scopus Google Scholar). were with The and proteins were by by pull-down of any in the of was of HEK-293 A of the P2X7 receptor the C terminus was of EMP-2 The receptor was expressed at the cell in a to the P2X7 receptor that EMPs with the C terminus of the P2X7 with the P2X7receptor C of EMPs in HEK-293 cell was by with cell were with to pull-down of and and with with was by whereas pull-down was for of cell to with in of EMP-2 with P2X7 but with the receptor with EMP-2 was by with the the was expressed in a to the receptor as by of and EMP-2 in cells shown to cell blebbing, and a in E. C. C. PubMed Scopus Google Scholar, C. S. E. C. C. PubMed Scopus Google Scholar, M. J. PubMed Scopus Google Scholar). We to overexpression of all four members of the family mediate effects in HEK-293 of protein led to a in cell death. A of in cells of in an in was as by cell with cells also in cell of HEK-293 cells that of the family in a of cells C and cells and cells with annexin The of cells in the is by the of The and in cells was by the of the A and of epithelial membrane proteins cell death. epithelial membrane protein were HEK-293 cells were in with in a of was and for HEK-293 cell was with and for which was as the at cells were and cells were by HEK-293 cells family were for annexin V binding by as a a cell of cells was and cells were gated four is the of is annexin is annexin and annexin are shown for and EMP-2 HEK-293 annexin V binding in the the of was to the of cells with in of HEK-293 cells with of the at the cell All cells with large membrane on of EMPs cell of the membrane was HEK-293 cells were with of the was the to P2X7 receptor is distinct with the other members of the ion channel family leads to membrane blebbing C. MacKenzie North R.A. Surprenant A. J. PubMed Scopus Google Scholar). have that the unique intracellular C terminus of the P2X7receptor is for this The of the C-terminal domain of P2X7 also the of as but it the channel (2Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1487) Google for proteins that with the C-terminal domain of and may be in the phenotype, led to the of EMP-2 of amino acids four predicted transmembrane domains 1996; PubMed Scopus Google and amino acid identity to two other proteins have identified by of a 1996; 36: PubMed Scopus Google and expressed 1996; PubMed Scopus Google interaction of EMP-2 and the C terminus of the P2X7 receptor was by and pull-down the proteins EMP-3, and were also found to with the same on of the with a in the of P2X7 and channel overexpression of EMP-2 in HEK-293 cells on a in the for found to associate with a proteins, proteins, and two integral membrane proteins, and the domains of the P2X7 receptor responsible for most of have M. North R.A. Surprenant A. J. PubMed Scopus Google Scholar). The C terminus of the P2X7 receptor is to be entirely whereas EMPs have a with a of the protein to be intracellular acids in this domain may be for the is a in the P2X7 receptor C terminus acids the that the interaction between the EMPs to the channel with and of the four transmembrane domains is of as the have shown to with neuronal channels J. PubMed Scopus Google Scholar, A. F. A. C. M. C. J. PubMed Scopus Google Scholar), that the P2X7 interaction is and a of ion channels may with membrane to the of of the and to and the the predicted transmembrane for and for and The the for the of EMPs is and the of have that it is responsible for a of in and type of a in F. R.A. M. PubMed Scopus Google Scholar), and which also is a of a the PubMed Scopus Google Scholar). of are with the to in S. PubMed Scopus Google Scholar). the of that it may a in was cells and was for C. PubMed Scopus Google Scholar). shown that of the is widely expressed 1996; PubMed Scopus Google Scholar, C. PubMed Scopus Google Scholar), and is with the of P2X7 receptors. epithelial cells express both EMPs and P2X7 receptors G. Neidhart S. Kawashima E. Kosco-Vilbois M. North R.A. Buell G. Neuropharmacology. 1997; 36: 1277-1283Crossref PubMed Scopus (438) Google Scholar). is highly expressed in cells G. C. G. J. PubMed Scopus Google Scholar, J. Neurosci. PubMed Scopus Google Scholar), is also for P2X7 receptors C. M. J. PubMed Scopus Google A. J. Neurosci. PubMed Google confirmed that all members of the family are in both HEK-293 cells and in THP-1 P2X7 receptors are expressed in THP-1 monocytes, and is an for interleukin-1β A. E. North R.A. Surprenant A. PubMed Scopus Google Scholar). P2X7 receptors mediate in J. J. PubMed Scopus Google Scholar, J. J. PubMed Google Scholar). cells, overexpression of also cell blebbing, and a in the E. C. C. PubMed Scopus Google Scholar, C. S. E. C. C. PubMed Scopus Google Scholar), and formation following overexpression of EMP-2 M. J. PubMed Scopus Google Scholar). We have and shown that membrane blebbing is and cell is any of the EMPs is in HEK-293 cells, as by annexin V binding, and The cell be by the of the that overexpression of EMP-3, in a caspase-dependent overexpression of EMPs leads to constitutive cell blebbing, the for an to this is that EMPs associate with other integral membrane proteins as the P2X7 which the ATP that and blebbing, the by which the cell blebbing is at EMPs are widely in cells this suggest may have additional integral membrane protein that proteins to of proteins be an for the with the in the constitutive cell blebbing proteins are 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 North R.A. Surprenant A. J. PubMed Scopus Google Scholar). of the P2X7 receptor in membrane blebbing to C. MacKenzie North R.A. Surprenant A. J. PubMed Scopus Google Scholar), and eventual cell C. MacKenzie North R.A. Surprenant A. J. PubMed Scopus Google Scholar, F. S. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar), are with other P2X receptors. We that the C terminus of the P2X7receptor might other proteins to mediate distinct of membrane blebbing and cell death. We this by a the C-terminal domain of the P2X7 protein as in a HEK-293 cells P2X7 receptors blebbing and eventual cell with ATP C. MacKenzie North R.A. Surprenant A. J. PubMed Scopus Google proteins a HEK-293 cell on the P2X7 receptor have shown that of the receptor by of most of the intracellular C terminus and channel (2Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1487) Google Scholar). We have that the C terminus is for the cell HEK-293 cells the cells membrane of with whereas cells with the any membrane a stimulation with the receptor and the receptor were expressed on the cell as by with (2Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1487) Google Scholar), the receptor was shown to a cells with the a stimulation with the P2X7 receptor C terminus is for cell this this intracellular domain to for proteins, which might mediate this was identified as a in the screen of a HEK-293 cell with the P2X7receptor C terminus The interaction was confirmed by of the EMP-2 domain cells and for on and The interaction between P2X7 receptor and EMP-2 was was with the receptor HEK-293 P2X7 receptor was cell with EMP-2 to a the of a of the A was was with an EMP-2 was in the of EMP-2 with an is a of a family of epithelial membrane proteins, which also and for family on of HEK-293 cells the of all four by is to a a of the of the and all were to in the were also confirmed by The was on THP-1 monocytes, and all four members were of the family members were in HEK-293 cells with the P2X7 The P2X7 receptor with of the proteins on the are expressed by HEK-293 cells and THP-1 and with P2X7 for and was by of HEK-293 The with is predicted of a of were as for and was by of THP-1 the same as for A. overexpression of four EMPs with P2X7 in HEK-293 cells was by a to the in the of P2X7 with EMP-3, and also to an interaction between the C terminus of the P2X7 receptor and the were HEK-293 cells with EMP-3, and of the proteins was confirmed by of of the by with the were for the EMPs J. PubMed Scopus Google Scholar). were with The and proteins were by by pull-down of any in the of was of HEK-293 A of the P2X7 receptor the C terminus was of EMP-2 The receptor was expressed at the cell in a to the P2X7 receptor that EMPs with the C terminus of the P2X7 with the P2X7receptor C of EMPs in HEK-293 cell was by with cell were with to pull-down of and and with with was by whereas pull-down was for of cell to with in of EMP-2 with P2X7 but with the receptor with EMP-2 was by with the the was expressed in a to the receptor as by of and EMP-2 in cells shown to cell blebbing, and a in E. C. C. PubMed Scopus Google Scholar, C. S. E. C. C. PubMed Scopus Google Scholar, M. J. PubMed Scopus Google Scholar). We to overexpression of all four members of the family mediate effects in HEK-293 of protein led to a in cell death. A of in cells of in an in was as by cell with cells also in cell of HEK-293 cells that of the family in a of cells C and cells and cells with annexin The of cells in the is by the of The and in cells was by the of the A and of epithelial membrane proteins cell death. epithelial membrane protein were HEK-293 cells were in with in a of was and for HEK-293 cell was with and for which was as the at cells were and cells were by HEK-293 cells family were for annexin V binding by as a a cell of cells was and cells were gated four is the of is annexin is annexin and annexin are shown for and EMP-2 HEK-293 annexin V binding in the the of was to the of cells with in of HEK-293 cells with of the at the cell All cells with large membrane on of EMPs cell of the membrane was HEK-293 cells were with of the was the to on the P2X7 receptor have shown that of the receptor by of most of the intracellular C terminus and channel (2Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1487) Google Scholar). We have that the C terminus is for the cell HEK-293 cells the cells membrane of with whereas cells with the any membrane a stimulation with the receptor and the receptor were expressed on the cell as by with (2Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1487) Google Scholar), the receptor was shown to a cells with the a stimulation with the P2X7 receptor C terminus is for cell this this intracellular domain to for proteins, which might mediate this EMP-2 was identified as a in the screen of a HEK-293 cell with the P2X7receptor C terminus The interaction was confirmed by of the EMP-2 domain cells and for on and The interaction between P2X7 receptor and EMP-2 was was with the receptor HEK-293 P2X7 receptor was cell with EMP-2 to a the of a of the A was was with an EMP-2 was in the of EMP-2 with an EMP-2 is a of a family of epithelial membrane proteins, which also and for family on of HEK-293 cells the of all four by is to a a of the of the and all were to in the were also confirmed by The was on THP-1 monocytes, and all four members were of the family members were in HEK-293 cells with the P2X7 The P2X7 receptor with of the proteins on the We also to an interaction between the C terminus of the P2X7 receptor and the were HEK-293 cells with EMP-3, and of the proteins was confirmed by of of the by with the were for the EMPs J. PubMed Scopus Google Scholar). were with The and proteins were by by pull-down of any in the of was of HEK-293 A of the P2X7 receptor the C terminus was of EMP-2 The receptor was expressed at the cell in a to the P2X7 receptor that EMPs with the C terminus of the P2X7 of and EMP-2 in cells shown to cell blebbing, and a in E. C. C. PubMed Scopus Google Scholar, C. S. E. C. C. PubMed Scopus Google Scholar, M. J. PubMed Scopus Google Scholar). We to overexpression of all four members of the family mediate effects in HEK-293 of protein led to a in cell death. A of in cells of in an in was as by cell with cells also in cell of HEK-293 cells that of the family in a of cells C and cells and cells with annexin The of cells in the is by the of The and in cells was by the of the A and of HEK-293 cells with of the at the cell All cells with large membrane on P2X7 receptor is distinct with the other members of the ion channel family leads to membrane blebbing C. MacKenzie North R.A. Surprenant A. J. PubMed Scopus Google Scholar). have that the unique intracellular C terminus of the P2X7receptor is for this The of the C-terminal domain of P2X7 also the of as but it the channel (2Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1487) Google for proteins that with the C-terminal domain of and may be in the phenotype, led to the of EMP-2 of amino acids four predicted transmembrane domains 1996; PubMed Scopus Google and amino acid identity to two other proteins have identified by of a 1996; 36: PubMed Scopus Google and expressed 1996; PubMed Scopus Google interaction of EMP-2 and the C terminus of the P2X7 receptor was by and pull-down the proteins EMP-3, and were also found to with the same on of the with a in the of P2X7 and channel overexpression of EMP-2 in HEK-293 cells on a in the for found to associate with a proteins, proteins, and two integral membrane proteins, and the domains of the P2X7 receptor responsible for most of have M. North R.A. Surprenant A. J. PubMed Scopus Google Scholar). The C terminus of the P2X7 receptor is to be entirely whereas EMPs have a with a of the protein to be intracellular acids in this domain may be for the is a in the P2X7 receptor C terminus acids the that the interaction between the EMPs to the channel with and of the four transmembrane domains is of as the have shown to with neuronal channels J. PubMed Scopus Google Scholar, A. F. A. C. M. C. J. PubMed Scopus Google Scholar), that the P2X7 interaction is and a of ion channels may with membrane of EMPs is and the of have that it is responsible for a of in and type of a in F. R.A. M. PubMed Scopus Google Scholar), and which also is a of a the PubMed Scopus Google Scholar). of are with the to in S. PubMed Scopus Google Scholar). the of that it may a in was cells and was for C. PubMed Scopus Google Scholar). shown that of the is widely expressed 1996; PubMed Scopus Google Scholar, C. PubMed Scopus Google Scholar), and is with the of P2X7 receptors. epithelial cells express both EMPs and P2X7 receptors G. Neidhart S. Kawashima E. Kosco-Vilbois M. North R.A. Buell G. Neuropharmacology. 1997; 36: 1277-1283Crossref PubMed Scopus (438) Google Scholar). is highly expressed in cells G. C. G. J. PubMed Scopus Google Scholar, J. Neurosci. PubMed Scopus Google Scholar), is also for P2X7 receptors C. M. J. PubMed Scopus Google A. J. Neurosci. PubMed Google confirmed that all members of the family are in both HEK-293 cells and in THP-1 P2X7 receptors are expressed in THP-1 monocytes, and is an for interleukin-1β A. E. North R.A. Surprenant A. PubMed Scopus Google Scholar). P2X7 receptors mediate in J. J. PubMed Scopus Google Scholar, J. J. PubMed Google Scholar). cells, overexpression of also cell blebbing, and a in the E. C. C. PubMed Scopus Google Scholar, C. S. E. C. C. PubMed Scopus Google Scholar), and formation following overexpression of EMP-2 M. J. PubMed Scopus Google Scholar). We have and shown that membrane blebbing is and cell is any of the EMPs is in HEK-293 cells, as by annexin V binding, and The cell be by the of the that overexpression of EMP-3, in a caspase-dependent overexpression of EMPs leads to constitutive cell blebbing, the for an to this is that EMPs associate with other integral membrane proteins as the P2X7 which the ATP that and blebbing, the by which the cell blebbing is at EMPs are widely in cells this suggest may have additional integral membrane protein that proteins to of proteins be an for the with the in the constitutive cell blebbing proteins are The P2X7 receptor is distinct with the other members of the ion channel family leads to membrane blebbing C. MacKenzie North R.A. Surprenant A. J. PubMed Scopus Google Scholar). have that the unique intracellular C terminus of the P2X7receptor is for this The of the C-terminal domain of P2X7 also the of as but it the channel (2Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1487) Google Scholar). The for proteins that with the C-terminal domain of and may be in the phenotype, led to the of EMP-2 of amino acids four predicted transmembrane domains 1996; PubMed Scopus Google and amino acid identity to two other proteins have identified by of a 1996; 36: PubMed Scopus Google and expressed 1996; PubMed Scopus Google interaction of EMP-2 and the C terminus of the P2X7 receptor was by and pull-down the proteins EMP-3, and were also found to with the same on of the with a in the of P2X7 and channel overexpression of EMP-2 in HEK-293 cells on a in the for P2X7 found to associate with a proteins, proteins, and two integral membrane proteins, and the domains of the P2X7 receptor responsible for most of have M. North R.A. Surprenant A. J. PubMed Scopus Google Scholar). The C terminus of the P2X7 receptor is to be entirely whereas EMPs have a with a of the protein to be intracellular acids in this domain may be for the is a in the P2X7 receptor C terminus acids the that the interaction between the The EMPs to the channel with and of the four transmembrane domains is of as the have shown to with neuronal channels J. PubMed Scopus Google Scholar, A. F. A. C. M. C. J. PubMed Scopus Google Scholar), that the P2X7 interaction is and a of ion channels may with membrane The of EMPs is and the of have that it is responsible for a of in and type of a in F. R.A. M. PubMed Scopus Google Scholar), and which also is a of a the PubMed Scopus Google Scholar). of are with the to in S. PubMed Scopus Google Scholar). the of that it may a in was cells and was for C. PubMed Scopus Google Scholar). shown that of the is widely expressed 1996; PubMed Scopus Google Scholar, C. PubMed Scopus Google Scholar), and is with the of P2X7 receptors. epithelial cells express both EMPs and P2X7 receptors G. Neidhart S. Kawashima E. Kosco-Vilbois M. North R.A. Buell G. Neuropharmacology. 1997; 36: 1277-1283Crossref PubMed Scopus (438) Google Scholar). is highly expressed in cells G. C. G. J. PubMed Scopus Google Scholar, J. Neurosci. PubMed Scopus Google Scholar), is also for P2X7 receptors C. M. J. PubMed Scopus Google A. J. Neurosci. PubMed Google Scholar). The confirmed that all members of the family are in both HEK-293 cells and in THP-1 P2X7 receptors are expressed in THP-1 monocytes, and is an for interleukin-1β A. E. North R.A. Surprenant A. PubMed Scopus Google Scholar). P2X7 receptors mediate in J. J. PubMed Scopus Google Scholar, J. J. PubMed Google Scholar). cells, overexpression of also cell blebbing, and a in the E. C. C. PubMed Scopus Google Scholar, C. S. E. C. C. PubMed Scopus Google Scholar), and formation following overexpression of EMP-2 M. J. PubMed Scopus Google Scholar). We have and shown that membrane blebbing is and cell is any of the EMPs is in HEK-293 cells, as by annexin V binding, and The cell be by the of the that overexpression of EMP-3, in a caspase-dependent phenotype. overexpression of EMPs leads to constitutive cell blebbing, the for an to this is that EMPs associate with other integral membrane proteins as the P2X7 which the ATP that and blebbing, the by which the cell blebbing is at EMPs are widely in cells this suggest may have additional integral membrane protein that proteins to of proteins be an for the with the in the constitutive cell blebbing proteins are We MacKenzie for for the for with cell and for