Analysis of Assembly and Trafficking of Native P2X4 and P2X7 Receptor Complexes in Rodent Immune Cells (2009)

P2X4 and P2X7 are the predominant P2X receptor subtypes expressed in immune cells. Having previously shown a structural and functional interaction between the two recombinant receptors, our aims here were to identify the preferred assembly pathway of the endogenous receptors in macrophage-like cells and to investigate the trafficking of these receptors between the plasma membrane and intracellular sites. We exploited the difference in size between the two subunits, and we used a combination of cross-linkers and blue native-PAGE analysis to investigate the subunit composition of complexes present in primary cultures of rat microglia and macrophages from wild type and P2X7–/– mice. Our results indicate that the preferred assembly pathway for both receptors is the formation of homotrimers. Homotrimers of P2X7 were able to co-immunoprecipitate with P2X4, suggesting that an interaction occurs between rather than within receptor complexes. In both macrophages and microglia, P2X7 receptors were predominantly at the cell surface, whereas P2X4 receptors were predominantly intracellular. There were clear cell type-dependent differences in the extent to which P2X4 receptors trafficked to and from the surface; trafficking was much more dynamic in microglia than in the macrophages, and further activation of cultured microglia with relatively short (3-h) incubations with lipopolysaccharide caused an ∼4-fold increase in the fraction of receptors at the surface with only a 1.2-fold increase in total expression. The redistribution of intracellular receptors is thus an efficient means of enhancing the functional expression of P2X4 at the plasma membrane of microglia. P2X4 and P2X7 are the predominant P2X receptor subtypes expressed in immune cells. Having previously shown a structural and functional interaction between the two recombinant receptors, our aims here were to identify the preferred assembly pathway of the endogenous receptors in macrophage-like cells and to investigate the trafficking of these receptors between the plasma membrane and intracellular sites. We exploited the difference in size between the two subunits, and we used a combination of cross-linkers and blue native-PAGE analysis to investigate the subunit composition of complexes present in primary cultures of rat microglia and macrophages from wild type and P2X7–/– mice. Our results indicate that the preferred assembly pathway for both receptors is the formation of homotrimers. Homotrimers of P2X7 were able to co-immunoprecipitate with P2X4, suggesting that an interaction occurs between rather than within receptor complexes. In both macrophages and microglia, P2X7 receptors were predominantly at the cell surface, whereas P2X4 receptors were predominantly intracellular. There were clear cell type-dependent differences in the extent to which P2X4 receptors trafficked to and from the surface; trafficking was much more dynamic in microglia than in the macrophages, and further activation of cultured microglia with relatively short (3-h) incubations with lipopolysaccharide caused an ∼4-fold increase in the fraction of receptors at the surface with only a 1.2-fold increase in total expression. The redistribution of intracellular receptors is thus an efficient means of enhancing the functional expression of P2X4 at the plasma membrane of microglia. Acting via P2X receptors, ATP has several effects on immune cells, including triggering the release of pro-inflammatory cytokines, programmed cell death, and mycobacterial killing (1Khakh B.S. North R.A. Nature. 2006; 442: 527-532Crossref PubMed Scopus (698) Google Scholar, 2North R.A. Physiol. Rev. 2002; 82: 1013-1067Crossref PubMed Scopus (2413) Google Scholar). Until recently, attention has focused on the P2X7 receptor as the relevant sensor at the cell surface, and there is evidence for its involvement in neuropathic and inflammatory pain (3Chessell I.P. Hatcher J.P. Bountra C. Michel A.D. Hughes J.P. Green P. Egerton J. Murfin M. Richardson J. Peck W.L. Grahames C.B. Casula M.A. Yiangou Y. Birch R. Anand P. Buell G.N. Pain. 2005; 114: 386-396Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar), arthritis (4Labasi J.M. Petrushova N. Donovan C. McCurdy S. Lira P. Payette M.M. Brissette W. Wicks J.R. Audoly L. Gabel C.A. J. Immunol. 2002; 168: 6436-6445Crossref PubMed Scopus (431) Google Scholar), and the control of mycobacterial infection (5Fairbairn I.P. Stober C.B. Kumararatne D.S. Lammas D.A. J. Immunol. 2001; 167: 3300-3307Crossref PubMed Scopus (205) Google Scholar). P2X7 has a low affinity for ATP compared with the other predominant subtype expressed by immune cells, P2X4. The role of P2X4 receptors is less well understood, although in microglia they were shown to be up-regulated following peripheral nerve injury and to play an important role in the development of neuropathic pain (6Tsuda M. Shigemoto-Mogami Y. Koizumi S. Mizokoshi A. Kohsaka S. Salter M.W. Inoue K. Nature. 2003; 424: 778-783Crossref PubMed Scopus (1229) Google Scholar). Regulation of the plasma membrane expression of these two receptors is not understood. Also, despite the considerable interest in both these receptor subtypes as potential targets in development of novel pain therapies, the subunit composition of the native receptors has not been determined. P2X receptor subunits associate to form trimeric complexes around a central conduction pore (7Nicke A. Baumert H.G. Rettinger J. Eichele A. Lambrecht G. Mutschler E. Schmalzing G. EMBO J. 1998; 17: 3016-3028Crossref PubMed Scopus (479) Google Scholar, 8Barrera N.P. Ormond S.J. Henderson R.M. Murrell-Lagnado R.D. Edwardson J.M. J. Biol. Chem. 2005; 280: 10759-10765Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). With the exception of P2X6, they form functional homomeric receptors, and several subtypes also form functional heterotrimers (9Murrell-Lagnado R.D. Qureshi O.S. Mol. Membr. Biol. 2008; 25: 321-331Crossref PubMed Scopus (37) Google Scholar). There is also evidence that P2X receptors form larger signaling complexes, interacting with other neighboring P2X receptors and also with ion channels that belong to different structural classes, including members of the Cys loop receptor family (10Khakh B.S. Zhou X. Sydes J. Galligan J.J. Lester H.A. Nature. 2000; 406: 405-410Crossref PubMed Scopus (158) Google Scholar) and the gap junction family (11Pelegrin P. Surprenant A. EMBO J. 2006; 25: 5071-5082Crossref PubMed Scopus (1110) Google Scholar). Higher order structures, with molecular mass corresponding to hexamers and nonamers, have been identified for heterologously expressed P2X2 and P2X4 receptors, suggesting that two or three receptors can form a stable association (12Aschrafi A. Sadtler S. Niculescu C. Rettinger J. Schmalzing G. J. Mol. Biol. 2004; 342: 333-343Crossref PubMed Scopus (103) Google Scholar). Consistent with this idea, P2X receptor channels within a patch of membrane have been shown to open in a non-independent manner and to display positive cooperativity (13Ding S. Sachs F. BMC Neurosci. 2002; 3: 17Crossref PubMed Scopus (35) Google Scholar, 14Fujiwara Y. Kubo Y. J. Physiol. (Lond.). 2004; 558: 31-43Crossref Scopus (71) Google Scholar). P2X4 and P2X7 are frequently co-expressed, not only in immune cells but also in epithelia and endothelia (15Surprenant A. North R.A. Annu. Rev. Physiol. 2009; 71: 333-359Crossref PubMed Scopus (430) Google Scholar). The P2X7 subtype differs from other members of the family in that it has a very long cytoplasmic C-terminal tail, a low affinity toward ATP, is preferentially activated by BzATP, and couples to the opening of a pore permeable to large molecules up to 900 Da (2North R.A. Physiol. Rev. 2002; 82: 1013-1067Crossref PubMed Scopus (2413) Google Scholar). P2X4 receptors have considerably higher affinity for ATP; they are up-regulated by the allosteric modulator ivermectin and are sensitive to the antagonist TNP-ATP 3The abbreviations used are: TNP-ATP, 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate; LPS, lipopolysaccharide; DSS, disuccinimidyl suberate; BS3, bis[sulfosuccinimidyl] suberate; BMDM, bone marrow-derived macrophage; PBS, phosphate-buffered saline; BN, blue native; HBS HEPES-buffered saline; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; WT, wild type. 3The abbreviations used are: TNP-ATP, 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate; LPS, lipopolysaccharide; DSS, disuccinimidyl suberate; BS3, bis[sulfosuccinimidyl] suberate; BMDM, bone marrow-derived macrophage; PBS, phosphate-buffered saline; BN, blue native; HBS HEPES-buffered saline; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; WT, wild type. at micromolar concentrations. It has been widely assumed that P2X7 does not form heteromeric assemblies with other members of the P2X family; however, recent evidence has indicated a structural and functional interaction between P2X4 and P2X7 receptors. First, P2X receptor currents recorded from airway-ciliated cells were reported to show a combination of P2X4-like and P2X7-like properties (16Ma W. Korngreen A. Weil S. Cohen E.B. Priel A. Kuzin L. Silberberg S.D. J. Physiol. (Lond.). 2006; 571: 503-517Crossref Scopus (71) Google Scholar). Second, we showed that P2X4 and P2X7 could be co-immunoprecipitated both from mouse bone marrow-derived macrophages (BMDMs) and also when heterologously co-expressed (17Guo C. Masin M. Qureshi O.S. Murrell-Lagnado R.D. Mol. Pharmacol. 2007; 72: 1447-1456Crossref PubMed Scopus (192) Google Scholar). In addition, we used two nonfunctional point mutants of P2X4 to demonstrate a functional interaction between the two receptors; one mutant exerted a dominant negative effect on P2X7 receptor currents, whereas another conferred P2X4-like properties on the whole cell currents, namely sensitivity to ivermectin and TNP-ATP. Our conclusion was that P2X4 and P2X7 form a heteromeric association but that it remained to be established whether or not they assemble as heterotrimers around the same conduction pore. In this study, we set out first to address the question of the preferred assembly pathway of native P2X4 and P2X7 receptors in immune cells, and second to compare how these receptors traffic within immune cells. We took advantage of the size difference between P2X4 and P2X7 subunits to analyze the composition of the predominant dimeric and trimeric forms. We found that in rodent macrophages and microglia, P2X4 and P2X7 receptors preferentially assemble as homotrimers. Comparison of the trafficking of these receptors indicated cell type-dependent differences in the extent to which the predominantly intracellular P2X4 receptor traffics to and from the surface. Materials—P2X4 and P2X7 antibodies were purchased from Alomone (Israel). Horseradish peroxidase-conjugated anti-rabbit IgG whole antibody was from Health Care Limited (United Kingdom). Culture media were obtained from Invitrogen. Cross-linkers, disuccinimidyl suberate (DSS) and bis[sulfosuccinimidyl] suberate (BS3), were supplied by Pierce. SDS and native gradient gels were purchased from Invitrogen. All other reagents were from Sigma, unless otherwise stated below. Cell Cultures—Primary cultures of microglial cells were prepared from cerebral cortices of newborn rats, as described previously (18Qureshi O.S. Paramasivam A. Yu J.C. Murrell-Lagnado R.D. J. Cell Sci. 2007; 120: 3838-3849Crossref PubMed Scopus (164) Google Scholar). Glial cultures were maintained for 9–14 days in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 50 units/ml streptomycin and then shaken to dislodge microglia. Microglial cells were seeded in 6-well plates at a density of 9 × 105 cells/well and used for experiments 16–24 h after plating. BMDMs were prepared from the femora of 5–6-week-old mice, as described previously (18Qureshi O.S. Paramasivam A. Yu J.C. Murrell-Lagnado R.D. J. Cell Sci. 2007; 120: 3838-3849Crossref PubMed Scopus (164) Google Scholar). Cells were cultured for 7–10 days in medium containing RPMI 1640 supplemented with 20% fetal bovine serum, 100 units/ml penicillin, 50 units/ml streptomycin, and 30% L-conditioned medium. L-conditioned medium was collected from cultures of confluent monolayers of L-cells (L929) grown in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cross-linking Receptors—To cross-link the total receptor population, cells were washed with ice-cold phosphate-buffered saline (PBS) and harvested in 1% Triton X-100 in PBS plus protease inhibitor mixture (Roche Applied Science). Samples were sonicated and incubated on ice for 30 min. Lysates were cleared by centrifugation at 14,000 rpm for 15 min at 4 °C. DSS was freshly prepared in DMSO, diluted to a final concentration of 4 mm, and incubated with the protein samples at room temperature for 30 min. The reaction was quenched by the addition of Tris-HCl, pH 7.5, to a final concentration of 50 mm, followed by incubation for 15 min at room temperature. To cross-link only surface receptors, cells were washed with ice-cold PBS and incubated with BS3 (1 mg/ml) in PBS for 1 h at 4 °C. 50 mm glycine was added to quench unreacted BS3. Cells were washed three times, lysed in Triple Extraction Buffer (TEB; 50 mm Tris-HCl, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS (Bio-Rad), pH 8), sonicated, and cleared by centrifugation at 14,000 rpm for 15 min at 4 °C. All samples were diluted in Laemmli buffer and analyzed by 4–12% SDS-PAGE and immunoblotting. Biotinylation of Surface Receptors—Confluent cells were rinsed five times with ice-cold DPBS (PBS, Invitrogen) (–CaCl2, –MgCl2) and incubated with freshly prepared Sulfo-NHS-SS-biotin (Pierce), diluted in DPBS (1 mg/ml), for 30 min at 12 °C. Nonreactive biotin was quenched with 50 mm glycine, followed by three washes with cold DPBS to ensure removal of unbound biotin. The cells were subsequently lysed in 150 μl of TEB and were sonicated to further and the cells. The was cleared by centrifugation at rpm for 15 min at 4 °C. the 50 μl was diluted in Laemmli buffer and used to total protein The in the 100 μl were by incubation with 30 μl of on a at 4 for three washes in to were in buffer in Laemmli and to SDS-PAGE and of biotin to surface receptors was by of to and total membrane protein for BMDMs were washed three times with HEPES-buffered saline buffer mm pH 100 mm NaCl, mm and then collected by The cell was in ice-cold buffer mm Tris-HCl, pH mm 1 mm and protease inhibitor and incubated for min on Cells were by the a and the was at 14,000 rpm for 15 min. The containing the protein fraction was by incubation in 1% in HBS buffer for 1 h on and the was at rpm for 1 total membrane protein were to DSS for 30 min at room and the reaction was quenched by the addition of Tris-HCl, pH 7.5, to a final concentration of 50 membrane protein were with anti-rabbit IgG for 30 min. The membrane protein were then incubated with of antibody in HBS buffer containing 1% for h at 4 on a IgG were subsequently added to the and the mixture was further incubated for 1 h at 4 °C. The protein complexes were washed times with HBS buffer containing 1% and were by for min in μl of Laemmli Samples were analyzed by 4–12% gradient SDS-PAGE and by the corresponding primary antibody and peroxidase-conjugated anti-rabbit IgG as was out as described previously (18Qureshi O.S. Paramasivam A. Yu J.C. Murrell-Lagnado R.D. J. Cell Sci. 2007; 120: 3838-3849Crossref PubMed Scopus (164) Google Scholar) that protein samples were on 4–12% gradient were washed three times with ice-cold PBS containing protease and lysed in buffer mm Tris-HCl, pH mm 1 mm and protease inhibitor mixture (Roche Applied Cells were further by and The was at rpm for min at 4 to and then at rpm for min at 4 °C. The was in buffer and in 1% for 1 h on samples were subsequently incubated with 0.1% SDS at for 1 membrane were supplemented with buffer and at rpm for min at 4 °C. Samples were on native and with analysis was out were with The molecular of P2X4 and P2X7 receptor complexes in primary cultures of rat microglia and mouse BMDMs were by with DSS and by SDS-PAGE followed by immunoblotting. In the of there was a predominant for P2X4 at and for P2X7 at which are the molecular for the of the In the of DSS, there were three for P2X4 and P2X7 in both microglia and The of the are shown in the density the and for P2X4 were of different from for The molecular were to the subunit with the of and these results that the preferred assembly pathway is formation of it is that a of with the not be as a in the P2X4 complexes with three P2X4 subunits be to from containing two P2X4 subunits and one P2X7 To this we compared from BMDMs obtained from wild type with obtained from P2X7–/– mice. We that there was a of heterotrimers in wild type cells, we a in the molecular mass of the by the as from the of the density incubation with DSS, however, the P2X4 at the same for both wild type and P2X7–/– cells and We also for to be in the P2X7–/– cells. There however, a very at the same molecular as P2X7 suggesting that there be expression of an although this was not when we for P2X7 following there is expression of a P2X7 in the mouse it is low as to not the of the P2X receptor complexes from wild type and P2X7–/– were in 1% and by The complexes at a higher molecular mass following than on as has been reported previously (12Aschrafi A. Sadtler S. Niculescu C. Rettinger J. Schmalzing G. J. Mol. Biol. 2004; 342: 333-343Crossref PubMed Scopus (103) Google Scholar) and Consistent with the results obtained the of native P2X4 complexes were the same in wild type and P2X7–/– there was a dominant at and after in 0.1% three were with with and homotrimers. The with the antibody were not as and In the of SDS there was a at with a large The complexes as an after with 0.1% corresponding to P2X7 and were Having previously shown that P2X7 with P2X4 in mouse BMDMs and when heterologously co-expressed, we to the of the P2X7 receptor complexes that with P2X4. We incubated with DSS to with the with in the in size to in the total namely and thus that an interaction occurs between P2X4 and P2X7 homotrimers. The BS3 has the same as DSS and can be used to cross-link receptors at the plasma well as to it a of the of receptors that are at the surface The and corresponding and show that in microglia the of P2X4 and P2X7 receptors is very P2X4 receptors were predominantly to the and as whereas the of P2X7 receptors were at the plasma membrane and as homotrimers. The fraction of P2X4 that was to BS3 was also in the form of homotrimers. In three experiments the in the form of was for P2X4 and for The intracellular of P2X4 was with our that these receptors with the for (18Qureshi O.S. Paramasivam A. Yu J.C. Murrell-Lagnado R.D. J. Cell Sci. 2007; 120: 3838-3849Crossref PubMed Scopus (164) Google Scholar). of receptors to BS3 was in BMDMs from wild type and P2X7–/– and the results were although surface expression of P2X4 was than in it was we could with this P2X7 the at the surface was to that in microglia To whether or not intracellular P2X4 receptors are trafficking to and from the plasma we the effects of two of and E. M. R. E. C. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar), on the of P2X4 receptors at the surface of microglia for was out as a control to ensure that intracellular were not also caused an increase in surface of P2X4 within min. The increase with was and incubations with than min not to further increase surface P2X4 suggesting that the intracellular was by this experiments were out the of of P2X4 at the surface of BMDMs and microglia in the of but this the surface BS3 The of P2X4 at the surface of microglia was within whereas in BMDMs we were not able to surface P2X4 receptors, after a incubation with We also the effects of on the surface of P2X7 but effect not suggesting that this receptor is expressed at the plasma membrane and not We that in cultured microglia, P2X4 receptors are trafficking to and from the surface predominantly whereas in cultured macrophages we have evidence for the dynamic trafficking of this in are to be in an activated however, be further activated by incubation with lipopolysaccharide S. L. P. R. J.J. Sci. S. A. 2003; PubMed Scopus Google Scholar). In a microglial cell a incubation with was shown to increase the of P2X4 receptor currents R. P. 2007; PubMed Scopus Google Scholar). We the effects of much incubations with on both the surface expression and total expression of P2X4 in our primary cultures of microglia. h there was an ∼4-fold increase in the of P2X4 receptors at the surface whereas expression of the protein by only 1.2-fold the redistribution of intracellular P2X4 receptors is a more for plasma membrane receptors than an increase in expression of this we and there was a very increase in the total expression of P2X4 but we were to surface cells as indicated by the of not It is the combination of and to the cells. P2X receptor subtypes are reported to form functional heteromeric and only for two subunit and is there evidence that is the preferred assembly pathway C. S. C. North R.A. Buell G. Surprenant A. Nature. PubMed Scopus Google Scholar, Y. North R.A. F. A. J. Neurosci. 2008; PubMed Scopus Google Scholar). Having previously reported a structural and functional interaction between heterologously expressed P2X4 and P2X7 receptors and the of the native receptors from mouse we to whether or not P2X4 and P2X7 preferentially form or heterotrimers when We from this that for P2X4 and P2X7 in immune cells, the preferred assembly pathway is the formation of homotrimers. Our results are in with a recent by A. 2008; PubMed Scopus Google Scholar), reported that the molecular of P2X4 and P2X7 receptor complexes when heterologously expressed in and from different are with complexes. We however, out the that a of heterotrimers the large of P2X7 that have been at for the J.C. S. 2005; PubMed Scopus Google Scholar), it is that the complexes a P2X7 the C-terminal It more however, that structural and functional interaction between these two receptors the association of neighboring P2X4 and P2X7 in to the that the and positive functional cooperativity by neighboring P2X2 receptors (13Ding S. Sachs F. BMC Neurosci. 2002; 3: 17Crossref PubMed Scopus (35) Google Scholar). Consistent with this idea, we found that of P2X7 co-immunoprecipitate with P2X4. of the plasma membrane expression of P2X4 and P2X7 differences in the of these two receptors. In microglia, P2X7 was predominantly in the form of at the cell surface, whereas P2X4 was intracellular. of however, show that P2X4 traffics to and from the surface. In addition, with to further microglia caused of P2X4 at the surface within a much more increase in the expression of the of P2X4 to is to be an important for the plasma membrane expression of this affinity receptor in microglia, and the role it in the development of neuropathic control of its at the surface of microglia is In the of P2X4 expression is and plasma membrane expression was the we could it with the used in this study, when was We the that the primary of of the P2X4 receptor is within intracellular rather than at the cell surface. We previously showed of P2X4 to and to the in freshly macrophages (18Qureshi O.S. Paramasivam A. Yu J.C. Murrell-Lagnado R.D. J. Cell Sci. 2007; 120: 3838-3849Crossref PubMed Scopus (164) Google Scholar). recent has shown signaling within the of of macrophages, and has that P2X7 receptors are activated within these to assembly around the Anand E. G. J. Cell Sci. 2009; PubMed Scopus Google Scholar). We a role for both P2X4 and P2X7 within the and that it is here that an interaction between these two subtypes is important for We for the P2X7 to C. We also our to A. for and