A truncated P2X7 receptor variant (P2X7-j) endogenously expressed in cervical cancer cells antagonizes the full-length P2X7 receptor through hetero-oligomerization
Ying-Hong Feng, Xin Li, Liqin Wang, Lingying Zhou, George I Gorodeski, Ying-Hong Feng, Xin Li, Liqin Wang, Lingying Zhou, George I Gorodeski
Abstract
A truncated naturally occurring variant of the human receptor P2X7 was identified in cancer cervical cells. The novel protein (P2X7-j), a polypeptide of 258 amino acids, lacks the entire intracellular carboxyl terminus, the second transmembrane domain, and the distal third of the extracellular loop of the full-length P2X7 receptor. The P2X7-j was expressed in the plasma membrane; it showed diminished ligand-binding and channel function capacities and failed to form pores and mediate apoptosis in response to treatment with the P2X7 receptor agonist benzoyl-ATP. The P2X7-j interacted with the full-length P2X7 in a manner suggesting heterooligomerization and blocked the P2X7-mediated actions. Interestingly, P2X7-j immunoreactivity and mRNA expression were similar in lysates of human cancer and normal cervical tissues, but full-length P2X7 immunoreactivity and mRNA expression were higher in normal than in cancer tissues, and cancer tissues lacked 205-kDa P2X7 immunoreactivity suggesting lack of P2X7 homo(tri)-oligomerization. These results identify a novel P2X7 variant with apoptosis-inhibitory actions, and demonstrate a distinct regulatory property for a truncated variant to antagonize its full-length counterpart through hetero-oligomerization. This may represent a general paradigm for regulation of a protein function by its variant.
Figures
A, nucleotide alignment of exons 7, 8, and 9 of the human wild-type P2X7 receptor (P2X7) and of the human truncated P2X7 receptor form (P2X7-j). Near complete deletion of exon 8, except adenine A888 (encircled), results in frameshift mutation with a new stop codon at bp 775–778. B, amino acid sequences of the region encoded by the end of exon 7 and beginning of exon 8 of the P2X7 and of the region of the frameshift at the new carboxyl terminus of the P2X7-j. TM-I and TM-II, transmembrane domains I and II, respectively.
A, RT-PCR analysis: WT, wild-type; NC, negative control; M, markers. B–D, heterologous expression of human P2X7 and P2X7-j proteins in MDCK cells. MDCK cells expressing tetracycline-regulated repressor were transfected with either the N-Myc-P2X7 or N-Myc-P2X7-j cDNAs. After treatment with doxycycline, lysates were immunoblotted (IB) with the anti-Myc antibody (B) or immunoprecipitated (IP) with the anti-Myc antibody and immunoblotted with the anti-Myc antibody or with the anti-P2X7 receptor (anti-P2X7-R) antibody (C). In C, lanes are total homogenates prior to the immunoprecipitation (b, “before”); the immunoprecipitate mixtures (IP); and post-immunoprecipitate mixtures (a, “after”). D, lysates were immunoblotted with the anti-P2X7-R antibody in the absence or presence of the P2X7-R antigen (used to generate the anti-P2X7-R antibody). E and F, heterologous expression of human P2X7 and P2X7-j proteins in HEK293 cells. HEK293 cells were transfected with P2X7, P2X7-j, or N-Myc-P2X7-j cDNAs. G and H, naturally occurring expression of the P2X7 and P2X7-j in human cancer cervical cell lines. All experiments were repeated three to six times with similar trends.
A, MDCK cells: distribution by cell fraction. B, HEK293 cells: immunoprecipitated (IP) and immunoblotted (IB) assays. C, laser confocal immunostaining. Assays in C: A and B utilized anti-Myc-antibody; C and D used anti-P2X7-R antibody. C: A and B, ×20; C and D, ×40.
A, upper panel: N-Myc-P2X7 or N-Myc-P2X7-j MDCK cells were pre-treated with doxycycline or vehicle. Lower panel, wild-type HEK293 cells (293), P2X7, or P2X7-j HEK293 cells were treated with 100 μm BzATP or vehicle. At days 1–4 sample plates were removed for cell number assays. Circles, statistically significant differences among the indicated groups (analysis of variance). B, changes in percent distribution cells in sub-G1 phase (determined in terms of DNA content measured by flow cytometry). Entry into cell cycle was synchronized by serum starvation for 6 h followed by shifting cells to serum-enriched medium plus keratinocyte growth supplement (KGS) for 18 h. Left panel, N-Myc-P2X7 or N-Myc-P2X7-j MDCK cells were pretreated with doxycycline (Dox +) or the vehicle (Dox −). Right panel, 293, wild-type HEK293 cells. When indicated, BzATP was added at 100 μm for 8 h. a and b, p < 0.01 compared with Dox −. c, p < 0.01 compared with W T. d, p < 0.05 compared with no-treatment with BzATP. C, apoptosis assays utilizing cells as in B, as well as HEK293 cells expressing P2X7 plus P2X7-j. BzATP was added at 100 μm for 8 h. a–d, p < 0.01–0.03. Shown are means of three to five experiments; variability ranged from 3 to 7%.
P2X7-j effects on BzATP-induced pore formation (A) and acute Ca2+ influx (B). Experiments utilized cells as described in the legend to Fig. 4C. A, pore formation was assayed in terms of BzATP-induced influx of ethidium bromide (EB) and the increase in EB fluorescence (A.U., arbitrary units). Cells attached on filters were shifted to medium containing 5 μm EB, and BzATP (arrows) was added at 100 μm to both the luminal and subluminal solutions. Quantified EB fluorescence intensity of snap shots collected at 30-s intervals are summarized in the figure. B, ligand recognition and channel properties were determined in terms of BzATP-induced acute calcium influx and the increase in cytosolic calcium. Cells attached on filters were treated with 100 μm BzATP (arrows), added to both the luminal and subluminal solutions. Quantified cytosolic Ca2+ fluorescence intensity of snap shots collected at intervals of 10 (MDCK cells) or 15 s (HEK293 cells) are summarized in the figure. C, co-expression of the P2X7-j in HEK293 cells (45–42 kDa) does not significantly modulate P2X7 protein expression (75 kDa). The experiments were repeated three times with similar trends.
Lysates of MDCK or HEK293 cells co-expressing Myc- or HA-tagged P2X7 or P2X7-j were immunoblotted, or immunoprecipitated (IP) and immunoblotted (IB) with the anti-Myc or anti-HA antibodies as described in the legends. Experiments were repeated 2–3 times with similar trends.
Lysates of HEK293 cells expressing P2X7 and P2X7-j alone or in combination were separated by 6–8% PAGE and immunoblotted with anti-P2X7 antibody. Experiments were repeated twice with similar trends. NS, nonspecific.
Tissues designated a–f were obtained from different women. Assays utilized discarded human tissues as described under “Materials and Methods.” A, total homogenates were resolved on 8% PAGE, Western blotted using the anti-P2X7 antibody, and reprobred with the anti-tubulin antibody. B, data of A were analyzed by densitometry and presented in terms of the ratios of the P2X7 and P2X7-j proteins relative to tubulin. AU, arbitrary units. C, total RNA obtained from lysates of specimens a–f were reverse transcribed and assayed by real time PCR. Shown are the ratios of P2X7 and P2X7-j mRNA relative to glyceraldehyde-3-phosphate dehydrogenase mRNA. Rq, relative quantification. The P2X7-j/GPDH mRNA levels are shown as × 10 (to fit in the figure).
Source: PubMed