The Pro-451 to Leu polymorphism within the C-terminal tail of P2X7 receptor impairs cell death but not phospholipase D activation in murine thymocytes.
The P2X family of ATP receptors (P2XR) are ligandgated channels that have been proposed to regulate cell death of immature thymocytes. However, the nature of the P2XR subtype involved has been controversial until recently. In agreement with previous studies, we found that extracellular ATP (ATPe) induces a caspase-dependent apoptosis of BALB/c thymocytes, as observed by DNA fragmentation. Additionally, ATPe induces a predominant caspase-independent thymocytes lysis characterized by plasma membrane disruption. Both responses to ATPe can be induced by a potent P2X7R agonist, benzoylbenzoyl-ATP, whereas P2X7R antagonists, oxidized ATP and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, inhibited the effect of ATPe. These results are further supported by observations where disruption of the P2X7R gene (P2X7R(-/-) mice) completely abolishes thymocytes death induced by ATPe. Interestingly, the natural P451L mutation in the C-terminal tail of P2X7R present in C57BL/6 mice, which impairs ATPe-dependent pore formation in T lymphocytes, significantly reduces thymocytes death triggered by ATPe. Furthermore, we found that P2X7R from BW5147 thymoma cells also harbors this point mutation, accounting for their insensitivity to ATPe-induced cell death. Concentrations of ATPe effective in inducing cell death also increase phosphatidylcholine-hydrolyzing phospholipase D (PC-PLD) activity in BALB/c thymocytes through the stimulation of P2X7R. However, in contrast to ATPe-induced cell death, PC-PLD activation is totally Ca(2+)-dependent. Moreover, the stimulation of PC-PLD by ATPe is not affected by the P451L mutation present in C57BL/6 thymocytes and BW5147 cells, suggesting that cell death and PC-PLD activity are regulated through distinct domains of the P2X7R. Finally, the inhibition of ATPe-induced PC-PLD stimulation does not affect thymocytes death. Altogether, these data suggest that P2X7R-induced thymocytes death is independent of the stimulation of PC-PLD activity.
Le Stunff
Hervé
H
Laboratoire d'activation Cellulaire et Transduction des Signaux, Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, UMR 8619 CNRS, Bâtiment 430, Université Paris-Sud, 91405 Orsay Cedex, France.
Auger
Rodolphe
R
Kanellopoulos
Jean
J
Raymond
Marie-Noëlle
MN
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D017304:Annexin A5 / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005434:Flow Cytometry; D020287:In Situ Nick-End Labeling; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007930:Leucine / Q000737:chemistry*; D008024:Ligands; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009154:Mutation; D010739:Phospholipase D / Q000378:metabolism*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011110:Polymorphism, Genetic; D011392:Proline / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000737:chemistry* / Q000235:genetics; D058486:Receptors, Purinergic P2X7; D013945:Thymoma / Q000473:pathology; D013950:Thymus Gland / Q000166:cytology* / Q000378:metabolism; D013997:Time Factors
D017304:Annexin A5; D008024:Ligands; C546469:P2rx7 protein, mouse; D010975:Platelet Aggregation Inhibitors; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D011732:Pyridoxal Phosphate; D000255:Adenosine Triphosphate; D011392:Proline; D007770:L-Lactate Dehydrogenase; D010739:Phospholipase D; D007930:Leucine