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

Abstract

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 caspasedependent 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.

Expression, signaling, and function of P2X7 receptors in bone

“…Polymorphisms that have been identified within the coding region of the human P2X7 receptor have a wide range of outcomes, such as gain-of-function [81], loss-of-function [82,83], impairment of cytokine release [84,85], and altered cell death [86]. The relatively common Glu496Ala polymorphism results in reduced pore-forming ability [82] without altering channel function [87], whereas the Ile568Asn polymorphism prevents normal channel trafficking and function [88].…”

Lipid metabolism modulation by the P2X7 receptor in the immune system and during the course of infection: new insights into the old view

“…This P2X7R-induced activation was also observed in human lymphocytes [81] and in murine thymocytes [48]. Supporting these data, Shemon et al used anti-P2X7R antibodies to abolish ATP-induced PLD stimulation in B-lymphocytes taken from patients with chronic lymphocytic leukemia (CLL), one of the most common types of leukemia.…”

“…However, the mutation of proline to leucine did not affect PLD activation [48] as much as it affected pore formation in P2X7R [48] (reviewed in Ref. [49]).…”

“…[49]). The activation of PLD by P2X7R has been described in other cell types, including lymphocytes [48], rat parotid acinar cells [36], rat brain-derived type 2 astrocyte cells [33], primary astrocytes and microglia [30,33,35], as well as macrophages [32,34,50]. In primary and macrophage cell lines, PLD has been described as part of an important killing mechanism against Mycobacterium [34] and Chlamydia [50,51], as discussed below.…”

Cell signaling via the P2X7 nucleotide receptor: linkage to ROS production, gene transcription, and receptor trafficking

“…In this regard, several single nucleotide polymorphisms (SNPs) have been identified in the promoter, introns, and coding sequences of P2X 7 [29,[32][33][34][35][36][37][38][39][40][41]. Investigations evaluating the significance of P2X 7 polymorphisms and overall protein expression levels support the idea that cells need to correctly respond to extracellular ATP via P2X 7 for the mediation of several functions, including neurotransmission, mycobacterial killing, and appropriate cell death.…”