Article

Differential Regulation of P2X(7) Receptor Activation by Extracellular Nicotinamide Adenine Dinucleotide and Ecto-ADP-Ribosyltransferases in Murine Macrophages and T Cells

Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44120, USA.
The Journal of Immunology (Impact Factor: 4.92). 08/2009; 183(1):578-92. DOI: 10.4049/jimmunol.0900120
Source: PubMed

ABSTRACT

Extracellular NAD induces the ATP-independent activation of the ionotropic P2X(7) purinergic receptor (P2X(7)R) in murine T lymphocytes via a novel covalent pathway involving ADP-ribosylation of arginine residues on the P2X(7)R ectodomain. This modification is catalyzed by ART2.2, a GPI-anchored ADP-ribosyltransferase (ART) that is constitutively expressed in murine T cells. We previously reported that ART2.1, a related ecto-ART, is up-regulated in inflammatory murine macrophages that constitutively express P2X(7)R. Thus, we tested the hypothesis that extracellular NAD acts via ART2.1 to regulate P2X(7)R function in murine macrophages. Coexpression of the cloned murine P2X(7)R with ART2.1 or ART2.2 in HEK293 cells verified that P2X(7)R is an equivalent substrate for ADP-ribosylation by either ART2.1 or ART2.2. However, in contrast with T cells, the stimulation of macrophages or HEK293 cells with NAD alone did not activate the P2X(7)R. Rather, NAD potentiated ATP-dependent P2X(7)R activation as indicated by a left shift in the ATP dose-response relationship. Thus, extracellular NAD regulates the P2X(7)R in both macrophages and T cells but via distinct mechanisms. Although ADP-ribosylation is sufficient to gate a P2X(7)R channel opening in T cells, this P2X(7)R modification in macrophages does not gate the channel but decreases the threshold for gating in response to ATP binding. These findings indicate that extracellular NAD and ATP can act synergistically to regulate P2X(7)R signaling in murine macrophages and also suggest that the cellular context in which P2X(7)R signaling occurs differs between myeloid and lymphoid leukocytes.

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    • "The corresponding Arg125 in the human P2X7R is in the head domain and indeed, as predicted, projects toward the ATP-binding site from the tip of the top half of the ATP-binding pocket (Figure 4), supporting the hypothesis that ADP-ribose binds to the same site as ATP (Young, 2010). Arg276 is not an ADP-ribosylation site, but the R276K mutation significantly increased the NAD sensitivity of the mouse P2X7R (Hong et al., 2009; Schwarz et al., 2012). As discussed above, this residue is located in the lower body domain and away from the ATP-binding site (Figure 4) and thus the mutational effect on the NAD sensitivity is likely to result from facilitation of conformational changes leading to receptor activation. "
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    • "The properties of these "gain of function" mutations were reminiscent of the P2X7 responses observed in murine T cells. In contrast, P2X7 on murine macrophages, akin to P2X7 on HEK cells, could be gated only by high concentrations of ATP but not by ADP-ribosylation [22]. "
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    • "mP2X7a receptors, but at low doses of ATP, to act synergistically. Hong et al. showed that at mP2X7a receptors, pre-stimulation with 100 mM NAD for 3–5 mins decreased the threshold concentration of ATP required to activate a P2X7 receptor-mediated rise in [Ca 2+ ] i (Hong et al., 2009). With at least two ATP molecules required to bind to fully activate the receptor an explanation for this result is that if a significant proportion of receptors are only partially occupied with ATP, Fig. 6. "
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