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


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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    ABSTRACT: The mammalian P2X7 receptors (P2X7Rs), a member of the ionotropic P2X receptor family with distinctive functional properties, play an important part in mediating extracellular ATP signaling in health and disease. A clear delineation of the molecular mechanisms underlying the key receptor properties, such as ATP-binding, ion permeation, and large pore formation of the mammalian P2X7Rs, is still lacking, but such knowledge is crucial for a better understanding of their physiological functions and contributions in diseases and for development of therapeutics. The recent breakthroughs in determining the atomic structures of the zebrafish P2X4.1R in the closed and ATP-bound open states have provided the long-awaited structural information. The human P2RX7 gene is abundant with non-synonymous single nucleotide polymorphisms (NS-SNPs), which generate a repertoire of human P2X7Rs with point mutations. Characterizations of the NS-SNPs identified in patients of various disease conditions and the resulting mutations have informed previously unknown molecular mechanisms determining the mammalian P2X7R functions and diseases. In this review, we will discuss the new insights into such mechanisms provided by structural modeling and recent functional and genetic linkage studies of NS-SNPs.
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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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    ABSTRACT: P2X7 is a homotrimeric ion channel with two transmembrane domains and a large extracellular ATP-binding domain. It plays a key role in the response of immune cells to danger signals released from cells at sites of inflammation. Gating of murine P2X7 can be induced by the soluble ligand ATP, as well as by NAD(+)-dependent ADP-ribosylation of arginine 125, a posttranslational protein modification catalyzed by the toxin-related ecto-enzymes ART2.1 and ART2.2. R125 is located at the edge of the ligand-binding crevice. Recently, an alternative splice variant of P2X7, designated P2X7(k), was discovered that differs from the previously described variant P2X7(a) in the N-terminal 42 amino acid residues composing the first cytosolic domain and most of the Tm1 domain. Here we compare the two splice variants of murine P2X7 with respect to their sensitivities to gating by ADP-ribosylation in transfected HEK cells. Our results show that the P2X7(k) variant is sensitive to activation by ADP-ribosylation whereas the P2X7(a) variant is insensitive, despite higher cell surface expression levels. Interestingly, a single point mutation (R276K) renders the P2X7(a) variant sensitive to activation by ADP-ribosylation. Residue 276 is located at the interface of neighboring subunits approximately halfway between the ADP-ribosylation site and the transmembrane domains. Moreover, we show that naive and regulatory T cells preferentially express the more sensitive P2X7(k) variant, while macrophages preferentially express the P2X7(a) variant. Our results indicate that differential splicing of alternative exons encoding the N-terminal cytosolic and transmembrane domains of P2X7 control the sensitivity of different immune cells to extracellular NAD(+) and ATP.
    PLoS ONE 07/2012; 7(7):e41269. DOI:10.1371/journal.pone.0041269 · 3.23 Impact Factor
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    • "In one study, where mice were infected intraperitoneally with tachyzoites of the type 2, avirulent ME49 strain of T. gondii, splenic parasite burdens in different mouse strains were in proportions consistent with their relative P2X7 receptor activity [54]; thus, P2X7 receptor knockout mice harboured more parasites than the parental C57BL/6J mice, whereas, in another study, using the same parasites and route of infection, no difference in parasite burden between receptor knockout mice and the parental strain was observed in vivo [56]. C57BL/6J mice are known to possess a proline-to-leucine polymorphism at amino acid 453 in their P2X7 receptor that affects some [22], [57]–[59], but not all [22], [60], [61], functions of the receptor and so, C57BL/6J mice, in turn, had higher parasite burdens than resistant BALB/c mice with fully functioning P2X7 receptors. It should be noted, however, that BALB/c and C57BL/6J mice display differing susceptibilities to T. gondii infection and multiple genes are associated with this [62]. "
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