Proteomic and functional evidence for a P2X7 receptor signalling complex

Abstract

P2X receptors are ATP-gated ion channels in the plasma membrane, but activation of the P2X 7 receptor also leads to rapid cytoskeletal re-arrangements such as membrane blebbing. We identi®ed 11 proteins in human embryonic kidney cells that interact with the rat P2X 7 receptor, by af®nity puri®cation followed by mass spectroscopy and immunoblotting [laminin a3, integrin b2, b-actin, a-actinin, supervillin, MAGuK, three heat shock proteins, phosphatidylinositol 4-kinase and receptor protein tyrosine phosphatase-b (RPTPb)]. Activation of the P2X 7 receptor resulted in its dephosphorylation. Whole-cell recordings from cells expressing P2X 7 receptors showed that this markedly reduced subsequent ionic currents and it also slowed membrane bleb formation. By mutagenesis, we identi®ed Tyr 343 in the putative second transmembrane domain as the site of phosphorylation. Thus, we have identi®ed a P2X 7 receptor signalling complex, some members of which may initiate cytoskeletal rearrangements following receptor activation. Others, such as RPTPb, might exert feedback control of the channel itself through its dephosphorylation. Keywords: ion channel/P2X receptors/receptor protein tyrosine phosphatase-b/signalling complex/tyrosine phosphorylation Introduction P2X receptors form a family of ATP-gated ion channels extensively distributed throughout the cells of vertebrates. They are ligand-gated ion channels, each with distinct pharmacological and/or physiological properties. They form as homomers and/or heteromers, and current biochemical evidence suggests that the channel has three or perhaps six subunits (Nicke et al., 1998; North and Surprenant, 2000). The P2X 7 receptor subunit has several features that set it apart from other members of the family (Surprenant et al., 1996). Co-immunoprecipitation experiments indicate that it is the only subunit that does not heteropolymerize with other P2X subunits (Torres et al., 1999). It is primarily localized to epithelia and immune cells, particularly antigen-presenting cells Mutini et al., 1999). Receptor activation requires concentrations of ATP that are 10±100 times higher than those required to activate other P2X receptors, but the agonist af®nity and maximum response can also be modulated 5-to 100-fold by alterations in external monovalent and divalent cations (Surprenant et al., 1996;Rassendren et al., 1997;Michel et al., 1999;Gudipaty et al., 2001). The ionic currents through P2X 7 receptors show considerable plasticity, in the sense that repeated applications of the agonist result in prominent changes in the amplitude and time course of the current elicited by subsequent applications (Surprenant et al., 1996;Rassendren et al., 1997;Hibell et al., 2000).Activation of native ATP receptors in macrophages and macrophage-like cell lines, in which P2X 7 subunits are predominately expressed, is fundamentally different from that observed for other ion channels because it initiates several cellular consequences further downstream. These include alterations in the cell morphology (Cohn...

P2X7 receptor and macrophage function

“…Low intracellular potassium results in inflammasome assembly (not shown) and hence caspase-1 activation; in number 2, LL-37 directly interacts with the P2X 7 receptor inducing a tyrosine phosphorylation event that is critical to the generation of a pannexin-1 pore that either allows potassium efflux (shown) or triggers a potassium-independent activation event [34]; finally possibility number 3 involves the P2X 7 receptor indirectly by means of an LL-37 pore that allows the activation of the P2X 7 receptor via autocrine ATP. AG126 is shown as a putative inhibitor either of the critical phosphorylation of the carboxy-terminal tyrosine 343 [18] of the P2X 7 receptor or of its extracellular ATP binding site be enhanced by increase understanding of the regulation of the purinergically regulated pores.…”

“…Of note, however, Shemon et al's protein tyrosine kinase inhibitor works on the extracellular side of the P2X 7 receptor and therefore may function by competing with ATP at the extracellular binding site. There is a highly conserved tyrosine residue in the carboxy terminus of the P2X 7 receptor near the second transmembrane region which appears to affect its channel function [18]. The role of AG126 in this regard is still unknown.…”

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

“…PI4K was identified, using a proteomic approach, as interacting with P2X7R [15]. PI4K is an enzyme involved in the phosphorylation of phosphatidylinositol to phosphatidylinositol 4-phosphate (PIP) [58,59].…”

“…P2X7R is 200 amino acids longer at its C terminus than other P2X family receptors. This longer C terminus allows P2X7R to interact with different proteins than other members of the P2X family [15]. ATP triggering P2X7R activity was described as a cation channel associated with a pore activity named P2Z [16].…”

“…ATP was described as inducing a potassium efflux, caspase activation, and interleukin-1β (IL-1β) maturation in resident mouse macrophages [17]. Later, the activation of P2X7R was directly associated with several cellular pathways, including the following: caspase-1 activation [18] and IL-1β maturation [19]; shedding of membrane proteins [20]; recruitment and activation of protein kinases, such as, the mitogen-activated protein kinase [21], protein kinase C (PKC) [22], Src [23], and glycogen synthase kinase 3 [24]; recruitment and activation of phosphatases [15]; and membrane blebbing via the activity of ROCK [25] and the activation of epithelial membrane proteins [26]. Despite this wide assortment of functions, this review will focus on the ability of P2X7R to modulate lipid pathways through the activation of different enzymes, such as, phospholipase A 2 (PLA2) [27,28], phospholipase C (PLC) [29][30][31], phospholipase D (PLD) [32][33][34][35][36][37][38][39], and sphingomyelinases [27,36].…”