The proinflammatory cytokine interleukin-1beta (IL-1beta) is a secreted protein that lacks a signal peptide and does not follow currently known pathways of secretion. Its efficient release from activated immune cells requires a secondary stimulus such as extracellular ATP acting on P2X(7) receptors. We show that human THP-1 monocytes shed microvesicles from their plasma membrane within 2-5 s of activation of P2X(7) receptors. Two minutes after such stimulation, the released microvesicles contained bioactive IL-1beta, which only later appeared in the vesicle-free supernatant. We conclude that microvesicle shedding is a major secretory pathway for rapid IL-1beta release from activated monocytes and may represent a more general mechanism for secretion of similar leaderless secretory proteins.

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...

We constructed a full-length human P2X 5 purinoceptor cDNA by incorporating a sequence corresponding to exon 10, which is missing in cDNAs cloned previously from human tissues. We studied the functional properties by patch-clamp recording and fluorescence imaging after expression in human embryonic kidney 293 cells. ATP (1-100 M; half-maximal current at 4 M) elicited inward currents at Ϫ60 mV; these persisted during brief (2 s) applications but declined during longer applications. The peak current was dependent on the holding potential and showed little rectification; however, both the desensitization during the application and the decline in the current when ATP was washed out were slower at ϩ30 mV than at Ϫ60 mV. 2Ј,3Ј-O-(4-Benzoyl)-benzoyl-ATP and ␣␤-methylene-ATP mimicked the action of ATP (half-maximal concentrations 6 and 161 M, respectively). The currents were inhibited by suramin, pyridoxal-5-phosphate-6-azo-2Ј,4Ј-disulfonic acid and Brilliant Blue G, with half-maximal inhibition at 3, 0.2, and 0.5 M, respectively; 2Ј,3Ј-O-(2Ј,4Ј,6Ј-trinitrophenol)-ATP (1 M) was ineffective. Removing divalent cations did not significantly alter ATP concentration-response curves. Reversal potential measurements showed that the human P2X 5 receptor was permeable to calcium (P Ca /P Na ϭ 1.5) and N-methyl-D-glucamine (NMDG) (P NMDG /P Na ϭ 0.4); it was also permeable to chloride (P Cl /P Na ϭ 0.5) but not gluconate (P gluc /P Na ϭ 0.01) ions. The permeability to NMDG developed as quickly as the channel opened, in contrast to the P2X 7 receptor where the NMDG permeability develops over several seconds. Cells expressing human P2X 5 receptors also rapidly accumulated the propidium dye YO-PRO-1 in response to ATP.A P2X 5 subunit cDNA was first isolated from libraries of rat sympathetic ganglia (Collo et al., 1996) and heart (Garcia-Guzman et al., 1996). The RNA is expressed predominantly in heart (Garcia-Guzman et al., 1996) but there is also expression in brain, spinal cord, and adrenal gland (GarciaGuzman et al., 1996). Immunoreactivity for P2X 5 subunits has also been described in developing skeletal muscle of the rat (Meyer et al., 1999;Ryten et al., 2001); recently, the receptor has been implicated in the differentiation of satellite cells into mature multinucleated muscle fibers (Ryten et al., 2002). Mammalian skin and endocrine (Glass and Burnstock, 2001) cells also show P2X 5 immunoreactivity.When expressed in HEK293 cells, the rat P2X 5 subunit cDNAs gave rise to currents activated by ATP, but these were very small and their properties not extensively studied. The current was elicited by ATP [half-maximal concentration (EC 50 ) about 10 M] but not by ␣␤meATP, and it was readily blocked by suramin and PPADS in the 1 to 10 M concentration range (Collo et al., 1996;Garcia-Guzman et al., 1996). A mouse cDNA has also been cloned and expressed; again, the currents recorded from HEK293 cells were only a few tens of picoamperes and they were not studied in detail . On the other hand, although these two mammalian recep...