The P2X4 receptor has a widespread distribution in the central nervous system and the periphery, and plays an important role in the function of immune cells and the vascular system. Its upregulation in microglia contributes to neuropathic pain following nerve injury. The mechanisms involved in its regulation are not well understood, although we have previously shown that it is constitutively retrieved from the plasma membrane and resides predominantly within intracellular compartments. Here, we show that the endogenous P2X4 receptors in cultured rat microglia, vascular endothelial cells and freshly isolated peritoneal macrophages are localized predominantly to lysosomes. Lysosomal targeting was mediated through a dileucine-type motif within the N-terminus, together with a previously characterized tyrosine-based endocytic motif within the C-terminus. P2X4 receptors remained stable within the proteolytic environment of the lysosome and resisted degradation by virtue of their N-linked glycans. Stimulation of phagocytosis triggered the accumulation of P2X4 receptors at the phagosome membrane. Stimulating lysosome exocytosis, either by incubating with the Ca2+ ionophore ionomycin, for normal rat kidney (NRK) cells and cultured rat microglia, or the weak base methylamine, for peritoneal macrophages, caused an upregulation of both P2X4 receptors and the lysosomal protein LAMP-1 at the cell surface. Lysosome exocytosis in macrophages potentiated ATP-evoked P2X4 receptor currents across the plasma membrane. Taken together, our data suggest that the P2X4 receptor retains its function within the degradative environment of the lysosome and can subsequently traffic out of lysosomes to upregulate its exposure at the cell surface and phagosome.

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We also compared the effects of the these IP 3 Ks with other enzymes that metabolize Ins(1,4,5)P 3 , including the Type I Ins(1,4,5)P 3 5-phosphatase, in both membrane-targeted and soluble forms, the human inositol polyphosphate multikinase, and the two isoforms of IP 3 K found in Drosophila. All reduce the Ca 2؉ signal but to varying degrees. We demonstrate that the activity of only one of two IP 3 K isoforms from Drosophila is positively regulated by calmodulin and that neither isoform associates with the cytoskeleton. Together the data suggest that IP 3 Ks evolved to regulate kinetic and spatial aspects of Ins (1,4,5)P 3 signals in increasingly complex ways in vertebrates, consistent with their probable roles in the regulation of higher brain and immune function.The metabolism of the calcium-mobilizing second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) 5 proceeds via two biochemical pathways. Type I Ins(1,4,5)P 3 5-phosphatase catalyzes the dephosphorylation of Ins(1,4,5)P 3 , producing the inactive (1, 2) metabolite Ins(1,4)P 2 (3). Alternatively, Ins(1,4,5)P 3 can be further phosphorylated to Ins(1,3,4,5)P 4 , in a reaction catalyzed by Ins(1,4,5)P 3 3-kinase (IP 3 K) (4). Ins(1,3,4,5)P 4 does not mobilize Ca 2ϩ , but is suggested to have a number of second messenger functions, most notably in the immune system (5, 6) and in neurons (7, 8) (reviewed in Ref. 9). Furthermore, IP 3 Ks may initiate a chain of reactions responsible for the production of higher inositol phosphates, having diverse intracellular roles (10), although this view has been challenged (11, 12).The human genome encodes three differentially expressed isoforms of IP 3 K, denoted IP 3 KA, -B, and -C (13, 14). In addition, inositol polyphosphate multikinase (IPMK; also described as IPK2, and Arg82 in yeast) harbors a range of inositol phosphate kinase activities, including Ins (1,4,5)P 3 3-kinase activity (15, 16). Two IP 3 Ks occur in Drosophila melanogaster, denoted dmIP 3 K␣ (17) and dmIP 3 K␤ (11). One IP 3 K gene occurs in Caenorhabditis elegans, and genetic evidence from both worms and flies strongly argues that IP 3 Ks function as negative regulators of Ins(1,4,5)P 3 Ca 2ϩ signals through their ability to remove Ins(1,4,5)P 3 (17, 18). Expression studies in mammalian cells further support the consensus view that IP 3 Ks negatively regulate Ins(1,4,5)P 3 Ca 2ϩ signals by their ability to remove Ins(1,4,5)P 3 (19 -25). The product of these enzymes, Ins(1,3,4,5)P 4 , may further regulate Ca 2ϩ homeostasis, although exactly how it may do that remains controversial (10).From the first studies of the IP 3 Ks, it has been assumed that because they are enzymes with a higher affinity for Ins(1,4,5)P 3 than the Type I Ins(1,4,5)P 3 5-phosphatase (26) and differ from the 5-phosphatase in that they are positively regulated by CaM and CaM kinase (see Refs. 13,14,27, and 28 for reviews), they must play a preferential role in regulating the pools of Ins (1,4,5)P 3 involved in signaling. However, the exact nature of this contribution is st...

Differentiation of PC12 cells by nerve growth factor (NGF) requires the activation of various mitogen-activated protein kinases (MAPKs) including p38 MAPK. Accumulating evidence has suggested cross-talk regulation of NGF-induced responses by G protein-coupled receptors, thus we examined whether NGF utilizes G(i/o) proteins to regulate p38 MAPK in PC12 cells. Induction of p38 MAPK phosphorylation by NGF occurred in a time- and dose-dependent manner and was partially inhibited by pertussis toxin (PTX). NGF-dependent p38 MAPK phosphorylation became insensitive to PTX treatment upon transient expressions of Galpha(z) or the PTX-resistant mutants of Galpha(i2) and Galpha(oA). Moreover, Galpha(i2) was co-immunoprecipitated with the TrkA receptor from PC12 cell lysates. To discern the participation of various signaling intermediates, PC12 cells were treated with a panel of specific inhibitors prior to the NGF challenge. NGF-induced p38 MAPK phosphorylation was abolished by inhibitors of Src (PP1, PP2, and SU6656) and MEK1/2 (U0126). Inhibition of the p38 MAPK pathway also suppressed NGF-induced PC12 cell differentiation. In contrast, inhibitors of JAK2, phospholipase C, protein kinase C and Ca(2+)/calmodulin-dependent kinase II did not affect the ability of NGF to activate p38 MAPK. Collectively, these studies indicate that NGF-dependent p38 MAPK activity may be mediated via G(i2) protein, Src, and the MEK/ERK cascade.