Tissue distribution of GAP1(IP4BP) and GAP1(m): two inositol 1,3,4,5-tetrakisphosphate-binding proteins.

Two members of the GAP1 family, GAP1(IP4BP) and GAP1(m), have been shown to bind the putative second messenger Ins(1,3,4,5)P4 with high affinity and specificity, though other aspects of their behaviour suggest that in vivo, whereas GAP1(IP4BP) may function as an Ins(1,3,4,5)P4 receptor, GAP1(m) may be a receptor for the lipid second messenger PtdIns(3,4,5)P3. As a step towards clarifying their cellular roles, we describe here how we have raised and characterised antisera that are specific for the two proteins, and used these to undertake a comprehensive study of their tissue distribution. Both proteins are widely expressed, but there are several clear differences between them in the tissues that show the highest levels of expression.

Inositol 1,3,4,5-tetrakisphosphate as a second messenger--a special role in neurones?

There has been much controversy over the possibility that inositol 1,3,4,5-tetrakisphosphate (InsP4) may have a second messenger function. A possible resolution to this controversy may stem from the recent cloning of two putative receptors for InsP4, GAP1IP4BP and GAP1m. Both these proteins are expressed at high levels in neurones, as is inositol 1,4,5-trisphosphate 3-kinase, the enzyme that makes InsP4. In this review we discuss the possible relevance of these high expression levels to the complex way in which neurones control Ca2+ and use it as a second messenger.

Extracellular heavy-metal ions stimulate Ca2+ mobilization in hepatocytes.

Populations of hepatocytes in primary culture were loaded with fura 2 and the effects of extracellular heavy-metal ions were examined under conditions that allowed changes in fura 2 fluorescence (R340/360, the ratio of fluorescence recorded at 340 and 360 nm) to be directly attributed to changes in cytosolic free [Ca2+] ([Ca2+]i). In Ca2+-free media, Ni2+ [EC50 (concentration causing 50% stimulation) approximately 24+/-9 microM] caused reversible increases in [Ca2+]i that resulted from mobilization of the same intracellular Ca2+ stores as were released by [Arg8]vasopressin. The effects of Ni2+ were not mimicked by increasing the extracellular [Mg2+], by addition of MnCl2, CoCl2 or CdCl2 or by decreasing the extracellular pH from 7.3 to 6.0; nor were they observed in cultures of smooth muscle, endothelial cells or pituitary cells. CuCl2 (80 microM), ZnCl2 (80 microM) and LaCl3 (5 mM) mimicked the ability of Ni2+ to evoke Ca2+ mobilization. The response to La3+ was sustained even in the absence of extracellular Ca2+, probably because La3+ also inhibited Ca2+ extrusion. Although Ni2+ entered hepatocytes, from the extent to which it quenched fura 2 fluorescence the free cytosolic [Ni2+] ([Ni2+]i) was estimated to be <5 nM at the peak of the maximal Ni2+-evoked Ca2+ signals and there was no correlation between [Ni2+]i and the amplitude of the evoked increases in [Ca2+]i. We conclude that extracellular Ni2+, Zn2+, Cu2+ and La3+, but not all heavy-metal ions, evoke an increase in [Ca2+]i in hepatocytes by stimulating release of the hormone-sensitive intracellular Ca2+ stores and that they may do so by interacting with a specific cell-surface ion receptor. This putative ion receptor may be important in allowing hepatocytes to contribute to regulation of plasma heavy-metal ions and may mediate responses to Zn2+ released into the portal circulation with insulin.

Tissue-specific expression and endogenous subcellular distribution of the inositol 1,3,4,5-tetrakisphosphate-binding proteins GAP1(IP4BP) and GAP1(m).

GAP1(IP4BP) and GAP1(m) belong to the GAP1 family of Ras GTPase-activating proteins that are candidate InsP4 receptors. Here we show they are ubiquitously expressed in human tissues and are likely to have tissue-specific splice variants. Analysis by subcellular fractionation of RBL-2H3 rat basophilic leukemia cells confirms that endogenous GAP1(IP4BP) is primarily localised to the plasma membrane, whereas GAP1(m) appears localised to the cytoplasm (cytosol and internal membranes) but not the plasma membrane. Subcellular fractionation did not indicate a specific co-localisation between membrane-bound GAP1(m) and several Ca2+ store markers, consistent with the lack of co-localisation between GAP1(m) and SERCA1 upon co-expression in COS-7 cells. This difference suggests that GAP1(m) does not reside at a site where it could regulate the ability of InsP4 to release intracellular Ca2+. As GAP1(m) is primarily localised to the cytosol of unstimulated cells it may be spatially regulated in order to interact with Ras at the plasma membrane.

Melatonin-sensitive, serum-stimulated signalling in ovine pars tuberalis.

In primary cultures of ovine pars tuberalis (oPT), serum acts through melatonin-sensitive mechanisms independent of cyclic AMP to increase the phosphorylation of the Ca2+/cyclic AMP response element binding protein (CREB). Immunocytochemical and biochemical assays were used to characterize the active components of serum and the signalling pathways through which they and melatonin function in oPT. The stimulatory effect of serum was heat-labile, sensitive to precipitation by methanol, and required components with a mass greater than 10 KDa implicating peptide or protein factors as the active agent. Serum increased the cytosolic free Ca2+ concentration ([Ca2+]i) of oPT cells. Serum also enhanced the release of [3H]-choline and [3H]-arachidonic acid from prelabeled cells, demonstrating that factors present in serum increase the breakdown of cellular phospholipids. This effect, however, was not blocked by melatonin (1 microM). Serum also caused a dose-dependent increase in levels of immediate early gene immunoreactivity, confirming that factors in serum have the ability to control transcription in the oPT. Down-regulation of protein kinase C (PKC) by treatment with 12-0-tetradecanoylphorbol-13-acetate (TPA, 100 nM) or treatment with a specific PKC inhibitor (RO-31-8220, 1 microM), did not affect protein kinase A-mediated stimulation of CREB phosphorylation. However, down-regulation of PKC blocked the acute stimulatory effects of TPA (100 nM) and of serum (1%). Moreover, RO-31-8220 abolished the stimulatory effect of TPA (100 nM) and strongly attenuated that of serum (1%). These results demonstrate that serum increases the phosphorylation of CREB by stimulating cyclic AMP-independent, PKC-dependent, signalling pathways within the oPT. PKC may be activated through increased phospholipid catabolism and/or raised [Ca2+]i.

Distinct subcellular localisations of the putative inositol 1,3,4,5-tetrakisphosphate receptors GAP1IP4BP and GAP1m result from the GAP1IP4BP PH domain directing plasma membrane targeting.

Inositol 1,3,4,5-tetrakisphosphate (IP4), is a ubiquitous inositol phosphate that has been suggested to function as a second messenger. Recently, we purified and cloned a putative IP4 receptor, termed GAP1(IP4BP)[1], which is also a member of the GAP1 family of GTPase-activating proteins for the Ras family of GTPases. A homologue of GAP1(IP4BP), called GAP1(m), has been identified [2] and here we describe the cloning of a GAP1(m) cDNA from a human circulating-blood cDNA library. We found that a deletion mutant of GAP1(m), in which the putative phospholipid-binding domains (C2A and C2B) have been removed, binds to IP4 with a similar affinity and specificity to that of the corresponding GAP1(IP4BP) mutant. Expression studies of the proteins in either COS-7 or HeLa cells showed that, whereas GAP1(IP4BP) is located solely at the plasma membrane, GAP1(m) seems to have a distinct perinuclear localisation. By mutational analysis, we have shown that the contrast in subcellular distribution of these two closely related proteins may be a function of their respective pleckstrin homology (PH) domains. This difference in localisation has fundamental significance for our understanding of the second messenger functions of IP4.

Endogenous heavy metal ions perturb fura-2 measurements of basal and hormone-evoked Ca2+ signals.

Using the membrane-permeant chelator of heavy metal ions, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylene diamine (TPEN), we demonstrate that in pancreatic acinar cells, hepatocytes, and a variety of mammalian cell lines, endogenous heavy metal ions bind to cytosolic fura-2 causing basal cytosolic free [Ca2+] ([Ca2+]i) to be overestimated. TPEN had most effect in cells lightly loaded with fura-2, suggesting the presence of a limited pool of heavy metal ions (> or = 12 microM in pancreatic acinar cells) that does not rapidly exchange across the plasma membrane. In fura-2-loaded hepatocytes, vasopressin failed to evoke a detectable change in fluorescence, but after preincubation of cells with TPEN, it caused fluorescence changes characteristic of an increase in [Ca2+]i. We conclude that in many mammalian cells, a slowly exchanging mixture of cytosolic heavy metal ions binds to fura-2 both to quench its fluorescence and to mimic the effects of Ca2+ binding, thereby causing basal [Ca2+]i to be overestimated. By chelating endogenous heavy metal ions, TPEN allows basal [Ca2+]i to be accurately measured and, by preventing competition between heavy metal ions and Ca2+ for binding to fura-2, unmasks the full effect of agonists in increasing [Ca2+]i.

Caffeine-stimulated Ca2+ release from the intracellular stores of hepatocytes is not mediated by ryanodine receptors.

Caffeine has been much used to examine the possibility that ryanodine receptors similar to those found in skeletal and cardiac muscle may be more widely distributed and perhaps contribute to regenerative Ca2+ signals in electrically inexcitable cells. In permeabilized hepatocytes loaded with 45Ca2+, caffeine (> or = 5 mM) decreased the 45Ca2+ content of the intracellular stores by up to 60%; the effect was substantially reversible and it was not mimicked by the closely related methylxanthine theophylline (20 mM). Ryanodine (5 microM) stimulated a far smaller Ca2+ mobilization (7 +/- 1%). Procaine (1 mM), Ruthenium Red (10 microM) and ryanodine (5 microM) did not affect the Ca2+ release evoked by InsP3 (3 microM) or caffeine (30 mM). We conclude that caffeine can specifically cause Ca2+ release from the intracellular stores of hepatocytes, but the effect is unlikely to be mediated by ryanodine receptors.