Search

The Golgi apparatus (GA) is a dynamic store of Ca(2+) that can be released into the cell cytosol. It can thus participate in the regulation of the Ca(2+) concentration in the cytosol ([Ca(2+) ](cyt) ), which might be critical for intra-Golgi transport. Secretory pathway Ca(2+) -ATPase pump type 1 (SPCA1) is important in Golgi homeostasis of Ca(2+) . The subcellular localization of SPCA1 appears to be GA specific, although its precise location within the GA is not known. Here, we show that SPCA1 is mostly excluded from the cores of the Golgi cisternae and is instead located mainly on the lateral rims of Golgi stacks, in tubular noncompact zones that interconnect different Golgi stacks, and within tubular parts of the trans Golgi network, suggesting a role in regulation of the local [Ca(2+) ](cyt) that is crucial for membrane fusion. SPCA1 knockdown by RNA interference induces GA fragmentation. These Golgi fragments lack the cis-most and trans-most cisternae and remain within the perinuclear region. This SPCA1 knockdown inhibits exit of vesicular stomatitis virus G-protein from the GA and delays retrograde redistribution of the GA glycosylation enzymes into the endoplasmic reticulum caused by brefeldin A; however, exit of these enzymes from the endoplasmic reticulum is not affected. Thus, correct SPCA1 functioning is crucial to intra-Golgi transport and maintenance of the Golgi ribbon.

The Phormidium sp. ETS-05 thermophile blue-green alga is one of the most typical and widespread species of cyanobacteria of the thermal muds of the Euganean hot springs, the therapeutic properties of which have been known since ancient times. The polar diacylglycerolipids of this cyanobacterium consists of monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulfoquinovosyldiacylglycerol and phosphatidylglycerol. We have isolated and purified these four diacylglycerolipids from ETS-05, and then analysed them for their quantitative and structural features and fatty acid contents. The monogalactosyldiacylglycerol and digalactosyldiacylglycerol show a marked presence of polyunsaturated fatty acids, of which C18 : 4 is the most common. We propose that these glycoglycerolipids can be used as markers for monitoring the thermal mud colonisation process.

Previously we identified MIR16 (membrane interacting protein of RGS16) as an integral membrane glycoprotein that interacts with regulator of G protein signaling proteins and shares significant sequence homology with bacterial glycerophosphodiester phosphodiesterases (GDEs), suggesting that it is a putative mammalian GDE. Here we show that MIR16 belongs to a large, evolutionarily conserved family of GDEs with a characteristic putative catalytic domain that shares a common motif (amino acids 92–116) with the catalytic domains of mammalian phosphoinositide phospholipases C. Expression of wild-type MIR16 (renamed GDE1), but not two catalytic domain mutants (E97A/D99A and H112A), leads to a dramatic increase in glycerophosphoinositol phosphodiesterase (GPI-PDE) activity in HEK 293T cells. Analysis of substrate specificity shows that GDE1/MIR16 selectively hydrolyzes GPI over glycerophosphocholine. The GPI-PDE activity of GDE1/MIR16 expressed in HEK 293T cells can be regulated by stimulation of G protein-coupled, α/β-adrenergic, and lysophospholipid receptors. Membrane topology studies suggest a model in which the catalytic GDE domain faces the lumen/extracellular space and the C terminus faces the cytoplasm. Our results suggest that by serving as a PDE for GPI with its activity regulated by G protein signaling, GDE1/MIR16 provides a link between phosphoinositide metabolism and G protein signal transduction.

Glycerophosphoinositol 4-phosphate (GroPIns-4P) is a biologically active, water-soluble phospholipase A metabolite derived from phosphatidylinositol 4-phosphate, whose cellular concentrations have been reported to increase in Ras-transformed cells. It is therefore important to understand its biological activities. Herein, we have examined whether GroPIns-4P can regulate the organization of the actin cytoskeleton, because this could be a Ras-related function involved in cell motility and metastatic invasion. We find that in serum-starved Swiss 3T3 cells, exogenously added GroPIns-4P rapidly and potently induces the formation of membrane ruffles, and, later, the formation of stress fibers. These actin structures can be regulated by the small GTPases Cdc42, Rac, and Rho. To analyze the mechanism of action of GroPIns-4P, we selectively inactivated each of these GTPases. GroPIns-4P requires active Rac and Rho, but not Cdc42, for ruffle and stress fiber formation, respectively. Moreover, GroPIns-4P induces a rapid translocation of the green fluorescent protein-tagged Rac into ruffles, and increases the fraction of GTP-bound Rac, in intact cells. The activation of Rac by GroPIns-4P was near maximal and long-lasting. Interestingly, this feature seems to be critical in the induction of actin ruffles by GroPIns-4P.

The lysophospholipids are integral components of the plasma membrane that have often been considered as side products of the phospholipase A2 (PLA2)-dependent production of arachidonic acid and the deacylation/reacylation processes involved in phospholipid homeostasis. Data indicating roles of these lipid derivatives in hormone responses and cell transformation have now led to a different view, and the understanding of their involvement in the modulation of cell function is building up. Here, we will summarise the current knowledge concerning the biological roles of the lysophosphoinositides and the glycerophosphoinositols (their fully deacylated counterparts) in the framework of their known effects, and those of the other lysophospholipids and glycerophospholipids.

Signaling pathways involving the inositol polyphosphates and the polyphosphoinositides have become intricately linked with a number of disease states. More recently, this has principally involved the 3-phosphorylated products of phosphoinositide 3-kinase, an enzyme that itself shows oncogenic activity and has hence become of interest in the design of antitumorigenic drugs. The downstream effectors of phosphoinositide 3-kinase are involved in different aspects of cellular signaling and cytoskeleton and trafficking events that are linked to specific polyphosphoinositide binding properties of specific protein domains, which themselves have emerging roles in specific disease states. Our recent findings have demonstrated that there is a selectivity of the intracellular effects of extracellularly applied inositol polyphosphates in their abilities to inhibit a range of growth-related in vivo assay conditions, and that these can themselves be linked to the inhibition of the membrane localization of a green fluorescent protein (GFP) -tagged PH domain. We propose that GFP fusions of the polyphosphoinositides binding domains of specific proteins of interest can be used in high-throughput investigations of the therapeutic value of specific inositol polyphosphates analogs. Inhibition of in vivo membrane targeting of these domains from proteins involved in cell growth and tumorigenesis can thus be used in the search for new anticancer drugs.

The glycerophosphoinositols, phosphoinositide metabolites formed by Ras-dependent activation of phospholipase A2 and a lysophospholipase, have been proposed to be markers of Ras-induced cell transformation. These compounds can have important cellular effects; GroPIns4P is an inhibitor of G protein-stimulated adenylate cyclase and is transiently produced in several cell types after growth factor receptor stimulation of phosphatidylinositol 3-kinase and the small G protein Rac, indicating the importance of defining further its cellular actions and metabolism. We show here that, in postnuclear membranes from Swiss 3T3 cells, there is no high-affinity 'receptor' binding of GroPIns4P. Instead, possibly through the interaction with a transporter, GroPIns4P rapidly equilibrates between medium and cell cytosol, and, at higher concentrations, can concentrate in the cell cytosol. GroPIns4P can be dephosphorylated to GroPIns in vitro by an enzyme that is membrane-associated, Ca2+-dependent, GroPIns4P-selective and has a specific pH profile. Under in vitro phosphorylating conditions, there is production of GroPIns(4,5)P2 and other inositol phosphates. As these in vitro enzyme activities do not fully correlate with the in vivo handling of GroPIns4P, the intracellular GroPIns4P levels may be controlled by its direct physical removal from the cells.

In order to investigate the cellular function of the mammalian phosphatidylinositol transfer protein alpha (PI-TPalpha), NIH3T3 fibroblast cells were transfected with the cDNA encoding mouse PI-TPalpha. Two stable cell lines, i.e. SPI6 and SPI8, were isolated, which showed a 2- and 3-fold increase, respectively, in the level of PI-TPalpha. Overexpression of PI-TPalpha resulted in a decrease in the duration of the cell cycle from 21 h for the wild type (nontransfected) NIH3T3 (wtNIH3T3) cells and mock-transfected cells to 13-14 h for SPI6 and SPI8 cells. Analysis of exponentially growing cultures by fluorescence-activated cell sorting showed that a shorter G(1) phase is mainly responsible for this decrease. The saturation density of the cells increased from 0.20 x 10(5) cells/cm(2) for wtNIH3T3 cells to 0.53 x 10(5) cells/cm(2) for SPI6 and SPI8 cells. However, anchorage-dependent growth was maintained as shown by the inability of the cells to grow in soft agar. Upon equilibrium labeling of the cells with myo-[(3)H] inositol, the relative incorporation of radioactivity in the total inositol phosphate fraction was 2-3-fold increased in SPI6 and SPI8 cells when compared with wtNIH3T3 cells. A detailed analysis of the inositol metabolites showed increased levels of glycerophosphoinositol, Ins(1)P, Ins(2)P, and lysophosphatidylinositol (lyso-PtdIns) in SPI8 cells, whereas the levels of phosphatidylinositol (PtdIns) and phosphatidylinositol 4, 5-bisphosphate were the same as those in control cells. The addition of PI-TPalpha to a total lysate of myo-[(3)H]inositol-labeled wtNIH3T3 cells stimulated the formation of lyso-PtdIns. The addition of Ca(2+) further increased this formation. Based on these observations, we propose that PI-TPalpha is involved in the production of lyso-PtdIns by activating a phospholipase A acting on PtdIns. The increased level of lyso-PtdIns that is produced in this reaction could be responsible for the increased growth rate and the partial loss of contact inhibition in SPI8 and SPI6 cells. The addition of growth factors (platelet-derived growth factor, bombesin) to these overexpressers did not activate the phospholipase C-dependent degradation of phosphatidylinositol 4,5-bisphosphate.

Many and various experimental techniques have been developed to fully analyze the intracellular signaling pathways of membrane phosphoinositides and their water‐soluble derivatives. This chapter concentrates mainly on the range of lipid‐derived, water‐soluble signaling molecules that can be produced in cells from these membrane phosphoinositides, for which we and others have proposed biological roles. These include lysophosphatidylinositol, produced via phospholipase A 1/2 activities on phosphatidylinositol; cyclic inositol phosphates, produced via phosphatidylinositol/lysophosphatidylinositol‐specific phospholipase C activities; and glycerophosphoinositols, produced via lysophospholipase A 2/1 activities on their corresponding lysophosphoinositides. While the methodologies described in this chapter are aimed more specifically at the separation, identification, and quantification of monophosphorylated glyceropho sphoinositols and other similarly charged inositol‐containing products of the membrane phosphoinositides in cell extracts, they can be equally well applied tothe full range of lysophosphoinositides, glycerophosphoinositols, inositol phosphates, and further inositol‐containing water‐soluble products of the phosphoinositides (e.g., cyclic inositol phosphates, methylphosphoinositol phosphates).

The thermophilic blue-green alga ETS-05 colonises the therapeutic thermal muds of Abano and Montegrotto, Italy. Following the isolation, purification and identification of monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulphoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol from ETS-05, we here examine their in vivo anti-inflammatory activities. MGDG, DGDG and SQDG inhibit croton-oil-induced ear oedema in the mouse in a dose-dependent manner. Inhibition by MGDG is greater than that of the reference drug, betamethasone 17,21-dipropionate, and is largely abrogated following acyl group saturation. SQDG is the least potent of these glycoglycerolipids, and shows an early transient effect. In the in vivo carrageenan-induced paw oedema model in the mouse, the inhibitory effects are again dose dependent, with an enhanced efficacy of MGDG over DGDG, SQDG and the reference drug, indomethacin. These compounds are all less toxic than indomethacin. The selective and enhanced inhibitory effects of MGDG over DGDG indicate the mechanisms behind these in vivo anti-inflammatory actions.

We have recently demonstrated that the beta subunit of the heterotrimeric G-proteins is endogenously mono-ADP-ribosylated in intact cells. The modified betagamma heterodimer loses its ability to inhibit calmodulin-stimulated type 1 adenylate cyclase and, remarkably, is de-ADP-ribosylated by a cytosolic hydrolase that completes an ADP-/de-ADP-ribosylation cycle of potential physiological relevance. In the present study, we show that this ADP-ribosylation might indeed be a general mechanism for termination of betagamma signalling, since the ADP-ribosylated betagamma subunit is also unable to activate both phosphoinositide 3-kinase-gamma and phospholipase C-beta2. Moreover, we show that beta subunit ADP-ribosylation is induced by G-protein-coupled receptor activation, since hormone stimulation of Chinese-hamster ovary plasma membranes leads to increases in beta subunit labelling. This occurs when betagamma is in its active heterodimeric conformation, since full inhibition of this modification can be achieved by binding of GDP-alphai3 to the betagamma heterodimer. Taken together, these findings delineate a pathway that arises from the activation of a G-protein-coupled receptor and leads to the inhibition of betagamma activity through its reversible mono-ADP-ribosylation.

Over the past ten years, our knowledge of the integral role that the phospho-inositide 3-kinases (PI3Ks) and their 3'-phosphorylated lipid products (3'-phosphorylated phosphoinositides; 3P-PIs) play in the mediation of signal transduction, cytoskeletal rearrangements and membrane trafficking has expanded considerably. They are now known to be involved in the regulation of cell growth, differentiation, mobility, proliferation and survival and hence they have become a potential target for the control of the growth and spread of cancer cells. More recently, the correlation of the multiplicity of isomers (both catalytic and regulatory) within the different classes of the PI3Ks with their functional relevance has become possible. This, combined with our further understanding of the protein recognition patterns for their different 3P-PIs and the newly-described pathways in the control of the levels of these by dephosphorylation, has provided new aspects and areas for interference in these multiple PI3K signalling pathways. However, in the search for effective, non-toxic, drugs for use in the treatment of cancers, these individual targets for PI3K inhibition need to be further correlated with the specific in vivo effects on cell survival, invasivity and metastatic potential. Here, the range of PI3K inhibition targets are discussed in the light of recent experimental findings, with a view to the exploitation of their specificities in new approaches to effective cancer treatments based on PI3K activity inhibition.

Upon engagement of TCR with peptide-MHC complexes displayed on the surface of antigen-presenting cells, T lymphocytes undergo a sustained elevation of intracellular Ca(2+) concentration([Ca(2+)](i)), which is required for cytokine production. In the present work, we investigate how inositol lipid metabolism can be activated for a prolonged time to ensure a sustained link between receptor triggering and downstream signaling effectors. Four lines of evidence indicate that an extensive phosphoinositide turnover induced by TCR and CD28 engagement allows this task to be accomplished: (i) continuous phosphoinositide breakdown is required for a sustained [Ca(2+)](i )increase in antigen-stimulated T cells; (ii) TCR triggering results in a continuous release of inositol phosphates from the cell membrane paralleled by a massive and sustained phosphoinositide re-synthesis due to free inositol re-incorporation; (iii) TCR-induced phosphoinositide turnover is strongly increased by CD28 ligation; and (iv) CD28 engagement augments and sustains the TCR-induced [Ca(2+)](i )increase. Our results show that the T cell pool of phosphoinositides is continuously re-formed during T cell-APC cognate interaction, thereby explaining how sustained receptor triggering can transduce an equally sustained [Ca(2+)](i) increase. Importantly, our data identify a novel step in the signaling cascade where co-stimulation converges with TCR-generated signals to sustain and amplify the activation process.

Previously it has been shown that injecting a cytosolic sperm protein factor into mammalian eggs induces sustained repetitive transients of cytosolic free Ca2+ ([Ca2+]i), or [Ca2+]i oscillations [Swann (1990) Development 110, 1295-1302]. These sperm-factor (SF)-induced [Ca2+]i oscillations are similar to those seen at fertilization. Here we demonstrate that injecting the same cytosolic extracts of mammalian sperm into single rat hepatocytes induces a series of [Ca2+]i oscillations, as measured by aequorin luminescence. SF injection into hepatocytes induced [Ca2+]i oscillations that were of longer duration, lower frequency and greater amplitude than those seen with the Ins(1,4,5)P3-generating agonist phenylephrine. The SF-induced [Ca2+]i responses appeared to be due to internal release of Ca2+, since transients could occur in Ca2+-free media. Addition of the phorbol ester phorbol 12,13-dibutyrate (PDBu) at low concentrations did not inhibit the SF-induced [Ca2+]i oscillations; high concentrations of PDBu led to a sustained increase in [Ca2+]i concentrations. These data demonstrate that sperm contain a protein factor capable of inducing a characteristic series of [Ca2+]i oscillations in a somatic cell, the hepatocyte. Along with previous observations in dorsal root ganglion neurons, the data suggest a widespread efficacy of the factor in triggering Ca2+ oscillations.

Cell Membrane Lipids and Signal Transduction Over the past two decades, the build-up of a complex understanding of the metabolism of cell membrane lipids has seen the development of the roles of these lipids from solely ‘passive’ structural components of the membrane bilayer to ‘active’ regulators of cell function. These roles have included the fields of lipid-protein interaction, membrane biogenesis and membrane transport (for reviews see Casey, 1995; Liscovitch and Cantley, 1995; Milligan et al., 1995), but it is in that of signal transduction (for reviews see Bell and Burns, 1991; Nishizuka, 1992, 1995; Billah, 1993; Divecha and Irvine, 1995; Liscovitch and Cantley, 1995; Milligan et al., 1995; Corda and Falasca, 1996) where novel lipid second messengers continue to be described (Panayotou and Waterfield, 1992; Cuadrado et al., 1993; Hannun, 1994; Su et al., 1994; Moolenaar, 1995; Pushkareva et al., 1995; Falasca et al., 1996a). Thus membrane lipids are acted upon by intracellular phospholipases (phospholipase A2 (PLA2), phospholipase C (PLC) and phospholipase D), leading to the production of lipid second messengers that are involved in the modulation of different functional cellular responses (Exton, 1990; Dennis et al., 1991; Cockcroft and Thomas, 1992; Nishizuka, 1992; Berridge, 1993; Billah, 1993; Glaser et al., 1993; Iacovelli et al., 1993; Mayer and Marshall, 1993; Dennis, 1994; Divecha and Irvine, 1995; Lee and Rhee, 1995). Activation of one of these phospholipases, PLA2, leads to the mobilisation of arachidonic acid from an sn-2 linkage of phosphatidylcholine (PtdCho), phosphatidyl-ethanolamine (PtdEth) and phosphatidylinositol (PtdIns) in numerous mammalian cell types (Van den Bosch, 1980; Irvine, 1982), which, in turn, serves as a substrate for the production of other cellular second messengers, such as the prostaglandins and leukotrienes (Piomelli and Greengard, 1990; Piomelli, 1993; Khan et al., 1995). In the case of PLA2 activation, this is also accompanied by the formation of biologically active lysolipids that have been shown to be mitogens in several cell lines, including lysophosphatidic acid (LysoPtdCOOH; Moolenaar, 1995), lysophosphatidylinositol (LysoPtdIns; Falasca and Corda, 1994) and lysophosphatidylcholine (LysoPtdCho; Asaoka et al., 1991). These lysolipids themselves are the substrates for lysophospholipases that release the fatty acid from the sn-1 linkage, thus producing glycerophospholipids.

We describe the reconstitution and purification of a membrane-associated phosphoinositide-specific phospholipase C (PIC) from turkey erythrocyte ghosts. This PIC is responsive to a G-protein coupled to P2y purinergic receptors which are expressed in turkey erythrocytes. Reconstitution is achieved by adding partially purified PIC to [3H]inositol-prelabelled turkey erythrocyte membranes depleted of their endogenous PIC (acceptor membranes). PIC activity is associated with a 52 kDa polypeptide on SDS-polyacrylamide gel electrophoresis. Addition of a 307-fold purified enzyme to the acceptor membranes has no effect on basal PIC activity, but markedly increases the response to GTPγS and P2y-purinergic receptor activation.

1 The binding properties of the muscarinic receptors in the ciliary muscle of cynomolgus monkeys have been evaluated. 2 The concentration of receptor binding sites is the highest yet reported. As found in many species and tissues, there are subclasses of agonist binding sites. Agonist binding is not affected by the non-hydrolysable guanosine triphosphate (GTP) analogue, GppNHp, suggesting that these receptors are not linked to adenylate cyclase. 3 Ciliary muscles made subsensitive by treatment with muscarinic agonists have a decreased receptor concentration but no other changes in the binding properties of the receptors could be detected.

Three distinct types of detergent have been found to be capable of solubilizing muscarinic binding activity from the rat forebrain. These are: (a) the alkylpolyoxyethylenes such as Lubrol PX [1]; (b) steroids such as cholate [2, 3], digitonin [4, 5] and the cholate sulphobetaine derivative CHAPS [6]; and (c) lysophosphatidylcholine [7]. In such studies, it has proved convenient to compare the overall yield of solubilized receptors from brain membranes (P2 and P3) pre-labelled with the irreversible alkylating affinity label [3H]propylbenzilylcholine (3H-PrBCM) [8] with the yield of active binding sites which can be assayed in the supernatant after extraction of non-pre-labelled membranes. Solubilized binding sites were conveniently measured by incubating 600,000 g-min supernatants with receptor-saturating concentrations (3 × 10-8 M) of (-)-N-[Me-3H]- scopolamine (3H-NMS) or (-)-[3H]quinuclidinyl-benzilate (3H-QNB), followed by separation of bound from free ligand by rapid gel filtration on small columns of Sephadex G-50 M [3].

The muscarinic acetylcholine receptor (mAChR) is a member of a class of neurotransmitter and hormone receptors which can activate more than one type of transmembrane signalling system. In the case of the mAChR, established mechanisms include (a) GTP-binding-proteinmediated inhibition of adenylate cyclase (Jakobs et al., 1979), (b) stimulation of the breakdown of triphosphoinositides (Jacobson et al., 1985), and (c) the opening of a certain category of K+ channels (Soejima & Noma, 1984). In each of these cases, there is evidence for the activation of distinct membrane-bound effector species by the agonistreceptor complex. The mAChR thus provides an example of a divergence of biochemical action at the level of effectuation of the response. The converse situation is also common, an example being the convergence of mAChR, crl -adrenoceptors and substance P receptors on the same pool of mobilizable intracellular Ca2+ in parotid gland cells (Putney , 1979). A likely implication of such functional diversity is that receptors such as the mAChR are, essentially, modular constructs, in which ligand-binding domains of different specificities exposed on the extracellular surface are linked to distinct effector recognition domains, or catalytic sites exposed to the intracellular milieu. Appropriate structural analogies may be provided by tyrosine kinases such as the epidermal growth factor and insulin receptors (Ullrich et al., 1984, 1985), by proteins of the major histocompatibility complex (Hood et al., 1982) or by immunoglobulins (Tonegawa, 1983). Conformational differences in the ligand-binding site induced by linkage to a variety of effector recognition sites might provide an explanation for the origin of the 'subtypes' detectable by selective ligands, in the case of the mAChR, by the selective antagonist pirenzepine (Pz) (Hammer et al., 1980; Birdsall & Hulme, 1983). However, conformational effects on the ligandbinding site could equally result from differences in effector coupling, or tissue-specific post-translational modifications, thus leading to the appearance of pharmacological subtypes without recourse to differences in the primary structure of the receptor. Convincing differentiation between these possibilities requires structure determination, and in particular the primary amino acid sequence of the receptor. This provides a major impetus for the purification, partial sequencing and cloning of receptors such as the mAChR. Further stimulus is provided by the prospect of obtaining monoclonal antibodies directed against different parts of the receptor sequence which may be used to detect structural homologies between different receptors and their subtypes (Andre et al., 1984; Venter et al., 1984), and which hold out the prospect of detailed immunohistochemical studies of receptor localization. To this end, we have purified the mAChR from rat forebrain by a straightforward procedure which exploits the properties of the o-atninobenzhydry