Two G-proteins act in series to control stimulus-secretion coupling in mast cells: use of neomycin to distinguish between G-proteins controlling polyphosphoinositide phosphodiesterase and exocytosis.

Provision of GTP (or other nucleotides capable of acting as ligands for activation of G-proteins) together with Ca2+ (at micromolar concentrations) is both necessary and sufficient to stimulate exocytotic secretion from mast cells permeabilized with streptolysin-O. GTP and its analogues, through their interactions with Gp, also activate polyphosphoinositide-phosphodiesterase (PPI-pde generating inositol 1,4,5-trisphosphate and diglyceride [DG]). We have used mast cells labeled with [3H]inositol to test whether the requirement for GTP in exocytosis is an expression of Gp activity through the generation of DG and consequent activation of protein kinase C, or whether GTP is required at a later stage in the stimulus secretion sequence. Neomycin (0.3 mM) inhibits activation of PPI-pde, but maximal secretion due to optimal concentrations of guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) can still be evoked in its presence. When ATP is also provided the concentration requirement for GTP-gamma-S in support of exocytosis is reduced. This sparing effect of ATP is nullified when the PPI-pde reaction is inhibited by neomycin. We argue that the sparing effect of ATP occurs as a result of enhancement of DG production and through its action as a phosphoryl donor in the reactions catalyzed by protein kinase C.

Essential synergy between Ca2+ and guanine nucleotides in exocytotic secretion from permeabilized rat mast cells.

Rat mast cells, pretreated with metabolic inhibitors and permeabilized by streptolysin-O, secrete histamine when provided with Ca2+ (buffered in the micromolar range) and nucleoside triphosphates. We have surveyed the ability of various exogenous nucleotides to support or inhibit secretion. The preferred rank order in support of secretion is ITP greater than XTP greater than GTP much greater than ATP. Pyrimidine nucleotides (UTP and CTP) are without effect. Nucleoside diphosphates included alongside Ca2+ plus ITP inhibit secretion in the order 2'-deoxyGDP greater than GDP greater than o-GDP greater than ADP approximately equal to 2'deoxyADP approximately equal to IDP. Secretion from the metabolically inhibited and permeabilized cells can also be induced by stable analogues of GTP (GTP-gamma-S greater than GppNHp greater than GppCH2p) which synergize with Ca2+ to trigger secretion in the absence of phosphorylating nucleotides. ATP enhances the effective affinity for Ca2+ and GTP analogues in the exocytotic process but does not alter the maximum extent of secretion. The results suggest that the presence of Ca2+ combined with activation of events controlled by a GTP regulatory protein provide a sufficient stimulus to exocytotic secretion from mast cells.

ARF1 mediates paxillin recruitment to focal adhesions and potentiates Rho-stimulated stress fiber formation in intact and permeabilized Swiss 3T3 fibroblasts.

Focal adhesion assembly and actin stress fiber formation were studied in serum-starved Swiss 3T3 fibroblasts permeabilized with streptolysin-O. Permeabilization in the presence of GTPgammaS stimulated rho-dependent formation of stress fibers, and the redistribution of vinculin and paxillin from a perinuclear location to focal adhesions. Addition of GTPgammaS at 8 min after permeabilization still induced paxillin recruitment to focal adhesion-like structures at the ends of stress fibers, but vinculin remained in the perinuclear region, indicating that the distributions of these two proteins are regulated by different mechanisms. Paxillin recruitment was largely rho-independent, but could be evoked using constitutively active Q71L ADP-ribosylation factor (ARF1), and blocked by NH2-terminally truncated Delta17ARF1. Moreover, leakage of endogenous ARF from cells was coincident with loss of GTPgammaS- induced redistribution of paxillin to focal adhesions, and the response was recovered by addition of ARF1. The ability of ARF1 to regulate paxillin recruitment to focal adhesions was confirmed by microinjection of Q71LARF1 and Delta17ARF1 into intact cells. Interestingly, these experiments showed that V14RhoA- induced assembly of actin stress fibers was potentiated by Q71LARF1. We conclude that rho and ARF1 activate complimentary pathways that together lead to the formation of paxillin-rich focal adhesions at the ends of prominent actin stress fibers.

Requirement for phosphatidylinositol transfer protein in epidermal growth factor signaling.

Stimulation of phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis is a widespread mechanism for receptor-mediated signaling in eukaryotes. Cytosolic phosphatidylinositol transfer protein (PITP) is necessary for guanosine triphosphate (GTP)-dependent hydrolysis of PIP2 by phospholipase C-beta (PLC-beta), but the role of PITP is unclear. Stimulation of phospholipase C-gamma (PLC-gamma) in A431 human epidermoid carcinoma cells treated with epidermal growth factor (EGF) required PITP. Stimulation of PI-4 kinase in cells treated with EGF also required PITP. Coprecipitation studies revealed an EGF-dependent association of PITP with the EGF receptor, with PI-4 kinase, and with PLC-gamma.

Phospholipase D: a downstream effector of ARF in granulocytes.

Activation of the phospholipase D (PLD) pathway is a widespread response when cells are activated by agonists that bind receptors on the cell surface. A 16-kD cytosolic component can reconstitute guanosine triphosphate (GTP)-mediated activation of phospholipase D in HL60 cells depleted of their cytosol by permeabilization. This factor was purified and identified as two small GTP-binding proteins, ARF1 and ARF3. Recombinant ARF1 substituted for purified ARF proteins in the reconstitution assay. These results indicate that phospholipase D is a downstream effector of ARF1 and ARF3. The well-established role of ARF in vesicular traffic would suggest that alterations in lipid content by PLD are an important determinant in vesicular dynamics.

Phosphatidylinositol transfer protein dictates the rate of inositol trisphosphate production by promoting the synthesis of PIP2.

BACKGROUND: Phosphatidylinositol transfer protein (PI-TP), which has the ability to transfer phosphatidylinositol (PI) from one membrane compartment to another, is required in the inositol lipid signalling pathway through phospholipase C-beta (PLC-beta) that is regulated by GTP-binding protein(s) in response to extracellular signals. Here, we test the hypothesis that the principal role of PI-TP is to couple sites of lipid hydrolysis to sites of synthesis, and so to replenish depleted substrate for PLC-beta. RESULTS: We have designed an experimental protocol that takes advantage of the different rates of release of endogenous PI-TP and PLC-beta from HL60 cells permeabilized with streptolysin O. We have examined the kinetics of stimulated inositol lipid hydrolysis in cells depleted of PI-TP, but not of endogenous PLC-beta, in the presence and absence of exogenous PI-TP. Linear time-courses were observed in the absence of any added protein, and the rate was accelerated by PI-TP using either guanosine 5'[gamma-thio]-triphosphate (GTP gamma S) or the receptor-directed agonist fMetLeuPhe as activators. In addition, depletion from the cells of both PI-TP and PLC-beta isoforms by extended permeabilization (40 minutes) allowed us to control the levels of PLC-beta present in the cells. Once again, PI-TP increased the rates of reactions. To identify whether the role of PI-TP was to make available the substrate phosphatidylinositol bisphosphate (PIP2) for the PLC, we examined the synthesis of PIP2 in cells depleted of PI-TP. We found that PI-TP was essential for the synthesis of PIP2. CONCLUSIONS: The predicted function of PI-TP in inositol lipid signalling is the provision of substrate for PLC-beta from intracellular sites where PI is synthesized. We propose that PI-TP is in fact a co-factor in inositol lipid signalling and acts by interacting with the inositol lipid kinases. We hypothesize that the preferred substrate for PLC-beta is not the lipid that is resident in the membrane but that provided through PI-TP.

ARF and PITP restore GTP gamma S-stimulated protein secretion from cytosol-depleted HL60 cells by promoting PIP2 synthesis.

BACKGROUND: In many cell types, including neutrophils and HL60 cells, there is an absolute requirement for a GTP-dependent step to elicit Ca(2+)-regulated secretion. Neutrophils and HL60 cells secrete lysosomal enzymes from azurophilic granules; this secretion is inhibited by 1% ethanol, indicating that phosphatidate (PA) produced by phospholipase D (PLD) activity may be involved. PLD can use primary alcohols in preference to water during the hydrolytic step, generating the corresponding phosphatidylalcohol instead of PA, its normal product. As ARF (ADP-ribosylation factor) proteins regulate PLD activity and are implicated in constitutive vesicular traffic, we have investigated whether ARF is also required for GTP-dependent secretion in HL60 cells. RESULTS: We have used a cell-permeabilization protocol that allows HL60 cells to become refractory to stimulation with GTP gamma S plus 10 microM Ca2+ with regard to secretion and PLD activity. Permeabilization with streptolysin O for 10 minutes permitted the loss of freely diffusable cytosolic proteins, including ARF proteins. Fractions derived from brain cytosol, enriched in ARF proteins, restored secretory function and PLD activity. The major contaminating protein present in these ARF-enriched fractions was identified as phosphatidylinositol transfer protein (PITP). Unexpectedly, PITP was also found to restore GTP gamma S-dependent secretion. Restoration of secretory function was characterized using recombinant proteins, rARF1 and rPITP alpha and rPITP beta. The rARF1 protein restored both secretory function and PLD activity, whereas PITP only restored secretory function. However, both ARF and PITP were capable of stimulating phosphatidylinositol bis phosphate (PIP2) synthesis. CONCLUSIONS: ARF and PITP restore secretory function in cytosol-depleted cells when stimulated with GTP gamma S plus Ca2+. We have previously shown that PITP participates in the synthesis of PIP2. In comparison, ARF1 activates PLD, producing PA, which is a known activator of phosphatidylinositol-4-phosphate 5 kinase, the enzyme responsible for PIP2 synthesis. We propose that ARF and PITP both restore exocytosis by a common mechanism-promoting PIP2 synthesis.

Ca2+-dependent conversion of phosphatidylinositol to phosphatidate in neutrophils stimulated with fMet-Leu-Phe or ionophore A23187.

Human and rabbit neutrophils stimulated with formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) and A23187 show a loss of phosphatidylinositol and an increase in phosphatidate. In cells prelabelled with 32Pi it would be expected that the newly synthesised phosphatidate would have the same specific activity as cellular ATP, provided that the loss of phosphatidylinositol is by phospholipase C attack and the resultant diacyglycerol is phosphorylated by ATP. Instead, it is demonstrated that the specific activity of newly-formed phosphatidate is less than a tenth of the specific activity of ATP initially followed by a gradual increase. The time-course of mass and of [3H]glycerol-labelled phosphatidate formation (from cells pulse-labelled with [3H]glycerol) is similar to enzyme release but differs from the generation of 32P-labelled phosphatidate (from cells prelabelled with 32Pi). The source of the newly formed phosphatidate is most likely from phosphatidylinositol because: (a) The increase in phosphatidate is always accompanied by a loss of phosphatidylinositol with no changes in the other lipids. (b) Cells pulse-labelled with [3H]glycerol lose label from phosphatidylinositol only and this is accompanied by an increase in label in phosphatidate. (c) The specific activity of the newly synthesised phosphatidate is closest to the specific activity of phosphatidylinositol. One plausible explanation for these results is that phosphatidylinositol is directly converted to phosphatidate by phospholipase D action and the resulting phosphatidate accumulates radioactivity by exchange of its phosphate group with ATP. It is also shown that enzyme secretion and conversion of phosphatidylinositol to phosphatidate can depend on both intra- as well as extracellular Ca2+. Depletion of the intracellular pool of Ca2+ is essential to inhibit totally the enzyme secretion and the conversion of phosphatidylinositol to phosphatidate in agreement with our previous results on rabbit neutrophils (Cockcroft, S., et al. (1981) Biochem. J. 200, 501-508).

Phospholipase D and membrane traffic. Potential roles in regulated exocytosis, membrane delivery and vesicle budding.

It is now well-established that phospholipase D is transiently stimulated upon activation by G-protein-coupled and receptor tyrosine kinase cell surface receptors in mammalian cells. Over the last 5 years, a tremendous effort has gone to identify the major intracellular regulators of mammalian phospholipase D and to the cloning of two mammalian phospholipase D enzymes (phospholipase D1 and D2). In this chapter, we review the physiological function of mammalian phospholipase D1 that is synergistically stimulated by ADP ribosylation factor, Rho and protein kinase Calpha. We discuss the function of this enzyme in membrane traffic, emphasising the possible integrated relationships between consumption of vesicles in regulated exocytosis, membrane delivery and constitutive membrane traffic.

Chemotactic peptide stimulation of arachidonic acid release in HL60 cells, an interaction between G protein and phospholipase C mediated signal transduction.

The mechanism of phospholipase A2 activation by chemotactic peptide was investigated in human promyelocytic HL60 cells. N-Formyl-methionyl-leucyl-phenylalanine (fMetLeuPhe) and the non-hydrolyzable GTP analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]) induced arachidonic acid release in permeabilized and metabolically inhibited HL60 cells, a preparation in which calcium was buffered and inositol phospholipid hydrolysis was inhibited. Inositol phosphate generation and arachidonic acid were shown to be temporally dissociated. These results suggest that receptor-dependent phospholipase C activity is not required for fMetLeuPhe to induce arachidonic acid release. However, fMetLeuPhe effects were highly calcium-dependent and inhibition of phospholipase C reduced fMetLeuPhe stimulation of arachidonic acid release even in the permeabilized cell preparation. We conclude that although phospholipase A2 activation is linked to the fMetLeuPhe receptor independent of phospholipase C, actions of phospholipase C to mobilize calcium and release diacylglycerol may be important to phospholipase A2 activation in the intact cell.

Use of cytochalasin B to distinguish between early and late events in neutrophil activation.

Cytochalasin B greatly enhances secretion of beta-glucuronidase and generation of superoxide on stimulation of rabbit peritoneal neutrophils with the soluble chemotactic factor N-formylmethionylleucylphenylalanine (f-Met-Leu-Phe). There are smaller changes due to cytochalasin B on binding of f-Met-Leu-[(3)H]-Phe, stimulation of phosphatidylinositol turnover and the stimulated increase in the permeability of the cell membrane to Ca(2+). These latter changes are probably artefactual and arise as secondary consequences of cell stimulation. Our observations support the notion that changes in Ca(2+) permeability of membranes and stimulation of phosphatidylinositol turnover reflect early stages in the sequence of events initiated by f-Met-Leu-Phe binding to its receptor and which lead to cell activation phenomena such as secretion and superoxide production.

Subcellular localisation of inositol lipid kinases in rat liver.

The subcellular distribution of the enzymes which phosphorylate phosphatidylinositol sequentially to form phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was investigated in rat liver. We demonstrate that whilst phosphatidylinositol kinase is present in Golgi, lysosomes and plasma membranes, the kinase that forms phosphatidylinositol 4,5-bisphosphate is localised predominantly at the plasma membrane. The role of the inositol lipid kinases in cell function is discussed.

Crystallization and preliminary X-ray diffraction studies on ADP-ribosylation factor 1.

ADP-ribosylation factor 1 (ARF-1) is a member of a family of small G-proteins that regulate both intracellular vesicle transport and phospholipase D activity. Crystals of ARF-1 suitable for X-ray diffraction analysis have been grown in the presence of GDP by the hanging drop vapour diffusion method. Crystals grow in space group C2 with cell dimensions a = 122.36 A, b = 45.01 A, c = 91.96 A and beta = 133.62 degrees and diffract to at least 2.3 A resolution. A second crystal form has been characterized (space group C2, a = 69.70 A, b = 45.25 A, c = 60.45 A, beta = 109.6 degrees) but does not grow reproducibly.

Effect of H-ras proteins on the activity of polyphosphoinositide phospholipase C in HL60 membranes.

The present study was undertaken to investigate whether purified ras proteins can affect the activity of polyphosphoinositide specific phospholipase C in a cell-free membrane system. For this purpose we used homogenous preparations of the proto-oncogenic (H-ras(gly 12)) and the oncogenic (H-ras(val 12)) forms of the human H-ras proteins and membranes prepared from the human leukemic HL60 cells. We demonstrate that both the proto-oncogenic and the oncogenic form of H-ras proteins stimulate phospholipase C activity only when coupled to non-hydrolysable analogues of GTP.

Phorbol ester inhibits polyphosphoinositide phosphodiesterase activity stimulated by either Ca2+, fluoride or GTP analogue in HL60 membranes and in permeabilized HL60 cells.

The effect of PMA (phorbol 12-myristate, 13-acetate) on PPI-pde (polyphosphoinositide phosphodiesterase) activity in the promyelocytic cell-line HL60 was examined. HL60 cells were pretreated with PMA in a time- and concentration-dependent manner and PPI-pde activity was monitored both in streptolysin O-permeabilized cells and in membranes. PPI-pde activity was stimulated by either GTP gamma S (guanosine 5'-[gamma-thio]triphosphate), fluoride or Ca2+. Both the Ca2(+)-stimulated and the G protein-mediated PPI-pde activity in permeabilized HL60 cells is maximally inhibited (70-90%) after 60 min pretreatment of intact cells with 10nM PMA. PPI-pde activity can also be observed in membranes prepared from HL60 cells although this activity represents only 10% of the total activity seen in permeabilized cells. In membranes, where PPI-pde activity can also be stimulated by either via the G-protein or directly by Ca2+, PMA pretreatment was also inhibitory regardless of the mode of activation. We suggest that both the membrane-bound PPI-pde activity and that present in the permeabilized cells are targets for protein phosphorylation by protein kinase C leading to inhibition of the catalytic function.

The differentiating agent, retinoic acid, causes an early inhibition of inositol lipid-specific phospholipase C activity in HL-60 cells.

Retinoic acid, a derivative of vitamin A, is shown to inhibit the levels of inositol phosphates and diacylglycerol by 25-30% when added to intact HL-60 cells at concentrations which induce differentiation. The onset of inhibition occurs after 10 min and reaches a maximum at 45 min. To study the mechanism and the site of action of retinoic acid, the activity of the phosphatidylinositol bisphosphate-specific phospholipase C was studied in cells permeabilized with streptolysin O and in membrane preparations. Phospholipase C activity was stimulated either via the guanine nucleotide regulatory protein (G-protein) or directly by Ca2+. Retinoic acid treatment, in a time- and concentration-dependent manner, led to a decrease in phospholipase C activity when stimulated with either GTP gamma S or NaF, both of which activate the enzyme via the G-protein. By contrast, it had no effect on the enzyme activity when stimulated with Ca2+ alone. This indicates that retinoic acid interferes with the coupling of the G-protein and phospholipase C. A relationship between the inhibition of phospholipase C activity and the induction of differentiation by retinoic acid was investigated. Only a small inhibition of GTP gamma S-stimulated phospholipase C activity was observed when an analogue of retinoic acid, etretine or Ro10-1670, with low differentiating activity, was used. Moreover, no inhibition of the GTP gamma S-stimulated phospholipase C activity was observed in an HL-60 sub-line resistant to retinoic acid. These results suggest that phospholipase C inhibition is an important step in the induction of differentiation.

The Role of Phosphatidylinositol Transfer Proteins (PITPs) in Intracellular Signalling.

Phosphatidylinositol transfer protein (PITP) has been identified as a key player in numerous signalling pathways relying on phosphatidylinositol (PI) metabolites. Although its cellular function is most likely linked to its PI/phosphatidylcholine (PC) transfer activity-an in vitro activity shared by all known PITPs-this feature cannot explain all findings from studies with PITP. Here, we review evidence suggesting that one of the main functions of PITP in cellular signalling is to present PI to lipid kinases for localized production of phosphatidylinositol (4,5)-bisphosphate (PIP(2)), either to be used as a signalling molecule (for example, in exocytosis) or as a substrate (for example, by phospholipases).

Phosphatidylinositol transfer proteins: requirements in phospholipase C signaling and in regulated exocytosis.

Phosphatidylinositol transfer proteins (PITP) are abundant cytosolic proteins originally identified because of their ability to act in vitro as specific transporters of phosphatidylinositol or phosphatidylcholine between membranes. However, the cellular function of mammalian PITP has remained enigmatic till recently. Due to the development of reconstitution assays in cytosol-depleted cells, PITP was found to be an essential component for phospholipase C-mediated hydrolysis of PIP2 and for regulated exocytosis. The exact mechanism how PITP exerts its effects is not known but the PI binding/transfer activity of PITP can partly explain its cellular function. PITP would enable the local synthesis of PIP2 by delivering PI to specialized signaling sites.

ATP stimulates secretion in human neutrophils and HL60 cells via a pertussis toxin-sensitive guanine nucleotide-binding protein coupled to phospholipase C.

Human neutrophils and HL60 cells respond to extracellular ATP by causing exocytotic secretion. Secretion is accompanied by increases in inositol phosphates and a rise in cytosol Ca2+. The responses to ATP are blocked by pertussis toxin pretreatment, indicating the involvement of a guanine nucleotide regulatory protein. Other nucleotides that are active in promoting secretion are ATP gamma S, UTP, ITP and AppNHp, whilst 8-bromo-ATP, AppCH2p, ADP, AMP and adenosine are inactive.

Undifferentiated HL60 cells respond to extracellular ATP and UTP by stimulating phospholipase C activation and exocytosis.

We have recently characterised the presence of a Ca2(+)-mobilising receptor for ATP which stimulates exocytosis in differentiated HL60 cells. Here we demonstrate that the undifferentiated HL60 cells also respond to extracellular ATP by stimulating an increase in inositol phosphates and exocytosis. Of the nucleotides (ATP, UTP, ITP, ATP gamma S, AppNHp, XTP, CTP, GTP, 8-Br-ATP and GTP gamma S) that were active in stimulating inositol phosphate formation, only UTP, ATP, ITP, ATP gamma S and AppNHp were active in stimulating secretion. On differentiation, the extent of secretion due to the purinergic agonists ATP, ITP, ATP gamma S and AppNHp remained unchanged whilst secretion due to UTP, a pyrimidine, was substantially increased. These results indicate that the effect of ATP and UTP may be mediated via separate purinergic and pyrimidinergic receptors, respectively.