guanosine-triphosphate and phosphatidylinositol-4-phosphate

ADP-ribosylation factors (Arfs) play central roles in the regulation of vesicular trafficking through the Golgi. Arfs are activated at the Golgi membrane by guanine-nucleotide-exchange factors (GEFs) that are recruited from cytosol. Here, we describe a novel mechanism for the regulation of recruitment and activity of the ArfGEF Golgi-specific BFA resistance factor 1 (GBF1). Conditions that alter the cellular Arf-GDP:Arf-GTP ratio result in GBF1 recruitment. This recruitment of GBF1 occurs selectively on cis-Golgi membranes in direct response to increased Arf-GDP. GBF1 recruitment requires Arf-GDP myristoylation-dependent interactions suggesting regulation of a membrane-bound factor. Once recruited, GBF1 causes increased Arf-GTP production at the Golgi, consistent with a feed-forward self-limiting mechanism of Arf activation. This mechanism is proposed to maintain steady-state levels of Arf-GTP at the cis-Golgi during cycles of Arf-dependent trafficking events.

Topics: ADP-Ribosylation Factors; Biocatalysis; Cell Polarity; Feedback, Physiological; Golgi Apparatus; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Guanosine Triphosphate; HeLa Cells; Humans; Intracellular Membranes; Models, Biological; Phosphatidylinositol Phosphates; Protein Isoforms

Several proteins at endoplasmic reticulum (ER)-Golgi membrane contact sites contain a PH domain that interacts with the Golgi phosphoinositide PI(4)P, a FFAT motif that interacts with the ER protein VAP-A, and a lipid transfer domain. This architecture suggests the ability to both tether organelles and transport lipids between them. We show that in oxysterol binding protein (OSBP) these two activities are coupled by a four-step cycle. Membrane tethering by the PH domain and the FFAT motif enables sterol transfer by the lipid transfer domain (ORD), followed by back transfer of PI(4)P by the ORD. Finally, PI(4)P is hydrolyzed in cis by the ER protein Sac1. The energy provided by PI(4)P hydrolysis drives sterol transfer and allows negative feedback when PI(4)P becomes limiting. Other lipid transfer proteins are tethered by the same mechanism. Thus, OSBP-mediated back transfer of PI(4)P might coordinate the transfer of other lipid species at the ER-Golgi interface.

Topics: ADP-Ribosylation Factor 1; Amino Acid Motifs; Amino Acid Sequence; Animals; Cytosol; Endoplasmic Reticulum; Golgi Apparatus; Guanosine Triphosphate; HeLa Cells; Humans; Hydrolysis; Molecular Sequence Data; Phosphatidylinositol Phosphates; Phosphoric Monoester Hydrolases; Receptors, Steroid; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sterols

Arf GAP2 is one of four Arf GAPs that function in the Golgi apparatus. We characterized the kinetics of Arf GAP2 and its regulation. Purified Arf GAP2 had little activity compared to purified Arf GAP1. Of the potential regulators we examined, coatomer had the greatest effect, stimulating activity one to two orders of magnitude. The effect was biphasic, with half-maximal activation observed at 50 nM coatomer and activation peaking at approximately 150 nM coatomer. Activation by coatomer was greater for Arf GAP2 than has been reported for Arf GAP1. The effects of phosphoinositides and changes in vesicle curvature on GAP activity were small compared to coatomer; however, both increased coatomer-dependent activity. Peptides from p24 cargo proteins increased Arf GAP2 activity by an additional 2- to 4-fold. The effect of cargo peptide was dependent on coatomer. Overexpressing the cargo protein p25 decreased cellular Arf1*GTP levels. The differential sensitivity of Arf GAP1 and Arf GAP2 to coatomer could coordinate their activities. Based on the common regulatory features of Arf GAP1 and 2, we propose a mechanism for cargo selection in which GTP hydrolysis triggered by cargo binding to the coat protein is coupled to coat polymerization.

Topics: ADP-Ribosylation Factors; Amino Acid Sequence; Animals; Biocatalysis; Chlorocebus aethiops; Coatomer Protein; COS Cells; GTPase-Activating Proteins; Guanosine Triphosphate; Humans; Hydrolysis; Kinetics; Molecular Sequence Data; Peptides; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Rats; Time Factors

Brain fodrin inhibited in a dose dependent manner the GTPgammaS-stimulated cytosolic PLA2 (cPLA2), PLC, and PLD activities in differentiated HL-60 cells permeabilized with streptolysin O. cPLA2 and PLD were inhibited by the same concentrations of fodrin (IC50=1.5-2 nM) but PLC was inhibited by lower concentrations (IC50=0.3 nM). Moreover, the rates of inhibition were different between the phospholipases. Spectrin, which shares 50% homology with fodrin, had similar effects on the three phospholipases. However, using cytosol-depleted cells or recombinant PLD1, we showed that fodrin was not a direct inhibitor. Studying the potential mechanisms of these inhibitions, we demonstrated that a major decrease in membrane phosphatidylinositol 4-monophosphate (PtdIns(4)P) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) amounts was induced by fodrin. Exogenous PtdIns(4,5)P2 partly reversed fodrin inhibition of GTPgammaS-stimulated phospholipase C activity. Hence, inhibition of PLC, cPLA2, and PLD activities observed with fodrin could be related to the decrease of PtdIns(4,5)P2, substrate of PLC, a cofactor of PLD and an enhancer of cPLA2 activity.

Topics: Carrier Proteins; Enzyme Activation; Enzyme Inhibitors; Guanosine Triphosphate; HL-60 Cells; Humans; Microfilament Proteins; Nerve Tissue Proteins; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phospholipase D; Phospholipases A; Phospholipases A2; Recombinant Proteins; Spectrin; Type C Phospholipases

1. The possible involvement of guanosine 5'-triphosphate (GTP)-binding proteins in the receptor mediated polyphosphoinositide (PPI) turnover event was investigated in rat cortical synaptosomes. 2. It was studied under the effects of guanine nucleotides on 32Pi incorporation into synaptosomal phospholipids in the absence or presence of carbachol. 3. The basal 32Pi incorporation into these phospholipids was altered by the presence of 1 mM carbachol: i.e. a decrease in 32Pi incorporation into phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-4-phosphate and an increase in the incorporation of 32Pi into phosphatidylinositol and phosphatidic acid. 4. In the presence of guanine nucleotides: GTP, Gpp(NH)p and GDP at suitable concentrations, there was a general decreasing effect on 32Pi incorporation into all 4 phospholipids, which are all involved in PPI turnover cycle, either in the basal or carbachol-stimulated levels. 5. There was no selective effect among the guanine nucleotides studied on this PPI turnover event. It is, therefore, likely that these nucleotides have a direct inhibitory effect on PPI turnover, and this action may not act through a GTP-binding protein.

Topics: Animals; Carbachol; Cerebral Cortex; GTP-Binding Proteins; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Phosphates; Phosphatidic Acids; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phosphatidylinositols; Rats; Rats, Inbred Strains; Synaptosomes

We have previously determined that human neutrophils and monocytes, as well as neutrophil/monocyte progenitor cells, express a subtype of P2-purinergic receptors (for ATP) which activate the inositol phospholipid signalling system. In the present study, membranes prepared from HL-60 promyelocytic leukemia cells were used to examine the mechanism by which these ATP receptors activate phosphatidylinositol-specific phospholipase C (PI-PLC) under defined in vitro conditions. Micromolar concentrations of the receptor agonists ATP, UTP, and ATP gamma S stimulated the GTP-dependent formation of inositol bisphosphate (IP2) and inositol trisphosphate (IP3) in washed membranes prepared from undifferentiated HL-60 cells prelabeled with [3H]inositol. The stimulatory effects of these nucleotides on PI-PLC appeared to be mediated through a GTP binding protein since minimal inositol polyphosphate accumulation was observed in the absence of guanine nucleotides. The increased inositol polyphosphate formation triggered by these nucleotide receptor agonists did not result from inhibition of GTP breakdown. Neither was it a consequence of increased [3H]polyphosphatidylinositol levels resulting from enhanced activity of membrane-associated PI- or PIP-kinases. Instead, the stimulated phospholipase activity was apparently receptor-mediated. The rank order of potency observed in these in vitro membrane assays (ATP = UTP greater than ATP gamma S much greater than TTP greater than CTP much greater than beta, gamma-CH-ATP) was similar to that observed with intact HL-60 cells. This order of potency appears to distinguish the P2-purinergic receptors expressed by human phagocytic leukocytes from the P2 gamma-purinergic receptors which activate PI-PLC in turkey erythrocyte membranes.

Topics: Animals; Cell Membrane; Enzyme Activation; Erythrocytes; Guanosine Triphosphate; Humans; Mitochondrial ADP, ATP Translocases; Monocytes; Neutrophils; Nucleotidyltransferases; Phosphatidylinositol Diacylglycerol-Lyase; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phosphoinositide Phospholipase C; Phosphoric Diester Hydrolases; Phosphorylation; Receptors, Cytoplasmic and Nuclear; Receptors, Purinergic; Tumor Cells, Cultured; Turkeys

Incorporation of 32P from [gamma-32P]ATP into phosphatidylinositol 4,5-bisphosphate (PIP2) in membranes isolated from rat brain was enhanced in a concentration-dependent manner by the GTP analogue guanosine 5'-O-(thio)triphosphate (GTP gamma S). In contrast, neither the labeling of phosphatidylinositol 4-phosphate in the same membranes nor PIP kinase activity in the soluble fraction were stimulated by GTP gamma S. Synthesis of [32P]PIP2 was not stimulated by GTP, GDP, GMP, or ATP; however, the stimulatory effects of GTP gamma S were antagonized by GTP, GDP, and guanosine 5'-O-thiodiphosphate (GDP beta S). The nucleotide-stimulated labeling of PIP2 was not due to protection of [gamma-32P] ATP from hydrolysis, activation of PIP2 hydrolysis by phospholipase C, or inhibition of PIP2 hydrolysis by its phosphomonoesterase. Therefore, phosphatidylinositol 4-phosphate kinase activity in brain membranes may be regulated by a guanine nucleotide regulatory protein. This system may enhance the resynthesis of PIP2 following receptor-mediated activation of phospholipase C.

Topics: Adenosine Triphosphate; Animals; Brain; Cell Membrane; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Monophosphate; Guanosine Triphosphate; Magnesium; Male; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phosphorylation; Phosphotransferases; Phosphotransferases (Alcohol Group Acceptor); Rats; Thionucleotides

The breakdown of exogenously added [3H]inositol-labeled phosphoinositides by rat brain cortical membranes was stimulated by the muscarinic cholinergic agonist carbachol. The stimulation required the presence of guanine nucleotide. Optimal conditions were similar to those described for guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) + carbachol stimulation of phosphoinositide breakdown in [3H]inositol-prelabeled brain membranes (Claro, E., Garcia, A., and Picatoste, F. (1989) Biochem J. 261, 29-35). Carbachol stimulated [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown was inhibited by atropine and guanosine 5'-O-(2-thiobisphosphate). The magnitude of the stimulation of exogenous PIP2 breakdown by carbachol and GTP gamma S (2- to 3-fold) was little affected over a PIP2 concentration range of 0.03-100 microM. Phosphatidylinositol 4-phosphate (PIP) was as good a substrate at all concentrations as PIP2 for carbachol stimulation of phospholipase C activity. There was appreciable phosphomonoesterase degradation of PIP to phosphatidylinositol (PI) over 10 min. There was also some conversion of added PIP to PIP2 in the presence of added ATP. The effect of calcium on PIP breakdown was similar to that on PIP2 breakdown, with an apparent EC50 for Ca2+ stimulation of 0.74 and 0.72 microM, respectively, under basal conditions. The stimulation of PIP2 and PIP breakdown by carbachol in the presence of GTP gamma S was greatest on a percentage basis at the lowest free Ca2+ concentrations. Above 1 microM free Ca2+, the stimulatory effect was lost, whereas 10 microM free Ca2+ gave a maximal stimulation of basal phospholipase C activity. Degradation of added PI was also stimulated by carbachol in the absence of ATP. PI breakdown had an EC50 for Ca2+ stimulation of 1.07 microM. The best stimulation of PI breakdown due to carbachol plus GTP gamma S was seen with 0.3 microM free Ca2+ and 100 microM PI. Maximal activation of PI breakdown was seen at 1 mM deoxycholate as was true for PIP2 and PIP breakdown. There was little effect, even of 30 microM GTP gamma S alone or of carbachol alone, on PI breakdown. Half-maximal activation of the carbachol response required only 0.2 microM GTP gamma S. These results indicate that the phospholipase C enzyme(s) activated by carbachol in the presence of GTP gamma S in rat brain cortical membranes can degrade PIP2, PIP, and PI to inositol phosphates and diacylglycerol.

Topics: Adenosine Triphosphate; Animals; Calcium; Carbachol; Cell Membrane; Cerebral Cortex; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Kinetics; Male; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phosphatidylinositols; Rats; Rats, Inbred Strains; Substrate Specificity; Thionucleotides; Type C Phospholipases

The effect of GTP on the hydrolysis of [3H]phosphatidylinositol (PI), [3H]phosphatidylinositol-4-phosphate (PIP) and [3H]phosphatidylinositol-4,5-bisphosphate (PIP2) by phospholipase C of rat brain plasma membrane, microsomes and cytosol was determined. Moreover the regulation of PI and PIP phosphorylation by GTP in brain plasma membrane was investigated. In the presence of EGTA PIP2 was actively degraded, opposite to PI and PIP which require Ca2+ for their hydrolysis. Addition of calcium ions in each case caused stimulation of inositide phosphodiesterase(s). GTP independently of calcium ions activates by about 3 times phospholipase C acting on PIP and PIP2 exclusively in the plasma membrane. PI degradation was unaffected by GTP. In the presence of Ca2+ guanine nucleotides have synergistic stimulatory effect on plasma membrane bound phospholipase C acting on PIP2. PIP kinase of brain plasma membrane was stimulated by GTP by about 20-100% in the presence of exogenous and endogenous substrate respectively. PI kinase was negligible activated by about 20% exclusively in the presence of endogenous substrate. These results indicated that guanine nucleotide modulates the level of second messengers as diacylglycerol and IP3 through the activation of phospholipase C acting on PIP2 exclusively in brain plasma membrane. The stimulation of phospholipase C by GTP may occur directly or through the enhancement of substrate level PIP2 due to stimulation of PIP kinase.

Topics: Animals; Brain; Calcium; Cell Membrane; Cytosol; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; In Vitro Techniques; Microsomes; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phosphorylation; Rats; Rats, Inbred Strains; Thionucleotides

In human placenta membranes the rate limiting enzyme for PIP2 formation from PI is PIP kinase. GTP gamma S is shown to activate PIP kinase by increasing Vmax of the enzyme. It is suggested that a guanine nucleotide regulatory protein is involved in the activation of PIP kinase although coupling with a specific receptor is not yet known. Since PIP2 is the preferred substrate of phospholipase C, the possibility exists that an increase of PIP2 due to activation of PIP kinase leads to an enhancement of phospholipase C activity and hence to an increased production of IP3 and DAG.

Topics: Cell Membrane; Enzyme Activation; Female; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Kinetics; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phosphorylation; Phosphotransferases; Phosphotransferases (Alcohol Group Acceptor); Placenta; Pregnancy; Thionucleotides; Type C Phospholipases