Real time fluorescence imaging of PLC gamma translocation and its interaction with the epidermal growth factor receptor.

The translocation of fluorescently tagged PLC gamma and requirements for this process in cells stimulated with EGF were analyzed using real time fluorescence microscopy applied for the first time to monitor growth factor receptor--effector interactions. The translocation of PLC gamma to the plasma membrane required the functional Src homology 2 domains and was not affected by mutations in the pleckstrin homology domain or inhibition of phosphatidylinositol (PI) 3-kinase. An array of domains specific for PLC gamma isoforms was sufficient for this translocation. The dynamics of translocation to the plasma membrane and redistribution of PLC gamma, relative to localization of the EGF receptor and PI 4,5-biphosphate (PI 4,5-P(2)), were shown. Colocalization with the receptor was observed in the plasma membrane and in membrane ruffles where PI 4,5-P(2) substrate could also be visualized. At later times, internalization of PLC gamma, which could lead to separation from the substrate, was observed. The data support a direct binding of PLC gamma to the receptor as the main site of the plasma membrane recruitment. The presence of PLC gamma in membrane structures and its access to the substrate appear to be transient and are followed by a rapid incorporation into intracellular vesicles, leading to downregulation of the PLC activity.

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.

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.

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.

ARF1(2-17) does not specifically interact with ARF1-dependent pathways. Inhibition by peptide of phospholipases C beta, D and exocytosis in HL60 cells.

The small GTP-binding protein ARF has been shown recently to regulate phospholipase D (PLD). In order to investigate the role of ARF proteins in regulated exocytosis, we have used the N-terminal peptide ARF1(2-17) of the ARF1 protein. ARF1 reconstituted PLD activity in cytosol-depleted HL60 cells was inhibited by ARF1(2-17). In the presence of endogenous cytosol, ARF1(2-17) also inhibited GTP-gamma-S-stimulated PLD activity and exocytosis. Mastoparan Politses jadwagae and mastoparan Vespula lewisii which exhibit similar structural properties to ARF1(2-17) also inhibited GTP-gamma-S-stimulated PLD and exocytosis. GTP-gamma-S-stimulated phospholipase C-beta (PLC-beta) was also inhibited by ARF(2-17) and mastoparan. In cytosol-depleted HL60 cells, the ARF(2-17) inhibited the reconstitution of GTP-gamma-S-stimulated PLC-beta activity with exogenously-added PLC-beta 1 and phosphatidylinositol transfer protein. We conclude that the widely-used ARF1(2-17) peptide inhibits both ARF-independent (i.e. PLC-beta) and ARF-dependent pathways (i.e. PLD) and therefore cannot be regarded as a specific inhibitor of ARF function.

In vitro and in vivo characterization of a novel nonsteroidal, species-specific progesterone receptor modulator, PRA-910.

The progesterone receptor (PR) is an important regulator of female reproduction. Consequently, PR modulators have found numerous pharmaceutical utilities in women's reproductive health. In the process of identifying more receptor-specific and tissue-selective PR modulators, we discovered a novel nonsteroidal, 6-aryl benzoxazinone compound, PRA-910, that displays unique in vitro and in vivo activities. In a PR/PRE reporter assay in COS-7 cells, PRA-910 shows potent PR antagonist activity with an IC50 value of approximately 20 nM. In the alkaline phosphatase assay in the human breast cancer cell line T47D, PRA-910 is a partial progesterone antagonist at low concentrations and is also an effective PR agonist at higher concentrations (EC50 value of approximately 700 nM). PRA-910 binds to the human PR with high affinity (Kd = 4 nM) and was previously shown to exhibit greater than 100-fold selectivity for the PR versus other steroid receptors. In the adult ovariectomized rat, PRA-910 is a potent PR antagonist. It inhibits progesterone-induced uterine decidual response with an ED50 value of 0.4 mg/kg, p.o., and reverses progesterone suppression of estradiol-induced complement C3 expression with potency similar to RU-486. In the nonhuman primate, however, PRA-910 is a PR agonist. The effect on endometrial histology strongly resembles that of progesterone. This unique compound also suppresses estradiol-induced epithelial cell proliferation and both estrogen and progesterone receptor expression in the uterine endometrium as a PR agonist would. In summary, PRA-910 is a structurally and biologically novel selective PR modulator with either PR agonist or antagonist activity, depending on context, concentration, and species.

Rat brain cytosol contains a factor which reconstitutes guanine-nucleotide-binding-protein-regulated phospholipase-D activation in HL60 cells previously permeabilized with streptolysin O.

We report that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) can stimulate phospholipase D (PLD) in HL60 cells acutely permeabilized with streptolysin O. The ability of GTP[S] to stimulate PLD is impaired if the cells are previously permeabilized such that the majority of the cytosol has leaked out. Rat brain and HL60 cytosols were both found to restore GTP[S]-stimulated PLD activity in a reconstitution assay consisting of previously permeabilized HL60 cells. Rat brain cytosol was fractionated on heparin agarose and assayed for reconstitution of GTP[S]-stimulated PLD activity. The active fractions were pooled, concentrated and chromatographed on gel filtration to assess its molecular mass. The molecular mass of the reconstituting factor was found to be 16 kDa. Reconstitution by the cytosolic factor was dependent on GTP[S]. Ca2+ (pCa 5), MgATP and MgCl2 enhanced GTP[S]-dependent reconstitution of PLD activity in the previously permeabilized HL60 cells. These results demonstrate the presence in rat brain cytosol of a factor which is an activator of GTP[S]-stimulated PLD.

ADP ribosylation factor 1 mutants identify a phospholipase D effector region and reveal that phospholipase D participates in lysosomal secretion but is not sufficient for recruitment of coatomer I.

The small GTP-binding protein, ADP-ribosylation factor 1 (ARF1) is essential for the formation of coatomer-coated vesicles from the Golgi and is also an activator of phospholipase D (PLD). Moreover, ARF1-regulated PLD is part of the signal-transduction pathway that can lead to secretion. In this study, substitution and deletion mutants of ARF1 were tested for their ability to activate PLD. These map the PLD effector region of ARF1 to the alpha2 helix, part of the beta2-strand and the N-terminal helix and its ensuing loop. ARF mutants with an increased or decreased ability to activate PLD showed similar characteristics when tested for their ability to stimulate secretion from HL60 cells. ARF1, deleted of the N-terminal 17 amino acid residues (Ndel17), did not support PLD activity or secretion, and neither did it inhibit the activity of wild-type myristoylated ARF1 (myrARF1). In contrast, Ndel17 effectively competed with wild-type myrARF1 to prevent coatomer binding to membranes. This appears to define a structural role for Ndel17, as it can bind a high-molecular mass complex in cytosol. In addition, ethanol has no effect on recruitment of coatomer to membrane. We conclude that the function of ARF-regulated PLD is in the signal-transduction pathway leading to secretion of lysosomal granules, and not as an essential component of ARF1-mediated coatomer binding.

A neutral magnesium-dependent sphingomyelinase isoform associated with intracellular membranes and reversibly inhibited by reactive oxygen species.

Activation of neutral sphingomyelinase(s) and subsequent generation of ceramide has been implicated in a wide variety of cellular responses. Although this enzyme(s) has not been purified and cloned from higher organisms, one mammalian cDNA has been previously isolated based on its similarity to the bacterial enzyme. To further elucidate the function of this neutral sphingomyelinase, we studied its relationship with enzymes present in mammalian cells and tissues, its subcellular localization, and properties that could be important for the regulation of its activity. Using specific antibodies, it is suggested that the enzyme could represent one of several forms of neutral sphingomyelinases present in the extract from brain particulate fraction. In PC12 cells, the enzyme is localized in the endoplasmic reticulum and is not present in the plasma membrane. The same result has been obtained in several cell lines transfected or microinjected with plasmids encoding this enzyme. The molecular and enzymatic properties of the cloned neutral magnesium-dependent sphingomyelinase, produced using baculovirus or bacterial expression systems, have been analyzed, demonstrating the expected ion dependence and substrate specificity. The enzyme activity also has a strong requirement for reducing agents and is reversibly inhibited by reactive oxygen species and oxidized glutathione. The studies demonstrate that the cellular localization and some properties of this enzyme are distinct from properties previously associated with neutral magnesium-dependent sphingomyelinases in crude or partially purified preparations.

ADP-ribosylation factor and Rho proteins mediate fMLP-dependent activation of phospholipase D in human neutrophils.

Activation of intact human neutrophils by fMLP stimulates phospholipase D (PLD) by an unknown signaling pathway. The small GTPase, ADP-ribosylation factor (ARF), and Rho proteins regulate the activity of PLD1 directly. Cell permeabilization with streptolysin O leads to loss of cytosolic proteins including ARF but not Rho proteins from the human neutrophils. PLD activation by fMLP is refractory in these cytosol-depleted cells. Readdition of myr-ARF1 but not non-myr-ARF1 restores fMLP-stimulated PLD activity. C3 toxin, which inactivates Rho proteins, reduces the ARF-reconstituted PLD activity, illustrating that although Rho alone does not stimulate PLD activity, it synergizes with ARF. To identify the signaling pathway to ARF and Rho activation by fMLP, we used pertussis toxin and wortmannin to examine the requirement for heterotrimeric G proteins of the Gi family and for phosphoinositide 3-kinase, respectively. PLD activity in both intact cells and the ARF-restored response in cytosol-depleted cells is inhibited by pertussis toxin, indicating a requirement for Gi2/Gi3 protein. In contrast, wortmannin inhibited only fMLP-stimulated PLD activity in intact neutrophils, but it has no effect on myr-ARF1-reconstituted activity. fMLP-stimulated translocation of ARF and Rho proteins to membranes is not inhibited by wortmannin. It is concluded that activation of Gi proteins is obligatory for ARF/Rho activation by fMLP, but activation of phosphoinositide 3-kinase is not required.

The structure of rat ADP-ribosylation factor-1 (ARF-1) complexed to GDP determined from two different crystal forms.

The ARFs are a family of 21,000 M(r) proteins with biological roles in constitutive secretion and activation of phospholipase D. The structure of ARF-1 complexed to GDP determined from two crystal forms reveals a topology that is similar to that of the protein p21 ras with two differences: an additional amino-terminal helix and an extra beta-strand. The Mg2+ ion in ARF-1 displays a five-coordination sphere; this feature is not seen in p21 ras, due to a shift in the relative position of the DXXG motif between the two proteins. The occurrence of a dimer in one crystal form suggests that ARF-1 may dimerize during its biological function. The dimer interface involves a region of the ARF-1 molecule that is analogous to the effector domain in p21 ras and may mediate interactions with its effectors.

Structural requirements for catalysis and membrane targeting of mammalian enzymes with neutral sphingomyelinase and lysophospholipid phospholipase C activities. Analysis by chemical modification and site-directed mutagenesis.

The sequence similarity with bacterial neutral sphingomyelinase resulted in the isolation of putative mammalian counterparts and, subsequently, identification of similar molecules in a number of other eukaryotic organisms. Based on sequence similarities and previous characterization of the mammalian enzymes, we have chemically modified specific residues and performed site-directed mutagenesis in order to identify critical catalytic residues and determinants for membrane localization. Modification of histidine residues and the substrate protection experiments demonstrated the presence of reactive histidine residues within the active site. Site directed mutagenesis suggested an essential role in catalysis for two histidine residues (His-136 and His-272), which are conserved in all sequences. Mutations of two additional histidines (His-138 and His-151), conserved only in eukaryotes, resulted in reduced neutral sphingomyelinase activity. In addition to sphingomyelin, the enzyme also hydrolyzed lysophosphatidylcholine. Exposure to an oxidizing environment or modification of cysteine residues using several specific compounds also inactivated the enzyme. Site-directed mutagenesis of eight cysteine residues and gel-shift analysis demonstrated that these residues did not participate in the catalytic reaction and suggested the involvement of cysteines in the formation/breakage of disulfide bonds, which could underlie the reversible inactivation by the oxidizing compounds. Cellular localization studies of a series of deletion mutants, expressed as green fluorescent protein fusion proteins, demonstrated that the transmembrane region contains determinants for the endoplasmic reticulum localization.

An essential role for phosphatidylinositol transfer protein in phospholipase C-mediated inositol lipid signaling.

Transmembrane signaling by the phospholipase C-beta (PLC-beta) pathway is known to require at least three components: the receptor, the G protein, and the PLC. Recent studies have indicated that if the cytosol is allowed to leak out of HL60 cells, then G protein-stimulated PLC activity is greatly diminished, indicating an essential role for a cytosolic component(s). We now report the complete purification of one component based on its ability to reconstitute GTP gamma S-mediated PLC activity and identify it as the phosphatidylinositol transfer protein (PI-TP). Based on the in vitro effects of PI-TP, we surmise that it is involved in transporting PI from intracellular compartments for conversion to PI bisphosphate (PIP2) prior to hydrolysis by PLC-beta 2/PLC-beta 3, the endogenous PLC isoforms present in these cells.