scholarly journals Erythropoietin stimulates G-protein-coupled phospholipase D in haematopoietic target cells

1996 ◽  
Vol 314 (3) ◽  
pp. 853-860 ◽  
Author(s):  
Sanda CLEJAN ◽  
Conrad MALLIA ◽  
David VINSON ◽  
Robert DOTSON ◽  
Barbara S. BECKMAN
Keyword(s):  
G Protein ◽  
Phospholipase D ◽  
Second Messengers ◽  
Haematopoietic Stem Cell ◽  
Target Cells ◽  
High Concentration ◽  
Guanine Nucleotide Binding Protein ◽  
Maximal Increase ◽  
Permeabilized Cells ◽  
Early Peak

A murine haematopoietic stem-cell line, B6SUt.EP, responsive to erythropoietin (EPO), has been found to exhibit both early and late changes in diacylglycerol (DAG) and phosphatidic acid (PA) as measured by HPLC and TLC. DAG levels peaked at 5 s with a 28.1% increase compared with control levels (from 17.3 to 22.2 pmol/106 cells) with a later peak at 30 min (84.2% increase from 17.3 to 31.9 pmol). These changes were concentration-dependent from 0.025 to 10 units/ml EPO (5 s, EC50 = 0.82 unit/ml; 30 min, EC50 = 0.10 unit/ml). In addition, PA levels increased 752.3% compared with control levels (from 8.6 to 64.7 μg/106 cells) with an early peak at 20 s, as measured by both HPLC and TLC (5 s, EC50 = 0.07 unit/ml). G-protein regulation was investigated by studying the effects of the non-hydrolysable GTP analogue guanosine 5´-[γ-thio]triphosphate (GTP[S]) on PA synthesis. The addition of GTP[S] (10 μM) in permeabilized cells increased PA content from 6.3 μg to 48.6 μg per 106 cells. In the presence of EPO and GTP[S], PA levels increased to 64.8 μg. An antagonist of G-proteins, guanosine 5´-[β-thio]diphosphate (GDP[S]), had no effect on control levels of PA (5.9 μg/106 cells) but blocked the effect of EPO on PA (30.6 μg/106 cells). Thus, EPO stimulated both lipid second messengers, DAG and PA. Our results demonstrate DAG kinetics to be biphasic, as observed with a high concentration of EPO, or monophasic, as observed with low concentrations of EPO. The PA accumulation preceding that of DAG in the slower and sustaining phase suggests that PA was not derived from DAG. This was confirmed by the stimulation of PA (without ATP) by GTP[S], effectively excluding phosphorylation of DAG by DAG kinase in the formation of PA. In addition, phospholipase D (PLD) activation was demonstrated with a maximal increase in phosphatidylethanol at 5 min, suggesting that EPO increases PA via a guanine nucleotide-binding protein coupled to PLD. The temporal relationship of the evolution of PA and DAG is further strengthened by experiments with ethanol and propranolol as inhibitors of the DAG/PA phosphohydrolase reaction and R59022 as an inhibitor of the DAG kinase reaction.

scholarly journals A pertussis-toxin-sensitive protein controls exocytosis in chromaffin cells at a step distal to the generation of second messengers

1991 ◽  
Vol 274 (2) ◽  
pp. 339-347 ◽  
Author(s):  
J M Sontag ◽  
D Thierse ◽  
B Rouot ◽  
D Aunis ◽  
M F Bader
Keyword(s):  
Arachidonic Acid ◽  
Cyclic Amp ◽  
G Protein ◽  
Pertussis Toxin ◽  
Chromaffin Cells ◽  
Second Messengers ◽  
Catecholamine Release ◽  
Secretory Process ◽  
Permeabilized Cells ◽  
Arachidonic Acid Concentration

The role of GTP-binding proteins (G-proteins) in the secretory process in chromaffin cells was investigated by studying the effects of pertussis toxin (PTX) on catecholamine release and generation of various second messengers. PTX was found to stimulate the catecholamine secretion induced by nicotine, 59 mM-K+ or veratridine. PTX also potentiated Ca2(+)-evoked catecholamine release from permeabilized chromaffin cells, suggesting that PTX substrate(s) regulate the exocytotic machinery at a step distal to the rise in intracellular Ca2+. We have investigated the possible intracellular pathways involved in the stimulation of secretion by PTX. PTX did not modify the translocation of protein kinase C (PKC) to membranes in intact or permeabilized cells; in addition, neither inhibitors nor activators of PKC had any effect on catecholamine release induced by PTX. Thus it seems unlikely that the effect of PTX on secretion is mediated by activation of PKC. The effect of PTX is also cyclic AMP-independent, as PTX did not change cytoplasmic cyclic AMP levels. The relationship between PTX treatment and arachidonic acid release was also examined. We found that an increase in cytoplasmic arachidonic acid concentration enhanced Ca2(+)-evoked catecholamine release in permeabilized cells, but arachidonic acid did not mimic the effect of PTX on the Ca2(+)-dose-response curve for secretion. Furthermore, PTX did not significantly modify the release of arachidonic acid measured in resting or stimulated chromaffin cells, suggesting that the stimulatory effect of PTX on secretion is not mediated by an activation of phospholipase A2. Taken together, these results suggest that PTX may modulate the intracellular machinery of secretion at a step distal to the generation of second messengers. In alpha-toxin-permeabilized cells, full retention of the PTX-induced activation of secretion was observed even 30 min after permeabilization. In contrast, when chromaffin cells were permeabilized with streptolysin-O (SLO), there was a marked progressive loss of the PTX effect. We found that SLO caused the rapid leakage of three G-protein alpha-subunits which are specifically ADP-ribosylated by PTX. We propose that a PTX-sensitive G-protein may play an inhibitory role in the final stages of the Ca2(+)-evoked secretory process in chromaffin cells.

Multiple kinases phosphorylate the pancreatic cholecystokinin receptor in an agonist-dependent manner

1993 ◽  
Vol 264 (5) ◽  
pp. G840-G847 ◽  
Author(s):  
L. K. Gates ◽  
C. D. Ulrich ◽  
L. J. Miller
Keyword(s):  
Protein Kinase ◽  
G Protein ◽  
Pancreatic Acinar Cell ◽  
Dependent Manner ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
Guanine Nucleotide Binding Protein ◽  
Permeabilized Cells ◽  
High Concentrations ◽  
G Protein Coupled

The cholecystokinin (CCK) receptor on the rat pancreatic acinar cell is a guanine nucleotide-binding protein (G protein)-coupled receptor, which was recently demonstrated to be phosphorylated in response to agonist stimulation (Klueppelberg et al., J. Biol. Chem. 266: 17744-17746, 1991). In this work, we establish that this receptor is phosphorylated in response to a variety of homologous and heterologous secretagogues and that these phosphorylation events represent action by more than one protein kinase. One subgroup of kinases includes one or more isotype of protein kinase C (PKC), and is capable of playing a role in homologous and heterologous desensitization. A second subgroup of kinases that acts on the CCK receptor was defined by its resistance to 10 microM staurosporine, which was shown to inhibit all PKC in these cells. The activity of the second group of kinases was observed only in response to occupation of the CCK receptor by high concentrations of native hormone, raising the possibility of a "receptor-specific kinase." Similar to the prototypical kinase, beta-adrenergic receptor kinase (beta-ARK), this activity was inhibited in permeabilized cells by heparin. Furthermore, like this enzyme activity, beta-ARK was shown to be resistant to staurosporine. Based on its action on a G protein-coupled receptor, its activation at high concentrations of native agonist, and its pattern of inhibition, we believe that the staurosporine-insensitive CCK receptor kinase activity represents either beta-ARK or a closely related member of the receptor-specific kinase enzyme family.

ACTION OF GTPγS [GUANOSINE 5∲-0-(3-THIOPHOSPHATE)] ON SAPONIN-PERMEABILISED PLATELETS: INVOLVEMENT OF 'G' PROTEINS IN PLATELET ACTIVATION

1987 ◽  
Author(s):  
K S Authi ◽  
B J Evenden ◽  
N Crawford
Keyword(s):  
G Protein ◽  
Shape Change ◽  
Second Messengers ◽  
Phosphatidyl Inositol ◽  
Protein S ◽  
Human Platelets ◽  
Dependent Manner ◽  
Guanine Nucleotide Binding Protein ◽  
Intact Cells ◽  
Dose Dependent

Certain ligand-receptor interactions at cell surfaces lead to the phospholipase-C (PLC) hydrolysis of phosphatidyl inositol (4.5) bisphosphate (PIP2). The products serve as intracellular second messengers, e.g. inositol (1.4.5) trisphosphate (IP3) releases Ca2+ from intracellular stores and diacylglycerol activates protein kinase-C. From studies using GTP and analogues (e.g. GTPγS) there is evidence of a key role for a guanine nucleotide binding protein(s) as a link between receptors and PIP2 hydrolysis. We report the actions of GTPγS on washed human platelets permeabilised with saponin (12-14 μg/ml) to allow penetration of low MWt polar substances. The responses to GTPγS are dose dependent (range 9-60 μM) and at 60 μM the agent induces shape change, aggregation and the secretion of 50% of previously incorporated [14C]-5HT. No effect of GTPγS is seen with intact cells. Shape change occurs 25-30 sec after GTPγS; aggregation and secretion is complete after 3 min. When GTP was used (up to 135 μM) with similarly permeabilised platelets no responses were initiated. Phosphatidylinositol turnover was monitored using 32P-labelling before permeabilisation. The addition of 90 μM GTPγS resulted in a 143 ± 23% (n=4) increase in 32P-phosphatidic acid (PA) with respect to the basal levels of “saponised control” cells. These findings suggest that GTPγS stimulates PLC activity through a ‘G’ protein interaction. The GDP analogue (GDPβS) produced no activation responses in saponised platelets but inhibited responses induced by GTPγS in a dose dependent manner (0-480 μM, max inhibition 480 μM). At 960 μM, GDPβS totally inhibited aggregation and secretion initiated by low doses of thrombin (0.1 U/ml) and collagen (1 μg/ml). Identical inhibition by GDPβS of thrombin and collagen-induced activation of intact platelets was observed indicating membrane penetration of this analogue. Shape change effects were not inhibited by GDPSS. The inhibitory effects of GDPSS towards thrombin and collagen induced secretion could be progressively overcome at higher doses of thrombin (0.2 U/ml - 2 U/ml) and collagen (5 μg/ml - 60 μg/ml) suggesting that at higher concentrations these agonists may exert effects through 'G' protein-independent mechanisms.

scholarly journals Association of ligand-receptor complexes with actin filaments in human neutrophils: a possible regulatory role for a G-protein.

1989 ◽  
Vol 109 (6) ◽  
pp. 2791-2799 ◽  
Author(s):  
E Särndahl ◽  
M Lindroth ◽  
T Bengtsson ◽  
M Fällman ◽  
J Gustavsson ◽  
...  
Keyword(s):  
G Protein ◽  
Second Messengers ◽  
Human Neutrophils ◽  
Receptor Complex ◽  
Chemotactic Peptide ◽  
Filamentous Actin ◽  
High Concentration ◽  
Intact Cells ◽  
Peptide Receptor ◽  
Receptor Complexes

Most ligand-receptor interactions result in an immediate generation of various second messengers and a subsequent association of the ligand-receptor complex to the cytoskeleton. Depending on the receptor involved, this linkage to the cytoskeleton has been suggested to play a role in the termination of second messenger generation and/or the endocytic process whereby the ligand-receptor complex is internalized. We have studied how the binding of chemotactic peptide-receptor complexes to the cytoskeleton of human neutrophils is accomplished. As much as 76% of the tritiated formylmethionyl-leucyl-phenylalanine (fMet-Leu-[3H]Phe) specifically bound to intact cells, obtained by a 30-s stimulation with 20 nM fMet-Leu-[3H]Phe, still remained after Triton X-100 extraction. Preincubating intact cells with dihydrocytochalasin B (dhCB) or washing the cytoskeletal preparation with a high concentration of potassium, reduced the binding of ligand-receptor complexes to the cytoskeleton by 46% or more. Inhibition of fMet-Leu-Phe-induced generation of second messengers by ADP-ribosylating the alpha-subunit of the receptor-coupled G-protein with pertussis toxin, did not reduce the binding of ligand-receptor complexes to the cytoskeleton. However, using guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) to prevent the dissociation of the fMet-Leu-Phe-associated G-protein within electrically permeabilized cells, led to a pronounced reduction (62%) of the binding between ligand-receptor complexes and the cytoskeleton. In summary, in human neutrophils the rapid association between chemotactic peptide-receptor complexes and the cytoskeleton is dependent on filamentous actin. This association is most likely regulated by the activation and dissociation of the fMet-Leu-Phe-associated G-protein.

scholarly journals Exocytosis from permeabilized bovine adrenal chromaffin cells is differently modulated by guanosine 5′-[γ-thio]triphosphate and guanosine 5′-[β γ-imido]triphosphate. Evidence for the involvement of various guanine nucleotide-binding proteins

1992 ◽  
Vol 284 (2) ◽  
pp. 321-326 ◽  
Author(s):  
G Ahnert-Hilger ◽  
U Wegenhorst ◽  
B Stecher ◽  
K Spicher ◽  
W Rosenthal ◽  
...  
Keyword(s):  
G Protein ◽  
Chromaffin Cells ◽  
Inhibitory Effect ◽  
Adrenal Chromaffin Cells ◽  
Prolonged Incubation ◽  
Permeabilized Cells ◽  
Alpha Subunits ◽  
Cytoplasmic Material ◽  
Streptolysin O ◽  
Adrenal Chromaffin

1. In bovine adrenal chromaffin cells made permeable either to molecules less than or equal to 3 kDa with alphatoxin or to proteins less than or equal to 150 kDa with streptolysin O, the GTP analogues guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5′-[gamma-thio]triphosphate (GTP[S]) differently modulated Ca(2+)-stimulated exocytosis. 2. In alphatoxin-permeabilized cells, p[NH]ppG up to 20 microM activated Ca(2+)-stimulated exocytosis. Higher concentrations had little or no effect. At a free Ca2+ concentration of 5 microM, 7 microM-p[NH]ppG stimulated exocytosis 6-fold. Increasing the free Ca2+ concentration reduced the effect of p[NH]ppG. Pretreatment of the cells with pertussis toxin prevented the activation of the Ca(2+)-stimulated exocytosis by p[NH]ppG. 3. In streptolysin O-permeabilized cells, p[NH]ppG did not activate, but rather inhibited Ca(2+)-dependent catecholamine release under all conditions studied. In the soluble cytoplasmic material that escaped during permeabilization with streptolysin O, different G-protein alpha-subunits were detected using an appropriate antibody. Around 15% of the cellular alpha-subunits were detected in the supernatant of permeabilized control cells. p[NH]ppG or GTP[S] stimulated the release of alpha-subunits 2-fold, causing a loss of about 30% of the cellular G-protein alpha-subunits under these conditions. Two of the alpha-subunits in the supernatant belonged to the G(o) type, as revealed by an antibody specific for G(o) alpha. 4. GTP[S], when present alone during stimulation with Ca2+, activated exocytosis in a similar manner to p[NH]ppG. Upon prolonged incubation, GTP[S], in contrast to p[NH]ppG, inhibited Ca(2+)-induced exocytosis from cells permeabilized by either of the pore-forming toxins. This effect was resistant to pertussin toxin. 5. The p[NH]ppG-induced activation of Ca(2+)-stimulated release from alphatoxin-permeabilized chromaffin cells may be attributed to one of the heterotrimeric G-proteins lost during permeabilization with streptolysin O. The inhibitory effect of GTP[S] on exocytosis is apparently not mediated by G-protein alpha-subunits, but by another GTP-dependent process still occurring after permeabilization with streptolysin O.

scholarly journals Induction of cytosolic phospholipase A2 activity by phosphatidic acid and diglycerides in permeabilized human neutrophils: interrelationship between phospholipases D and A2

1997 ◽  
Vol 322 (2) ◽  
pp. 353-363 ◽  
Author(s):  
Sue A. BAULDRY ◽  
Rhonda E. WOOTEN
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase A2 ◽  
Second Messengers ◽  
Platelet Activating Factor ◽  
Human Neutrophils ◽  
Partial Restoration ◽  
Permeabilized Cells ◽  
Cpla2 Activation ◽  
Factor Α ◽  
Cytosolic Pla2

Relationships between phospholipases are poorly understood, but phosphatidic acid (PA) and diglycerides (DGs), produced by phospholipase D (PLD) and phosphatidate phosphohydrolase actions, might function as second messengers coupling cell stimulation to cellular responses. This study investigates the role of PLD-mediated PA and DG formation in inducing phospholipase A2 (PLA2) activity in intact human neutrophils (PMNs) and in PMNs permeabilized with Staphylococcus aureusα-toxin. PMNs were labelled with [3H]arachidonic acid (AA) to assess AA release and metabolism and diacylglycerol formation, or with [3H]1-O-hexadecyl-2-lyso-glycerophosphatidylcholine for the determination of platelet-activating factor (PAF), PA and alkylacylglycerol production. In intact PMNs primed with tumour necrosis factor α before stimulation with N-formyl-Met-Leu-Phe, AA release and metabolism and PAF formation increased in parallel with enhanced PA and DG formation, and inhibition of PA and DG production led to a decrease in both AA release and PAF accumulation. In α-toxin-permeabilized PMNs, AA release and PAF production result from the specific activation of cytosolic PLA2 (cPLA2). In this system, PA and DG formation were always present when cPLA2 activation occurred; blocking PA and DG production inhibited AA release and PAF accumulation. Adding either PA or DG back to permeabilized cells (with endogenous PA and DG formation blocked) led to a partial restoration of AA release and PAF formation; a combination of PA and DGs reconstituted full cPLA2 activity. These results strongly suggest that products of PLD participate in activating cPLA2 in PMNs.

CCK receptor phosphorylation exposes regulatory domains affecting phosphorylation and receptor trafficking

2000 ◽  
Vol 279 (6) ◽  
pp. C1986-C1992 ◽  
Author(s):  
Rammohan V. Rao ◽  
Eileen L. Holicky ◽  
Susan M. Kuntz ◽  
Laurence J. Miller
Keyword(s):  
G Protein ◽  
Chinese Hamster ◽  
G Protein Coupled Receptors ◽  
Receptor Internalization ◽  
Ovary Cell ◽  
Guanine Nucleotide Binding Protein ◽  
Receptor Phosphorylation ◽  
Regulatory Domains ◽  
Ovary Cell Line ◽  
G Protein Coupled

Agonist-stimulated phosphorylation of guanine nucleotide-binding protein (G protein)-coupled receptors has been recognized as an important mechanism for desensitization by interfering with coupling of the activated receptor with its G protein. We recently described a mutant of the CCK receptor that modified two of five key sites of phosphorylation (S260,264A) and eliminated agonist-stimulated receptor phosphorylation, despite normal ligand binding and signaling (20). As expected, this nonphosphorylated mutant had impaired rapid desensitization but was ultimately able to be desensitized by normal receptor internalization. Here we demonstrate that this mutant receptor is also defective in resensitization, with abnormal recycling to the cell surface. To explore this, another receptor mutant was prepared, replacing the same serines with aspartates to mimic the charge of serine-phosphate (S260,264D). This mutant was expressed in a Chinese hamster ovary cell line and shown to bind CCK normally. It had accelerated kinetics of signaling and desensitization and was phosphorylated in response to agonist occupation, with all other normal sites of phosphorylation modified. It was internalized like wild-type receptors and was resensitized and trafficked normally. This provides evidence for an additional important function for phosphorylation of G protein-coupled receptors. Phosphorylation may induce a conformational change in the receptor to expose other potential sites of phosphorylation and to expose domains involved in the targeting and trafficking of endosomes. The hierarchical phosphorylation of these sites may play a key role in receptor regulation.

scholarly journals Regulation of Ca2+ current in frog ventricular cardiomyocytes by guanosine 5'-triphosphate analogues and isoproterenol.

1993 ◽  
Vol 102 (3) ◽  
pp. 525-549 ◽  
Author(s):  
T D Parsons ◽  
H C Hartzell
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Ventricular Myocytes ◽  
Calcium Currents ◽  
Internal Perfusion ◽  
High Concentration ◽  
Patch Clamp Technique ◽  
Intact Cells ◽  
Molecule Interactions ◽  
Gamma S

Calcium currents (ICa) were measured in frog ventricular myocytes using the whole-cell patch clamp technique and a perfused pipette. To gain insight into the role of G proteins in the regulation of ICa in intact cells, the effect of internal perfusion with hydrolysis-resistant GTP analogues, guanylyl 5'-imidodiphosphate (GppNHp) or guanosine 5'-thiotriphosphate (GTP gamma S), on ICa stimulated by isoproterenol (Iso) or forskolin (Forsk) was examined. Significant differences were observed between the effects of the two GTP analogues. Internal perfusion of GppNHp resulted in a near-complete (approximately 80%) and irreversible inhibition of Iso-stimulated ICa. In contrast, internal perfusion with GTP gamma S resulted in only a partial (approximately 40%) inhibition of Iso- or Forsk-stimulated ICa. The fraction of the current not inhibited by GTP gamma S remained persistently elevated after the washout of Iso but declined to basal levels upon washout of Forsk. Excess internal GTP or GppNHp did not reduce the persistent ICa. Internal adenosine 5'-thiotriphosphate (ATP gamma S) mimicked the GTP gamma S-induced, persistent ICa. GppNHp sometimes induced a persistent ICa, but only if GppNHp was present at high concentration before Iso exposure. Inhibitors of protein kinase A inhibited both the GTP gamma S- and ATP gamma S-induced, persistent ICa. We conclude that: (a) GTP gamma S is less effective than GppNHp in inhibiting adenylyl cyclase (AC) via the inhibitory G protein, Gi; and (b) the persistent ICa results from a long-lived Gs-GTP gamma S complex that can activate AC in the absence of Iso. These results suggest that different hydrolysis-resistant nucleotide analogues may behave differently in activating G proteins and imply that the efficacy of G protein-effector molecule interactions can depend on the GTP analogue with which the G protein is activated.

scholarly journals Respiratory Syncytial Virus Grown in Vero Cells Contains a Truncated Attachment Protein That Alters Its Infectivity and Dependence on Glycosaminoglycans

2009 ◽  
Vol 83 (20) ◽  
pp. 10710-10718 ◽  
Author(s):  
Steven Kwilas ◽  
Rachael M. Liesman ◽  
Liqun Zhang ◽  
Edward Walsh ◽  
Raymond J. Pickles ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
G Protein ◽  
Cell Cultures ◽  
Vero Cells ◽  
Target Cells ◽  
Apical Surface ◽  
Virus Attachment ◽  
C Terminus ◽  
Syncytial Virus

ABSTRACT Human respiratory syncytial virus (RSV) contains a heavily glycosylated 90-kDa attachment glycoprotein (G). Infection of HEp-2 and Vero cells in culture depends largely on virion G protein binding to cell surface glycosaminoglycans (GAGs). This GAG-dependent phenotype has been described for RSV grown in HEp-2 cells, but we have found that it is greatly reduced by a single passage in Vero cells. Virions produced from Vero cells primarily display a 55-kDa G glycoprotein. This smaller G protein represents a post-Golgi compartment form that is lacking its C terminus, indicating that the C terminus is required for GAG dependency. Vero cell-grown virus infected primary well-differentiated human airway epithelial (HAE) cell cultures 600-fold less efficiently than did HEp-2 cell-grown virus, indicating that the C terminus of the G protein is also required for virus attachment to this model of the in vivo target cells. This reduced infectivity for HAE cell cultures is not likely to be due to the loss of GAG attachment since heparan sulfate, the primary GAG used by RSV for attachment to HEp-2 cells, is not detectable at the apical surface of HAE cell cultures where RSV enters. Growing RSV stocks in Vero cells could dramatically reduce the initial infection of the respiratory tract in animal models or in volunteers receiving attenuated virus vaccines, thereby reducing the efficiency of infection or the efficacy of the vaccine.

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