The mitogenic signaling pathway of fibroblast growth factor is not mediated through polyphosphoinositide hydrolysis and protein kinase C activation in hamster fibroblasts

Basic or acidic fibroblast growth factor (FGF), alone, was found to be as potent as alpha-thrombin to reinitiate DNA synthesis in G0-arrested Chinese hamster lung fibroblasts (CCL39). Basic FGF at 50 ng/ml or thrombin at 1 unit/ml rapidly initiated early events such as cytoplasmic alkalinization (0.2-0.3 pH units), rise in cytoplasmic Ca2+, phosphorylation of ribosomal protein S6 and increased c-myc expression, followed by a 30-40-fold increase in labeled nuclei. Whereas thrombin is a potent activator of phospholipase C as judged by the rapid release of inositol trisphosphate, inositol bisphosphate and by the massive accumulation of total inositol phosphate (IP) in the presence of 20 mM Li+, FGF failed to induce the breakdown of polyphosphoinositides in quiescent CCL39 cells. Indeed, no inositol trisphosphate nor inositol bisphosphate could be detected in response to FGF; in presence of Li+ the total IP release never exceeded 8% of the IP released by the action of thrombin. Two additional findings indicated that FGF and thrombin activate different signaling pathways. First, we found that, in contrast to thrombin, the FGF-induced rise in the cytoplasmic free Ca2+ concentration measured by quin-2 fluorescence, is strictly dependent upon the presence of Ca2+ in the external medium. Second, we found that FGF failed to activate protein kinase C as judged by the epidermal growth factor-receptor binding assay. Treatment of the cells with either thrombin or phorbol esters, rapidly inhibited 125I-labeled epidermal growth factor binding (50-60%). Basic or acidic FGF had no effect. We conclude that: the FGF-receptor signaling pathway is not coupled to phospholipase C activation, and early mitogenic events and reinitiation of DNA synthesis can be initiated independently of inositol lipid breakdown and protein kinase C activation.     

Pertussis toxin inhibits thrombin-induced activation of phosphoinositide hydrolysis and Na+/H+ exchange in hamster fibroblasts.

Prior treatment with pertussis toxin of G0-arrested hamster fibroblasts (CCL39) results in a dose-dependent inhibition of two early events of the mitogenic response elicited by alpha-thrombin: accumulation of inositol phosphates in Li+-treated cells, and activation of the Na+/H+ antiport, measured either by the amiloride-sensitive 22Na+ influx or by the increase in intracellular pH. At 10(-1) U/ml of alpha-thrombin, the maximal inhibition was approximately 50% for these two early cellular responses, but the pertussis toxin effect was more pronounced at lower thrombin concentrations. In contrast, pertussis toxin does not affect the Na+/H+ antiport activation induced by phorbol esters or EGF, the action of which is not mediated by the phosphoinositide-metabolizing pathway in CCL39 cells. Therefore, our data suggest the following. A GTP-binding regulatory protein is probably involved in signal transduction between thrombin receptors and the phosphatidylinositol 4,5-bisphosphate-specific phospholipase C. This regulation does not seem to be exerted via modulations of cyclic AMP levels. The thrombin-induced activation of Na+/H+ antiport is, at least in part, mediated by the protein kinase C, as a consequence of stimulation of phosphatidylinositol turnover.     

Homologous desensitization of thrombin-induced phosphoinositide breakdown in hamster lung fibroblasts

Activation of phosphoinositide breakdown is thought to be an important signaling pathway involved in the mitogenic effects of alpha-thrombin in Chinese hamster lung fibroblasts. We have previously shown that the initial strong stimulation of inositol phosphate formation induced by thrombin in quiescent hamster cells (CCL39 line) is rapidly attenuated. We now report that this desensitization of phospholipase C to thrombin 1) is independent of protein kinase C activation, because thrombin-induced desensitization normally occurs in cells that have been depleted in protein kinase C by a prolonged treatment with a phorbol ester, and 2) is even independent of phosphoinositide hydrolysis because the desensitization still occurs, although at a lesser degree, at 4 degrees C, in the absence of any phospholipase C activity. Furthermore, phospholipase C desensitization to thrombin is homologous. It does not affect the response to thrombin-free serum or the direct activation by A1F-4 of the GTP-binding protein (G-protein) coupled to phospholipase C. We therefore conclude that the desensitization of phospholipase C to thrombin does not result from an impairment of the G-protein-phospholipase C complex, or from a depletion in phosphoinositides, but rather from a modification of thrombin receptors leading to their uncoupling from G-protein. This modification is slowly reversible because, upon thrombin removal, a prolonged incubation (approximately 2 h) restores responsiveness of the cells to thrombin. Although the desensitization seems to depend on thrombin receptor occupancy, it cannot be accounted for by an internalization of the occupied receptors, because it is not blocked at 4 degrees C. The exact mechanism underlying this homologous desensitization of thrombin receptors remains to be elucidated.     

Tyrosine kinase-activating growth factors potentiate thrombin- and AIF4- -induced phosphoinositide breakdown in hamster fibroblasts. Evidence for positive cross-talk between the two mitogenic signaling pathways

Basic fibroblast growth factor (FGF) and alpha-thrombin can stimulate DNA synthesis in Chinese hamster fibroblasts (CCL39) by two separate signaling pathways (Chambard, J.C., Paris, S., L'Allemain, G., and Pouysségur, J. (1987) Nature 326, 800-803) but can also act synergistically. We have examined whether this synergism might depend upon changes in inositol lipid metabolism. Indeed, FGF, which has no effect on its own on phosphoinositide hydrolysis, potentiates (by up to 2-fold) thrombin-induced formation of inositol phosphates. This enhancing effect is also observed upon direct activation by AIF4- of the GTP-binding protein coupled to phospholipase C, and is best revealed when phospholipase C is weakly stimulated. With low thrombin concentrations or with AIF4-, the formation of inositol phosphates is immediately increased with a marked reduction of the initial lag, whereas at high thrombin concentrations, the stimulation by FGF becomes pronounced only after desensitization of phospholipase C to thrombin. FGF-induced potentiation is not mimicked by calcium ionophores, but is likewise elicited by epidermal growth factor, platelet-derived growth factor, and to a lesser extent by insulin, other growth factors known to activate receptor tyrosine kinases. We therefore propose that the tyrosine kinase-activating growth factors enhance the coupling between GTP-binding protein and phospholipase C, presumably through the phosphorylation of one of these two proteins. Treatment of cells with pertussis toxin attenuates thrombin-induced phospholipase C activity but does not impede the potentiation by FGF. Comparison of the potentiating effects of FGF on inositol phosphate formation and on DNA synthesis suggests than an increased production of second messengers by the inositol lipid pathway in the first hours of stimulation might be, at least in part, responsible for the synergistic actions of FGF and thrombin on DNA synthesis.     

Activation of phospholipase C in platelets by platelet activating factor and thrombin causes hydrolysis of a common pool of phosphatidylinositol 4,5-bisphosphate

Despite their physicochemical and mechanistic differences platelet activating factor (or acetylglycerylether phosphorylcholine; AGEPC) and thrombin, both platelet stimulatory agents, induce phosphoinositide turnover in platelets. We therefore investigated the stimulation of the phosphoinositide phosphodiesterase by these agents and questioned whether they evoked hydrolysis of the same or different pools of phosphoinositides. [3H]Inositol-labelled rabbit platelets were challenged with thrombin and/or AGEPC under a variety of protocols, and the phospholipase C mediated production of radioactive inositol monophosphate (IP); inositol bisphosphate (IP2) and inositol trisphosphate (IP3) was used as the parameter. AGEPC (1 X 10(-9) M) caused a transient maximum (5 to 6-fold) increase in [3H]IP3 at 5 s followed by a decrease. Thrombin (2 U/ml) elicited an increase in [3H]IP3 at a much slower rate than AGEPC; 2 fold at 5 s, 5 fold at 30 s and a maximum 6 to 8-fold at 2-5 min. Compared to AGEPC, thrombin stimulated generation of [3H]IP2 and [3H]IP were severalfold higher. When thrombin and AGEPC were added together to platelets there was no evidence for an additive increase in inositol polyphosphate levels except at earlier time points where increases were submaximal. When AGEPC was added at various time intervals after thrombin pretreatment, no additional increases in [3H]IP3 were observed over that maximally seen with thrombin or AGEPC alone. In another set of experiments, submaximal increases (about 1/4 and 1/2 of maximum) in [3H]IP3 were achieved by using selected concentrations of thrombin (0.1 U and 0.3 U, respectively) and then AGEPC (1 X 10(-9) M) was added for 5 s. Once again the increase in [3H]IP3 was close to the maximal level seen with thrombin or AGEPC individually. It is concluded that thrombin and AGEPC differentially activated phosphoinositide phosphodiesterase (phospholipase C) in rabbit platelets and that the stimulation of the phospholipase C by these two stimuli causes IP3 production via hydrolysis of a common pool of phosphatidylinositol 4,5-bisphosphate.     

Thrombin-induced phosphoinositide hydrolysis in platelets. Receptor occupancy and desensitization.

The relationship between occupancy of thrombin receptors on platelets and enhanced phosphoinositide hydrolysis was analysed by examination of the dose-response relationship, the effects of thrombin inhibitors and the contribution of secondary effects. Washed human platelets were labelled with [3H]inositol, and agonist-induced accumulation of labelled inositol phosphates was measured. The dose-response curves and the time courses for alpha-thrombin- or gamma-thrombin-induced accumulation of inositol phosphates were similar to those for dense-granule secretion. Addition of the thrombin inhibitor hirudin to thrombin-activated platelets revealed that the continuous presence of active thrombin was required to maintain the accumulation of labelled inositol phosphates; the total production of inositol phosphates increased with longer periods of exposure to thrombin, reaching a maximum between 5 and 10 min. After activation with thrombin, the ability of a second, greater, addition of thrombin to induce additional phosphoinositide hydrolysis decreased with time; it was absent within 10 min after the first addition. The failure to sustain accumulation of labelled inositol phosphates or to respond to a second addition of thrombin beyond 10 min was not due to depletion of the pool of labelled precursors, because the platelets retained their ability to respond to collagen. Addition of ADP-consuming enzymes decreased sensitivity to thrombin, but inhibition of cyclo-oxygenase with indomethacin did not impair the thrombin-induced hydrolysis of phosphoinositides. It was concluded that thrombin-induced hydrolysis of phosphoinositides has characteristics consistent with mediation by a receptor that is similar to that that triggers dense-granule secretion, requires continuous presence of active thrombin to be maintained, is mediated by a receptor that displays thrombin-induced desensitization, and is only partially enhanced by secondary agents.     

Synergism between thrombin and adrenaline (epinephrine) in human platelets. Marked potentiation of inositol phospholipid metabolism.

We have studied synergism between adrenaline (epinephrine) and low concentrations of thrombin in gel-filtered human platelets prelabelled with [32P]Pi. Suspensions of platelets, which did not contain added fibrinogen, were incubated at 37 degrees C to measure changes in the levels of 32P-labelled phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and phosphatidate (PA), aggregation and dense-granule secretion after stimulation. Adrenaline alone (3.5-4.0 microM) did not cause a change in any parameter (phosphoinositide metabolism, aggregation and dense-granule secretion), but markedly enhanced the thrombin-induced responses over a narrow range of thrombin concentrations (0.03-0.08 units/ml). The thrombin-induced hydrolysis of inositol phospholipids by phospholipase C, which was measured as the formation of [32P]PA, was potentiated by adrenaline, as was the increase in the levels of [32P]PIP2 and [32P]PIP. The presence of adrenaline caused a shift to the left for the thrombin-induced changes in the phosphoinositide metabolism, without affecting the maximal levels of 32P-labelled compounds obtained. A similar shift by adrenaline in the dose-response relationship was previously demonstrated for thrombin-induced aggregation and dense-granule secretion. Also, the narrow range of concentrations of thrombin over which adrenaline potentiates thrombin-induced platelet responses is the same for changes in phosphoinositide metabolism and physiological responses (aggregation and dense-granule secretion). Our observations clearly indicate that adrenaline directly or indirectly influences thrombin-induced changes in phosphoinositide metabolism.     

A rapid attenuation of muscarinic agonist stimulated phosphoinositide hydrolysis precedes receptor sequestration in human SH-SY-5Y neuroblastoma cells

Agonist occupancy of muscarinic cholinergic receptors in human SH-SY-5Y neuroblastoma cells elicited two kinetically distinct phases of phosphoinositide hydrolysis when monitored by either an increased mass of inositol 1,4,5-trisphosphate, or the accumulation of a total inositol phosphate fraction. Within 5s of the addition of the muscarinic agonist, oxotremorine-M, the phosphoinositide pool was hydrolyzed at a maximal rate of 9.5%/min. This initial phase of phosphoinositide hydrolysis was short-lived (t_1/2=14s) and after 60s of agonist exposure, the rate of inositol lipid breakdown had declined to a steady state level of 3.4%/min which was then maintained for at least 5–10 min. This rapid, but partial, attenuation of muscarinic receptor stimulated phosphoinositide hydrolysis occurred prior to the agonist-induced internalization of muscarinic receptors.Abbreviations I(1,4,5)P₃ inositol 1,4,5-trisphosphate - IP total inositol phosphate fraction - IPL total inositol lipid fraction - mAChR muscarinic acetylcholine receptor - NMS N-methylscopolamine - Oxo-M oxotremorine-M - PI phosphatidylinositol - PIP phosphatidylinositol 4-phosphate - PIP₂ phosphatidylinositol 4,5-bisphosphate - PPI phosphoinositide - QNB quinuclidinyl benzilate Special issue dedicated to Dr. Bernard W. Agranoff     

Induction of the fibrinogen receptor on human platelets by intracellular mediators

We have used platelets permeabilized with saponin to examine the mechanism by which platelet activation causes the exposure of surface receptors for fibrinogen. Receptor exposure was detected using 125I-fibrinogen and 125I-PAC1, a monoclonal antibody specific for the activated form of the fibrinogen receptor. The potential mediators that were studied included guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and guanosine 5'O-(thiotriphosphate) (GTP gamma S), which cause G protein-dependent phospholipase C activation in platelets; inositol 1,4,5-triphosphate (IP3), which causes Ca2+ release from the platelet dense tubular system; and diacylglycerol and phorbol ester, which activate protein kinase C. Each of these molecules caused fibrinogen and PAC1 binding. The effect of IP3 was mimicked by raising the cytosolic free Ca2+ concentration in the permeabilized platelets. However, IP3 and Ca2+-induced PAC1 binding were abolished by indomethacin or aspirin, which had no effect on PAC1 binding caused by Gpp(NH)p, phorbol ester, or diacylglycerol. This suggests that the response to IP3 and Ca2+ is due to the formation of metabolites of arachidonic acid. One such metabolite, TxA2, is believed to activate platelets by stimulating G protein-dependent phosphoinositide hydrolysis. Indeed, we found that the G protein inhibitor guanyl-5'-yl thiophosphate (GDP beta S) inhibited PAC1 binding caused by a thromboxane A2 analog (U46619), IP3, and Ca2+, but had no effect on diacylglycerol or phorbol ester-induced PAC1 binding. Thrombin-induced PAC1 binding and phosphoinositide hydrolysis were also inhibited by GDP beta S and by pertussis toxin. Increasing the thrombin concentration overcame the inhibition of PAC1 binding caused by GDP beta S but did not overcome the inhibition of phosphoinositide hydrolysis. These observations demonstrate that fibrinogen receptor exposure occurs by at least two routes. One of these, in response to agonists such as thrombin and U46619, is initiated by G protein-dependent phosphoinositide hydrolysis and involves the formation of IP3 and diacylglycerol. IP3 appears to act by stimulating Ca2+-dependent arachidonic acid metabolism which, in turn, triggers further phosphoinositide hydrolysis. Diacylglycerol acts by stimulating protein kinase C. A second route is activated by high concentrations of thrombin and is independent of phosphoinositide hydrolysis.     

GTP and cytosol stimulate phosphoinositide hydrolysis in isolated platelet membranes

Hydrolysis of polyphosphoinositides by phospholipase C was examined in isolated membranes prepared from [32P]labelled platelets. In the presence of GTP gamma S, thrombin increased the release of inositol triphosphate and inositol biphosphate approximately 500%. GTP gamma S alone stimulated release 2 fold. Maximal activation of thrombin-induced phosphoinositide hydrolysis was observed at 10 uM GTP. Although addition of calcium had no effect, 2 mM EGTA completely inhibited inositolphosphate release. Addition of high speed supernatant to [32P]labelled membranes stimulated the release of inositolphosphates. This hydrolysis was further enhanced by the addition of GTP. These data demonstrate that the breakdown of polyphosphoinositides in isolated platelet membranes is dependent on GTP and stimulated by platelet cytosol.     

EGF and insulin action in fibroblasts. Evidence that phosphoinositide hydrolysis is not an essential mitogenic signalling pathway

Chinese hamster lung fibroblasts (CHL) arrested in G0 by serum starvation reinitiate DNA synthesis in response to either EGF, thrombin or serum. Arrested cells, prelabelled to equilibrium with [3H]inositol and receiving 20 mM LiCl prior stimulation, released rapidly large amounts of inositol phosphates when stimulated with thrombin or serum. In sharp contrast, EGF alone, or in association with insulin, failed to induce phosphoinositide breakdown at either early or late stages of EGF stimulation or in growing cells in EGF-supplemented serum-free medium. Phospholipase C remained, however, highly activatable by thrombin at all stages of EGF stimulation. Since EGF and thrombin are equally potent mitogens for CHL, we conclude that hydrolysis of polyphosphoinositides is not an exclusive signalling pathway for commitment to DNA replication and cell division.     

Excitatory Amino Acid Agonist-Antagonist Interactions at 2-Amino-4-Phosphonobutyric Acid-Sensitive Quisqualate Receptors Coupled to Phosphoinositide Hydrolysis in Slices of Rat Hippocampus

Studies were carried out to define the relative affinities and intrinsic activities of excitatory amino acid agonists that activate receptor sites coupled to phosphoinositide hydrolysis in brain. Slices of rat hippocampus were prelabeled with myo-[3H]inositol, and agonist stimulation was indexed by measuring the accumulation of [3H]inositol monophosphate [( 3H]IP) in the presence of Li+. It was observed that ibotenic (IBO) and quisqualic (QUIS) acids both elicit highly significant, concentration-dependent stimulation of phosphoinositide hydrolysis. Whereas maximal stimulation by IBO (10(-3) M) was four- to fivefold over basal values, the maximal effect of QUIS (10(-4) M) was less (about twofold). Based on the relative concentrations required for 50% maximal stimulation, QUIS was 20 times more potent than IBO. Stimulation of phosphoinositide hydrolysis by either IBO or QUIS was additive to the effects of nonexcitatory amino acid agonists (carbachol and norepinephrine) in this tissue. However, the stimulatory effects of IBO plus QUIS were not additive. At greater than or equal to 10(-4) M, QUIS significantly inhibited phosphoinositide hydrolysis by a maximal stimulatory concentration of IBO (10(-3) M) to a level observed with QUIS alone. Other excitatory amino acid agonists, including kainate, N-methyl-D-aspartate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), had no stimulatory effects at concentrations as high as 10(-3) M. The D,L or L forms of 2-amino-4-phosphonobutyric acid (AP4), but not D-AP4, significantly enhanced [3H]IP levels to approximately 135% of basal values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coupling between phosphoinositide breakdown and early mitogenic events in fibroblasts. Studies with fluoroaluminate, vanadate, and pertussis toxin

In the preceding paper (Paris, S., and Pouysségur J. (1987) J. Biol. Chem. 262, 1970-1976), AlF4- and vanadate have been shown to induce inositol phosphate formation in resting hamster fibroblasts (CCL39). In this study, we show that these two phosphate analogs are good tools to explore the causal relationship between phosphoinositide breakdown and early mitogenic events. AlF4- can activate, very similarly to the mitogen alpha-thrombin: the amiloride-sensitive Na+/H+ antiport, the bumetanide-sensitive Na+/K+/Cl- co-transport, and the expression of c-myc mRNA. The link between phospholipase C activation and these early events of the mitogenic response is demonstrated by the similarity of all dose-response curves for NaF and AlCl3 and by the common sensitivity of the four events to pertussis toxin. Vanadate likewise stimulates the Na+/H+ antiport through a pertussis toxin-sensitive pathway. On longer incubations, both fluoride and vanadate were found to be toxic and failed to induce DNA synthesis. Therefore, we have used pertussis toxin to investigate the link between phospholipase C activation and commitment to DNA synthesis. We show that pertussis toxin strikingly inhibits thrombin-induced reinitiation of DNA synthesis but does not affect the stimulation by the epidermal or fibroblast growth factors, two mitogens that do not stimulate phosphoinositide breakdown in CCL39 cells. In conclusion, these studies demonstrate that activation of phospholipase C, if not an obligatory step in the action of all growth factors, plays a crucial role in the mitogenic signaling pathway of alpha-thrombin.     

Guanosine 5'-O-(3-thiotrisphosphate) potentiates both thrombin- and platelet-derived growth factor-induced inositol phosphate release in permeabilized vascular smooth muscle cells. Signaling mechanisms distinguished by sensitivity to pertussis toxin and phorbol esters

We compared the mechanisms by which thrombin and platelet-derived growth factor (PDGF) activate phospholipase C in cultured vascular smooth muscle cells. Thrombin caused a transient (less than 5 min) increase in inositol trisphosphate (IP3) while PDGF caused a sustained (greater than 10 min) increase. Both pertussis toxin and phorbol 12-myristate 13-acetate (PMA) inhibited the thrombin-induced increase in IP3 but neither agent affected the PDGF-induced increase in IP3. To examine the role of GTP binding (G) proteins in the activation of phospholipase C by these two hormones, GTP analogues were introduced into saponin-permeabilized cells. In the absence of hormones, guanosine 5'-O-(3-thiotrisphosphate) (GTP gamma S) caused a progressive increase in IP3 release which was inhibited 55% by PMA (200 ng/ml). In the presence of thrombin, GTP gamma S caused synergistic increase in IP3 release. The synergism between GTP gamma S and thrombin was virtually eliminated by 10 min prior exposure to PMA (200 ng/ml). When PDGF was the hormonal agonist, GTP gamma S also caused synergistic increase in IP3 release and guanosine 5'-O-(2-thiodiphosphate) blunted PDGF-induced IP3 release. However, in contrast to thrombin, the synergism between GTP gamma S and PDGF was unaffected by PMA. Thus, thrombin and PDGF activate phospholipase C by signal transduction systems which differ in kinetic properties and in sensitivity to PMA and pertussis toxin. Despite these differences, both systems appear to involve GTP binding proteins at some step.     

Characterization of Bradykinin-Induced Phosphoinositide Turnover in Neurohybrid NCB-20 Cells

Phosphoinositide hydrolysis was studied in neurohybrid NCB-20 cells prelabeled with myo-[3H]inositol. Among nearly 20 neurotransmitters and neuromodulators examined, only bradykinin, carbachol, and histamine significantly increased the accumulation of [3H]inositol monophosphate (IP1) in the presence of lithium. The EC50 of bradykinin was 20 nM and the saturating concentration was approximately 1 microM. The bradykinin response was robust (10-fold) and was potently and selectively blocked by a bradykinin antagonist, B 4881 [D-Arg-(Hyp3, Thi, D-Phe)-bradykinin], with a Ki of 10 nM. This effect of bradykinin appeared to be additive to that mediated by activation of muscarinic cholinergic and histamine H1 receptors. The accumulation induced by bradykinin or carbachol was dependent on the presence of calcium in the incubation medium; less than twofold stimulation was observed in the absence of exogenous calcium. Bradykinin-induced [3H]IP1 accumulation required high concentration of lithium to elicit its maximal stimulation; the concentration of lithium required for half maximal effect was about 13 mM, similar to the value reported previously for carbachol-induced accumulation in the same cell line. In contrast, using related neurohybrid NG108-15 cells, bradykinin-induced [3H]IP1 accumulation was found to require much less lithium. IN the presence of lithium, bradykinin also evoked a transient increase in the production of [3H]-inositol bis- and trisphosphate. Basal and bradykinin-induced phosphoinositide breakdown was inhibited by 4 beta-phorbol 12,13-dibutyrate, but was unaffected by the biologically inactive 4 beta-phorbol. Pretreatment of cells with pertussis toxin induced only about 30% loss of the bradykinin-induced [3H]IP1 accumulation, without affecting basal activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Shc adaptor proteins are key transducers of mitogenic signaling mediated by the G protein-coupled thrombin receptor.

The serine protease thrombin activates G protein signaling systems that lead to Ras activation and, in certain cells, proliferation. Whereas the steps leading to Ras activation by G protein-coupled receptors are not well defined, the mechanisms of Ras activation by receptor tyrosine kinases have recently been elucidated biochemically and genetically. The present study was undertaken to determine whether common signaling components are used by these two distinct classes of receptors. Here we report that the adaptor protein Shc, is phosphorylated on tyrosine residues following stimulation of the thrombin receptor in growth-responsive CCL39 fibroblasts. Shc phosphorylation by thrombin or the thrombin receptor agonist peptide is maximal by 15 min and persists for > or = 2 h. Following thrombin stimulation, phosphorylated Shc is recruited to Grb2 complexes. One or more pertussis toxin-insensitive proteins appear to mediate this effect, since (i) pertussis toxin pre-treatment of cells does not blunt the action of thrombin and (ii) Shc phosphorylation on tyrosine can be stimulated by the muscarinic m1 receptor. Shc phosphorylation does not appear to involve protein kinase C, since the addition of 4-beta-phorbol-12,13-dibutyrate has no effect. Rather, thrombin-induced Shc phosphorylation is enhanced in cells depleted of phorbol ester-sensitive protein kinase C isoforms. Expression of mutant Shc proteins defective in Grb2 binding displays a dominant-negative effect on thrombin-stimulated p44 MAP kinase activation, gene induction and cell growth. From these data, we conclude that Shc represents a crucial point of convergence between signaling pathways activated by receptor tyrosine kinases and G protein-coupled receptors.     

Carbachol and histamine stimulation of guanine-nucleotide-dependent phosphoinositide hydrolysis in rat brain cortical membranes.

Guanine nucleotides have been shown to stimulate phosphoinositide breakdown in brain membranes, but no potentiation of such an effect by agonist was demonstrated. We have studied the effect of carbachol and histamine on guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulation of inositol phosphates formation in [3H]inositol-labelled rat brain cortical membranes. In this preparation, GTP[S] enhancement of phosphoinositide hydrolysis required the presence of MgATP and low Ca2+ concentration (100 nM). Carbachol potentiation of the GTP[S] effect was only observed when 1 mM-deoxycholate was also added. Under these conditions, stimulated production of [3H]inositol phosphates was linear for at least 15 min, and [3H]inositol bisphosphate [( 3H]IP2) accounted for approx. 80%, whereas the amount of [3H]inositol trisphosphate [( 3H]IP3) was very low. Stimulation by GTP[S] was concentration-dependent (half-maximal effect at 0.86 microM), and its maximal effect (815% over basal) was increased by 1 mM-carbachol (1.9-fold) and -histamine (1.7-fold). Both agonists decreased the slope index of the GTP[S] concentration/effect curve to values lower than unity, suggesting the appearance of some heterogeneity in the population of guanine-nucleotide-binding proteins (G-proteins) involved. The carbachol and histamine effects were also concentration-dependent, and were inhibited by atropine and mepyramine respectively. Fluoroaluminate stimulated phosphoinositide hydrolysis to a higher extent than GTP[S] plus carbachol, and these stimulations were not additive, indicating that the same polyphosphoinositide phospholipase C-coupled G-protein mediates both effects.     

Mechanism of thrombin-induced arachidonic acid release in osteoblast-like cells

In a previous study, we have reported that thrombin stimulates phosphatidylcholine hydrolysis by phospholipase (PL) D, but has little effect on phosphoinositide hydrolysis by PLC in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the mechanism of the thrombin-induced arachidonic acid (AA) release in MC3T3-E1 cells. Thrombin stimulated AA release dose dependently in the range between 0.1 and 1 U/ml. Quinacrine, a PLA₂ inhibitor, suppressed the thrombin-induced AA release. In addition, quinacrine also suppressed the thrombin-induced prostaglandin E₂ synthesis in these cells. On the other hand, propranolol, which is known to inhibit phosphatidic acid phosphohydrolase, did not affect the thrombin-induced AA release. 1(6-((17β-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-d |ione (U-73122), a PLC inhibitor, had no effect on the AA release by thrombin. In addition, 1,6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC-80267), a selective inhibitor of diacylglycerol lipase, had little effect on the thrombin-induced AA release. Neither propranolol, U-73122 nor RHC-80267 affect the thrombin-induced prostaglandin E₂ synthesis. These results strongly suggest that thrombin induces AA release not by phosphatidylcholine hydrolysis by PLD nor phosphoinositide hydrolysis by PLC but mainly by PLA₂ in osteoblast-like cells.     

The formation of inositol 1,2-cyclic phosphate on agonist stimulation of phosphoinositide breakdown in mouse pancreatic minilobules. Evidence for direct phosphodiesteratic cleavage of phosphatidylinositol

It is generally thought that formation of inositol 1,2-cyclic phosphate (IcP) on agonist-stimulated "breakdown" of endogenous phosphatidylinositol in intact cells would provide strong evidence for the direct phosphodiesteratic cleavage of phosphatidylinositol. We report here that on ionophoresis of extracts of pancreatic minilobules incubated with the cholecystokinin/pancreozymin congener, caerulein, the usual inositol phosphates, i.e. inositol 1-phosphate (IP), inositol 4,5-bisphosphate (IP2), and inositol 1,4,5-trisphosphate (IP3) were seen. In addition, an [3H]inositol-labeled unknown was present with the correct electrophoretic mobility of IcP. There was only a trace of "IcP" in the unstimulated pancreatic minilobules. Several lines of evidence indicate that the unknown peak was IcP. 1) It ran on ionophoresis with standard [14C]IcP, and the ratio of 3H to 14C for each point on the peak was a constant within experimental error. 2) The putative IcP peak which had been eluted from the electropherogram also coincided with standard [14C]IcP on paper chromatography. 3) On mild acid hydrolysis in the presence of standard 14C-labeled IP, the putative [3H] IcP peak disappeared and appeared in the exact position of the standard [14C]IP peak, as to be predicted of IcP. The formation of IcP on agonist stimulation supports direct phosphodiesteratic cleavage of phosphatidylinositol on stimulation of phosphoinositide breakdown in pancreatic minilobules.