New developments in phospholipase D.
Explore the source record for details and available documents.
ADP-Ribosylation Factors↗
Biomedical subjects
J H Exton
Publications and source records attributed to J H Exton.
Dissociation of tyrosine phosphorylation and activation of phosphoinositide phospholipase C induced by the protein kinase C inhibitor Ro-31-8220 in Swiss 3T3 cells treated with platelet-derived growth factor.
Platelet-derived growth factor (PDGF) stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate (Ptd InsP2) via phospholipase C-gamma1 (PLC-gamma1) in Swiss 3T3 cells. Treatment of cells with the protein kinase C (PKC) inhibitor Ro-31-8220 greatly decreased PDGF-induced tyrosine phosphorylation of PLC-gamma1, but paradoxically enhanced the production of inositol phosphates (InsPs). The inhibitor also caused an increase of PDGF receptor tyrosine phosphorylation at later times. The changes in phosphorylation of the receptor were correlated with alterations in PLC-gamma1 translocation to the particulate fraction. Thus, although activation of PLC-gamma1 was associated with phosphorylation of the receptor and translocation of the enzyme to the particulate fraction, it was dissociated from its tyrosine phosphorylation. A non-receptor-associated, cytosolic tyrosine kinase also was found to phosphorylate PLC-gamma1 in a PDGF-dependent manner, but was not inhibited by Ro-31-8220 in vitro. PKC depletion by phorbol ester treatment decreased the tyrosine phosphorylation of PLC-gamma1 induced by PDGF and slowed the translocation of PLC-gamma1, but Ro-31-8220 produced further effects. The effect of Ro-31-8220 to enhance the production of InsPs could not be attributed to inhibition of PKC since InsPs production with PDGF was decreased in PKC-depleted cells and a stimulatory effect of the inhibitor was still evident. Interestingly, Ro-31-8220 decreased the radioactivity in phosphatidylinositol and increased that in phosphatidylinositol 4-phosphate and PtdInsP2 in cells labeled with myo[3H]inositol. The increased synthesis of PtdInsP2 could contribute to the increased production of InsPs induced by Ro-31-8220. In summary, these results support the conclusion that the activation of PLC-gamma1 in response to PDGF requires autophosphorylation of the receptor and membrane association of PLC-gamma1, but not phosphorylation of the enzyme. Furthermore, the effects of Ro-31-8220 on the tyrosine phosphorylation and activity of PLC-gamma1, and on PtdInsP2 synthesis cannot be attributed to inhibition of PKC.
3T3 Cells↗
Arfaptin 1, a putative cytosolic target protein of ADP-ribosylation factor, is recruited to Golgi membranes.
ADP-ribosylation factors (ARFs) have been implicated in vesicle transport in the Golgi complex. Employing yeast two-hybrid screening of an HL60 cDNA library using a constitutively active mutant of ARF3 (ARF3.Q71L), as a probe, we have identified a cDNA encoding a novel protein with a calculated molecular mass of 38.6 kDa, which we have named arfaptin 1. The mRNA of arfaptin 1 was ubiquitously expressed, and recombinant arfaptin 1 bound preferentially to class I ARFs, especially ARF1, but only in the GTP-bound form. The interactions were independent of myristoylation of ARF. Arfaptin 1 in cytosol was recruited to Golgi membranes by ARF in a guanosine 5'-O-(3-thiotriphosphate)-dependent and brefeldin A-sensitive manner. When expressed in COS cells, arfaptin 1 was localized to the Golgi complex. The yeast two-hybrid system yielded another clone, which encoded a putative protein, which we have named arfaptin 2. This consisted of the same number of amino acids as arfaptin 1 and was 60% identical to it. Arfaptin 2 was also ubiquitously expressed and bound to the GTP-, but not GDP-liganded form of class I ARFs, especially ARF1. These results suggest that arfaptins 1 and 2 may be direct target proteins of class 1 ARFs. Arfaptin 1 may be involved in Golgi function along with ARF1.
ADP-Ribosylation Factor 1↗
Dependence of activated Galpha12-induced G1 to S phase cell cycle progression on both Ras/mitogen-activated protein kinase and Ras/Rac1/Jun N-terminal kinase cascades in NIH3T3 fibroblasts.
We evaluated the roles of mitogen-activated protein kinase (MAPK) and Jun N-terminal kinase (JNK) signaling cascades in Galpha12-induced G1 to S phase cell cycle progression in NIH3T3(M17) fibroblasts. Transient expression of a constitutively active mutant of Galpha12, Galpha12(R203C), resulted in a 2-fold increase in the number of bromodeoxyuridine-positive S phase cells over vector control level under serum-deprived conditions. Consistent with the ability of Galpha12(R203C) to induce G1/S transition, its expression led to a 2-fold increase in cyclin A promoter activity, which showed a marked synergism with a low concentration of serum, resulting in up to a 15-fold elevation over the basal level. In addition, Galpha12(R203C) caused a 2-fold stimulation in E2F-mediated transactivation. Wild type Galpha12 showed similar stimulatory effects on cyclin A promoter activity and E2F-mediated transactivation, although of lesser magnitude. We observed a modest but constitutive activation of MAPK in cells transfected with Galpha12(R203C), which was abolished by a dominant negative form of Ras. Galpha12(R203C) also induced a 3-fold increase in JNK activity, which was abolished by dominant negative forms of either Rac1 or Ras. The expression of dominant negative forms of Ras, MAPK, Rac1, or JNK inhibited Galpha12(R203C)-induced increases in bromodeoxyuridine-positive cells. Also, the dominant negative forms of Ras, MAPK, and JNK strongly inhibited Galpha12(R203C)-induced stimulation of cyclin A promoter activity. These results demonstrate that both the Ras/MAPK and Ras/Rac1/JNK pathways convey necessary, if not sufficient, mitogenic signals induced by Galpha12 activation.
3T3 Cells↗
Role of Rho family proteins in phospholipase D activation by growth factors.
Treatment of fibroblasts with growth factors results in activation of phospholipase D (PLD). In order to determine the role of the Rho family of small GTPases in growth factor-mediated PLD activation, we used cells transfected with wild type and mutant Rac1. In response to epidermal growth factor (EGF), PLD activity was greatly increased in Rat1 fibroblasts expressing wild type Rac1 (wtRac1), and completely abrogated in cells expressing dominant negative N17Rac1, consistent with Rac1 mediating the action of this growth factor. In contrast, in cells treated with platelet-derived growth factor (PDGF) or phorbol ester, the wtRac1 cells showed little or no enhancement of PLD activity, and the response was not affected in the N17Rac1 cells, implying that Rac1 played a minimal role in the activation of PLD by PDGF or protein kinase C. Both growth factors produced an attenuated PLD response in cells expressing constitutively active V12Rac1, but these cells showed other changes, including altered morphology, increased basal PLD, and decreased growth factor receptor autophosphorylation. The effects of EGF and PDGF on phosphoinositide phospholipase C activity were not enhanced in cells expressing wtRac1 or inhibited in those expressing N17Rac1. In cells expressing constitutively active V12Rac1, basal phosphoinositide phospholipase C was elevated, but there were no significant effects of EGF or PDGF. We used C3 transferase of Clostridium botulinum, which ADP-ribosylates and inactivates RhoA, to investigate the involvement of RhoA in the activation of PLD by PDGF. Cells expressing wtRac1 and N17Rac1 showed a decreased PLD in response to PDGF when treated with C3 transferase, indicating a role for RhoA. In summary, these data indicate a major role for Rac1 in the activation of PLD by EGF, but not PDGF or protein kinase C.
Animals↗
Cell signalling through guanine-nucleotide-binding regulatory proteins (G proteins) and phospholipases.
Phospholipases are important enzymes in cell signal transduction since they hydrolyze membrane phospholipids to generate signalling molecules. Heterotrimeric guanine-nucleotide-binding regulatory proteins (G proteins) play a major role in their regulation by a variety of agonists that activate receptors with seven membrane-spanning domains. Phospholipases of the C type, which hydrolyze inositol phospholipids to yield inositol trisphosphate and diacylglycerol, are regulated by the alpha and betagamma subunits of certain heterotrimeric G proteins as well as by receptor-associated and non-receptor-associated tyrosine kinases. Phospholipases of the D type, which hydrolyze phosphatidylcholine to phosphatidic acid, are regulated by members of the ADP-ribosylation factor and Rho subfamilies of small G proteins, and by protein kinase C and other factors. This review presents recent information concerning the molecular details of G protein regulation of these phospholipases.
ADP-Ribosylation Factors↗
Regulation of phosphoinositide phospholipases by G-proteins.
Explore the source record for details and available documents.
Animals↗
Arf and RhoA regulate both the cytosolic and the membrane-bound phospholipase D from human placenta.
In this paper we demonstrate for the first time that human placenta contains a cytosolic phospholipase D (PLD) activity. This activity had a pH optimum of 7.0 and was stimulated by PIP2 and inhibited by oleate. Furthermore, cytosolic PLD was stimulated by 30 microM GTP gamma S (6-14-fold) and by the small G proteins 1 microM mArf3 (2-fold) and 0.37 nM RhoA (2-fold). This is the first report to show RhoA activation of a cytosolic PLD. The activation by mArf3 was maintained after partial purification on DEAE Sepharose of the enzyme. We have previously reported the existence of a membrane-bound PLD from human placenta, which is stimulated by PIP2, but not by oleate (Vinggaard, A. M. & Hansen, H. S. (1995) Biochim. Biophys. Acta 1258, 169-176). Here we show that oleic acid and alpha-linolenic acid both dose-dependently inhibited solubilized membrane PLD (65% inhibition at 4 mM), whereas stearic acid (4 mM) had no effect. Thus, the presence of double bonds in the fatty acid is important for the inhibitory effect. Furthermore, placental membrane PLD was activated by 30 microM GTP gamma S (4-fold) and by mArf3 (1 microM) and RhoA (0.37 nM) by a factor of 3 and 2, respectively. The solubilized membrane phospholipase D was partially purified to a basal specific activity of 25-37 nmol/min/mg. This preparation was devoid of endogenous RhoA and Arf and could not be stimulated by GTP gamma S. However, mArf3 (1 microM) still activated this partially purified membrane PLD, whereas RhoA (0.37 nM) was not able to activate this PLD fraction. In conclusion, our results suggest that the human placenta contains a PLD that is located both in the cytosol and the membranes, and that is activated by PIP2, mArf3 and RhoA but inhibited by oleate.
ADP-Ribosylation Factors↗
Phospholipase D: enzymology, mechanisms of regulation, and function.
Phospholipase D exists in various forms that differ in their regulation but predominantly hydrolyze phosphatidylcholine. The Ca(2+)-dependent isozymes of protein kinase C regulate phospholipase D in vitro and play a major role in its control by growth factors and G protein-linked agonists in vivo. Recent studies have demonstrated that small G proteins of the ADP-ribosylation factor (ARF) and Rho families activate the enzyme in vitro, and evidence is accumulating that they also are involved in its control in vivo. Both types of G protein play important roles in cellular function, and the possible mechanisms by which they are activated by agonists are discussed. There is also emerging evidence of the control of phospholipase D and Rho proteins by soluble tyrosine kinases and novel serine/threonine kinases. The possible role of these kinases in agonist regulation of phospholipase D is discussed. The function of phospholipase D in cells is still poorly defined. Postulated roles of phosphatidic acid produced by phospholipase D action include the activation of Ca(2+)-independent isoforms of protein kinase C, the regulation of growth and the cytoskeleton in fibroblasts, and control of the respiratory burst in neutrophils. Another important function of phosphatidic acid is to act as a substrate for a specific phospholipase A2 to generate lysophosphatidic acid, which is becoming increasingly recognized as a major intercellular messenger. Finally, it is possible that the phospholipid changes induced in various cellular membranes by phospholipase D may per se play an important role in vesicle trafficking and other membrane-associated events.
Animals↗
Differential translocation of rho family GTPases by lysophosphatidic acid, endothelin-1, and platelet-derived growth factor.
The small GTPases of the Rho family play a key role in a number of signaling pathways activated by lysophosphatidic acid (LPA). However, little is known concerning the mechanism of regulation of these proteins. In this study we demonstrate that in Swiss 3T3 fibroblasts, LPA induces a sustained, time-dependent relocalization of RhoA to the Triton X-100-soluble low speed membrane fraction, which can be reversed by removal of LPA from the medium. Translocation was only observed with micromolar concentrations of LPA and was inhibited by pretreating the cells with pertussis toxin but not with tyrosine kinase inhibitors. LPA also induced translocation of CDC42Hs to the membranes but had no effect on the distribution of Rac1, RhoB, or Rho-GDI. Translocation of RhoA was also induced by endothelin-1. Conversely, platelet-derived growth factor did not cause the translocation of RhoA to any membrane fraction but stimulated relocalization of Rac1 to the high speed membrane fraction. Significantly, incubation of cell lysates with guanosine 5'-O-(thiotriphosphate) was sufficient to translocate RhoA, Rac1, and CDC42Hs from the cytosol to the membranes, whereas incubation with GDP had the opposite effect. These data suggest that the translocation of the Rho family proteins to the membrane fraction is controlled by their activation state and that agonists show selectivity in inducing the activation/translocation of these proteins.
3T3 Cells↗
Tissue-specific distribution and subcellular distribution of phospholipase D in rat: evidence for distinct RhoA- and ADP-ribosylation factor (ARF)-regulated isoenzymes.
Phospholipase D (PLD) is regulated by many factors including the small G-proteins, RhoA and ADP-ribosylation factor (ARF). The present study examined the distribution of RhoA- and ARF-responsive PLD in membranes, microsomes and cytosol of rat tissues and in rat liver subcellular fractions. PLD was present in all tissue fractions examined and was stimulated by guanosine 5'-[gamma-thio]triphosphate (GTP[S]), with the highes: specific activities being in lung, kidney and spleen. When myristoylated recombinant ARF (mARF) was added with GTP[S], the PLD activity was stimulated further, but the addition of RhoA was without effect. However, in extracts from crude membranes both mARF and RhoA enhanced the stimulation by GTP[S], with high specific activities of PLD being observed in all tissues except muscle. The response to mARF was usually greater than to RhoA, and the responses were additive, except for liver, which showed synergism. When the PLD activity of subcellular fractions of liver was examined, GTP[S] caused increases in all fractions except microsomes and mitochondria, which exhibited low activity. All fractions except mitochondria showed responses to RhoA and mARF, with the response to RhoA being greater in plasma membranes and that to mARF being greater in Golgi and nuclei. Western blotting showed that RhoA was located mainly in the cytosol and plasma membranes, whereas ARF was principally in the cytosol. These findings demonstrate the widespread occurrence of significant activity of both Rho- and ARF-responsive forms of PLD in membranes from all tissues except muscle, and the presence of both forms in liver subcellular fractions except mitochondria. The large variations in the relative responses of PLD to Rho and ARF observed in different tissues and fractions support the existence of different isoforms of the enzyme.
ADP-Ribosylation Factors↗
Enhanced phospholipase D activity and altered morphology in RhoA-overexpressing RAT1 fibroblasts.
Small GTP-binding proteins of the Rho family are implicated in the regulation of phospholipase D (PLD). However, few studies have addressed their role in agonist-stimulated PLD activity in vivo. Stable lines of Rat1 fibroblasts overexpressing RhoA were shown to have altered morphology. Moreover, they demonstrated increased PLD activity when stimulated with lysophosphatidic acid, platelet-derived growth factor, and phorbol ester, compared with vector-transfected cells. However, phosphoinositide phospholipase C activity was unaltered by overexpression of RhoA. These data indicate a critical downstream role for RhoA in agonist-stimulated PLD activity in intact cells.
Animals↗
Purification of a lysophospholipase from bovine brain that selectively deacylates arachidonoyl-substituted lysophosphatidylcholine.
A high activity lysophospholipase A (lysoPLA) was purified from the soluble fraction of bovine brain. The separation included sequential DEAE-Sephacel, phenyl-Sepharose FF, heparin-Sepharose CL-6B, and Q-Sepharose FF column chromatography. Mono Q, Sephacryl S300HR, and hydroxylapatite column chromatography in the presence of the detergent CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate) and glycerol further purified the activity to 17,000-fold. The enzyme was purified to homogeneity by polyacrylamide gel electrophoresis using nondenaturing conditions. The pure enzyme migrated as a single polypeptide of 95 kDa mass by SDS-polyacrylamide gel electrophoresis and deacylated arachidonoyl-lysophosphatidylcholine (ara-lysoPC) at rate of 70 micromol/(min mg). The enzyme showed selectivity for arachidonoyl-substituted lysoPC, since palmitoyl-lysoPC was deacylated at a much lower rate (7 micromol/(min mg)). LysoPLA activity was maximal at pH 7.4-8.0 and was increased 1.3-fold by MgCl2 (5 mM). By including MgCl2, however, the range of optimal activity was expanded to pH values up to 9.0. The 95-kDa protein also deacylated arachidonoyl groups from 1-O-hexadecyl-2-arachidonoyl-PC (PLA2 activity) at a rate of 15 micromol/(min mg). Moreover, the deacylation of arachidonoyl groups from diacylPC was greatly increased by including purified bovine brain PLA1 in the reaction mixture. Thus, the same 95-kDa polypeptide catalyzed both lysoPLA and PLA2 activities, but the rate of arachidonoyl group deacylation was increased by prior sn-1 deacylation. Finally, the 95-kDa polypeptide cross-reacted with antibodies raised against a human recombinant cPLA2, implying that the 95-kDa protein is structurally similar to cPLA2. Additionally, these data suggest that the combined actions of PLA1 and the 95-kDa protein generate significant amounts of free arachidonic acid in the brain.
Animals↗
Evidence for Rho-mediated agonist stimulation of phospholipase D in rat1 fibroblasts. Effects of Clostridium botulinum C3 exoenzyme.
Small GTP-binding proteins of the Rho family are implicated in the in vitro regulation of phosphatidylcholine hydrolysis by phospholipase D (PLD). However, their role in agonist-stimulated PLD activity in whole cells is not clear. The ribosyltransferase C3 from Clostridium botulinum modifies Rho proteins and inhibits their function. When introduced into rat1 fibroblasts by scrape-loading, C3 inhibited PLD activity stimulated by lysophosphatidic acid (LPA), endothelin-1, or phorbol ester. Neither the time course nor agonist dose response for LPA-stimulated PLD activity was altered in C3-treated cells. In contrast to the effects of C3 on PLD activity, agonist-stimulated phosphatidylinositol-phospholipase C activity was not altered in C3-treated cells. Surprisingly, C3 treatment led to a decrease in the amount of RhoA protein, indicating that the loss of PLD activity in response to agonist was partly due to the loss of Rho proteins. As described previously, C3 treatment led to the inhibition of LPA-stimulated actin filament formation. However, disruption of actin filaments with cytochalasin D caused only a minor inhibition of LPA-stimulated PLD activity. Interestingly, stimulation of cells with LPA caused a rapid enrichment of RhoA in the particulate fraction of cell lysates. These data support an in vivo role for RhoA in agonist-stimulated PLD activity that is separate from its role in actin fiber formation.
ADP Ribose Transferases↗
Identification of determinants in the alpha-subunit of Gq required for phospholipase C activation.
A series of chimeras between a constitutively active mutant of the alpha-subunit of Gq and the alpha-subunit of Gs was constructed to identify the domains in alphaq specifically involved in interaction with its effector phosphoinositide phospholipase C (PLC). Transient expression of the chimeric proteins and measurement of the production of inositol phosphates and cAMP in HEK-293 cells revealed that the Ile217-Lys276 sequence of alphaq contained the PLC interaction sites, whereas the residues for activation of adenylyl cyclase were in the Ile235-Leu294 sequence of alphas. Alanine scanning mutagenesis of the Ile217-Lys276 region of alphaq further identified two clusters of amino acids (Asp243,Asn244,Glu245 and Arg256,Thr257) that were specifically required for interaction with PLC. Comparison of the sequences of alphaq, alphas, and alphat showed that the PLC-interacting residues identified in alphaq are different from the corresponding residues in alphas and alphat that are involved in effector activation. Alignment of the sequences of alphaq and alphat, based on the crystal structure of alphat (Noel, J. P., Hamm, H. E., and Sigler, P. D. (1993) Nature 366, 654-663), indicated that the PLC-activating residues of alphaq are located in alpha-helix 3 and its linker to beta-sheet 4, which are adjacent to a switch region whose conformation changes with activation. It is proposed that the selectivity of alphaq for PLC involves relatively few amino acids, but that the effector may interact with other nonselective sequences in the alpha-subunit.
Alanine↗
Subcellular fractions of bovine brain degrade phosphatidylcholine by sequential deacylation of the sn-1 and sn-2 positions.
Phosphatidylcholine (PC) metabolism was investigated using cytosol (fraction I) and particulate fractions of bovine brain that were enriched with microsomes (fraction II), plasma membranes (fraction III) or mitochondria (fraction IV). Fractions I-III incubated with 1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphocholine yielded [14C]arachidonic acid at near equal rates, whereas only fraction I accumulated significant amounts of 2-[14C]arachidonoyl-sn-glycero-3-phosphocholine. Much slower rates of arachidonic acid release were observed using an ether PC (1-O-hexadecyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine). Moreover, arachidonic acid yield from the diacyl, but not ether PC was slowed by pretreating fractions I-III, but not IV, with phenylmethylsulfonyl fluoride (PMSF). Coincident with this decreased arachidonic acid, 2-[14C]arachidonoyl-sn-glycero-3-phosphocholine was increased, indicating high PLA1 activity. Taken together these data suggest that arachidonic release was largely dependent on initial deacylation of position sn-1. Incubating each untreated fraction with 2-[3-H]arachidonoyl-sn-glycero-3-phosphocholine yielded [3H]arachidonic acid (lysophospholipase A2 activity) at rate that was substantially greater than that using the comparable PMSF-treated fraction. Thus, the large effect of PMSF on arachidonic acid release can be accounted for if much of the fatty acid formation arose from the sequential sn-1 and sn-2 deacylation of diacyl-PC by phospholipase A1 and lysophospholipase A2. When PMSF-treated fractions were incubated with 2-[3H]arachidonoyl-sn-glycero-3-phosphocholine, [3H]PC accumulated at low rates that were enhanced by adding coenzyme A or stearoyl-coenzyme A. Thus, the lysophospholipid was also reacylated to form PC, but this reaction was negligible in the absence of PMSF and added cofactors. In summary, we conclude that, in brain subcellular fractions, deacylation of the sn-1 position of diacyl-PC proceeded more rapidly than sn-2 hydrolysis. There was substantial further metabolism of 2-acyl lysophospholipids due to the combined activities of a PMSF-sensitive and -insensitive lysophospholipase. Finally, the sequential deacylation of diacyl-PC by phospholipase A1 and lysophospholipase A2 probably accounted for the major portion of arachidonic acid produced.
Acylation↗
Regulation of phosphoinositide phospholipases by hormones, neurotransmitters, and other agonists linked to G proteins.
The actions of many hormones, neurotransmitters, and growth factors are mediated by the hydrolysis of phosphatidylinositol 4,5-bisphosphate catalyzed by specific isozymes of phospholipase C. This hydrolysis releases inositol 1,4,5-trisphosphate, which mobilizes Ca2+ ions from components of the endoplasmic reticulum, and 1,2-diacylglycerol, which activates isozymes of protein kinase C. The hormones and neurotransmitters activate beta-isozymes of phospholipase C through receptors that have seven transmembrane segments and couple to G proteins of the Gq and Gi/o families. Activation of phospholipase C by the Gq family involves their alpha-subunits, whereas activation by the Gi/o family involves their beta gamma-subunits. The growth factors activate gamma-isozymes of phospholipase C through receptors that become autophosphorylated due to their stimulated tyrosine kinase activity and provide binding sites for the Src homology domains of the isozymes. The molecular mechanisms by which agonists activate phopholipase isozymes are described in detail.
Animals↗
Purinergic agonist and G protein stimulation of phospholipase D in rat liver plasma membranes. Independence from phospholipase C activation.
Hormonal regulation of phospholipase D (PLD) was studied in isolated rat liver plasma membranes. Purinergic agents and a submaximal concentration of guanosine 5'-0-(3-thiotriphosphate) (GTP gamma S), a non-hydrolyzable analog of GTP, synergistically stimulate phosphatidylethanol formation, a measure of PLD activity. The rank order of efficacy for stimulation of PLD activity in the presence of 0.2 microM GTP gamma S was beta, gamma-methylene-ATP > adenosine 5'-0-(3-thiotriphosphate) = ATP = ADP = 2-methylthio-ATP > alpha, beta-methylene-ATP = UTP. This pattern of activation does not conform to the series at known P2 receptors. GTP gamma S stimulated PLD activity in a dose-dependent manner, and the GTP gamma S dose-response curve for phosphatidylethanol formation was shifted to the left by an analog of ATP. Activation of PLD by purinergic agents in the presence of GTP gamma S supports the involvement of a purinergic receptor of the P2 class and a GTP-binding protein. Purinergic agents competitively inhibited [35S]adenosine 5'-0-(3-thiotriphosphate) binding to plasma membranes in the rank order adenosine 5'-0'(3-thiotriphosphate) > ATP > alpha,beta-methylene-ATP = UTP >> beta, gamma-methylene-ATP = ADP. Stimulation of phosphoinositide phospholipase C (PI-PLC) by purinergic agents, as measured by release of radioactivity from endogenously myo[3H]inositol-labeled plasma membranes, occurred in the order alpha, beta-methylene-ATP >> 2-methylthio-ATP. Beta, gamma-methylene-ATP had little effect on PI-PLC activity. Different dose-response relationships for agonist-stimulation of PI-PLC and PLD indicate that activation of PI-PLC is not involved in stimulation of PLD in rat liver plasma membranes, and suggest that purinergic activation of PLD occurs via a pathway involving a G protein and a heretofore uncharacterized P2 receptor.
Adenosine Triphosphate↗