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Biomedical subjects

D R Manning

Publications and source records attributed to D R Manning.

At least 37 records · Page 2Linked to original sources
PubMed1993

Epidermal growth factor-mediated signaling of G(i)-protein to activation of phospholipases in rat-cultured hepatocytes.

Hepatocytes were established in tissue culture in order to study the effects of pertussis toxin (PT) on epidermal growth factor (EGF)-mediated cellular responses under in vitro conditions. EGF caused a 3-fold increase of myo-inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) mass and a 50% increase of diacylglycerol mass within the first minute, with the change of diacylglycerol content being 100-fold greater than that of Ins-1,4,5-P3. Diacylglycerol, but not Ins-1,4,5-P3, continued to accumulate over several hours, indicating that EGF increased the hydrolysis of lipids other than phosphatidylinositol 4,5-bisphosphate (PIP2). EGF increased phosphoinositide-specific phospholipase C-gamma (PLC-gamma) tyrosine phosphorylation within 1 min, but no effect was observed with vasopressin, insulin, or glucagon after 5 min. EGF also caused a rapid, tyrosine kinase-dependent association of G(i) alpha with PLC-gamma, which was maximal within 10 min. In contrast to our previous data on fresh hepatocytes, PT had no effect on the EGF-induced tyrosine phosphorylation of PLC-gamma, although Ins-1,4,5-P3 and diacylglycerol production were inhibited. The role of G-proteins in EGF signaling was investigated further by microinjection of G alpha antibodies into single fura-2-loaded hepatocytes. Anti-G(i) alpha (common) antibodies prevented EGF-induced but not vasopressin-induced Ca2+ transients. These results strengthen previous observations that a PT-sensitive G-protein is involved in EGF-mediated phospholipid metabolism in hepatocytes and show that tyrosine phosphorylation of PLC-gamma is an insufficient signal for activation of PIP2 hydrolysis.

Adenosine Diphosphate Ribose↗
PubMed1993

Agonist receptors and G proteins as mediators of platelet activation.

Recent studies have helped to define the earliest events of signal transduction in platelets, particularly those involved in the generation of second messengers. The best-understood of these events are those which involve guanine nucleotide binding regulatory proteins. G proteins are heterotrimers comprised of alpha, beta and gamma subunits, each of which can exist in multiple forms. Some, but not all, of the known variants of G alpha are substrates for ADP-ribosylation by pertussis toxin, a modification which disrupts the flow of information from receptor to effector. The G proteins that have been identified in platelets to date are Gs, Gi1, Gi2, Gi3, Gz and Gq. Gs and one or more of the Gi family members regulate cAMP formation by adenylylcyclase. Gi may also be responsible for the pertussis toxin-sensitive activation of phospholipase C which occurs when platelets are activated by thrombin. Gq is thought to be responsible for the pertussis toxin-resistant activation of phospholipase C by TxA2. Gz does not have an established role, but has the unique property of being phosphorylated by protein kinase C during platelet activation. Recent efforts to clone the receptors that interact with G proteins in platelets have been successful for epinephrine, thrombin, TxA2 and platelet activating factor. Each of these resembles other G protein-coupled receptors, being comprised of a single polypeptide with 7 transmembrane domains. In the case of thrombin, receptor activation is thought to involve a unique mechanism in which thrombin cleaves its receptor, creating a new N-terminus that can serve as a tethered ligand. Peptides corresponding to the tethered ligand can mimic the effects of thrombin, while antibodies to the same domain inhibit platelet activation. Shortly after activation, thrombin receptors become resistant to re-activation by thrombin. This desensitization, which appears to be due to a combination of proteolysis, phosphorylation and internalization, provides a potential mechanism for limiting the duration of thrombin-initiated signals in platelets.

Adenosine Diphosphate↗
PubMed1991

Phosphorylation of Gz in human platelets. Selectivity and site of modification.

We have demonstrated previously that the G protein alpha subunit Gz alpha (or Gx alpha) in human platelets is subject to phosphorylation by agents that activate protein kinase C, including phorbol 12-myristate 13-acetate, thrombin, and the thromboxane A2 analog U46619. We examine here the site and selectivity of phosphorylation both in vitro using recombinant G protein alpha subunits and in situ using permeabilized and intact platelets. Protein kinase C catalyzes the rapid and nearly stoichiometric phosphorylation of recombinant Gz alpha, with the modification occurring preferentially for the GDP-bound form of the subunit. Under the same conditions, phosphorylation of recombinant Gi alpha 1, Gi alpha 2, Gi alpha 3, Gs alpha-S, Gs alpha-L, and Go alpha 1 was minimal. Phosphorylation of both rGz alpha and platelet Gz alpha occurs at a serine residue near the amino terminus. This conclusion is supported by phosphoamino acid analysis and the incorporation of radiolabel from [gamma-32P]ATP into the amino-terminal CNBr peptide (residues 2-53 of the encoded protein). One of the antisera used in this study (6354, directed toward residues 24-33) recognizes only the nonphosphorylated form of Gz alpha, providing strong evidence that Ser25 or Ser27 is the site of phosphorylation. Results obtained with 6354 also suggest that phorbol ester-promoted phosphorylation of Gz alpha approaches 1 mol of phosphate per mol of subunit in permeabilized platelets.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1991

The role of GTP-binding proteins in platelet activation.

Platelet activation begins with the binding of an agonist to the cell surface and culminates in the events of platelet aggregation, secretion and clot formation. Recent studies have identified two large families of GTP-binding proteins in platelets that are thought to participate in the events of platelet activation. The first of these are the G proteins, heterotrimeric proteins which are best known for their ability to mediate the interaction between agonist receptors and intracellular enzymes such as adenylyl cyclase, phospholipase C and phospholipase A2. To date, at least six G proteins have been identified in platelets: Gs, Gz, three variants of Gi and either Gq or G11 (or both). An additional, pertussis toxin-resistant G protein, Gq, may also be present. The second group of GTP-binding proteins present in platelets is substantially smaller than the heterotrimeric G proteins, ranging in size from 21 to 28 kDa. At least 15 such low molecular weight GTP-binding proteins have been identified in platelets, many of which are homologous to the products of the ras proto-oncogenes. In cells other than platelets, low molecular weight GTP-binding proteins have been implicated in protein transport, cell activation events and malignant transformation. Their role in platelets is unknown.

Adenylyl Cyclases↗
PubMed1991

Identification of Gz alpha as a pertussis toxin-insensitive G protein in human platelets and megakaryocytes.

G proteins mediate the interaction between cell surface receptors and intracellular effectors. Recent studies have shown that human retina and rat brain contain mRNA encoding a novel 40-Kd G protein alpha subunit referred to as Gz alpha. Studies with an antiserum selective for the predicted sequence of this protein have suggested that a similar protein is present in human platelets and is phosphorylated during platelet activation. To better understand the structure and function of this protein, the present studies examine its sequence in platelets and compare its abundance in human platelets, megakaryocytes, and two megakaryoblastic cell lines, HEL cells and Dami cells. Three different Gz alpha-selective antisera reacted with a 40-Kd protein in platelet membranes. None of these detected a corresponding protein in HEL or Dami cells, despite the presence in both cell lines of proteins recognized by antisera selective for three members of the Gi alpha family. Northern blotting with a Gz alpha-specific probe prepared from retinal Gz alpha showed two hybridizing species in platelet RNA: a major band at 3.5 kb and a minor band at 2.2 kb. Both were detectable in HEL and Dami cells, but at greatly reduced levels compared with platelets. RNA encoding Gz alpha was also detected in individual human megakaryocytes by in situ hybridization. The amount present approached that of Gi alpha 2' the most abundant of the Gi alpha species present in platelets. The complete sequence of the platelet homolog to Gz alpha was determined from platelet RNA amplified by the polymerase chain reaction. The encoded protein was the same as those obtained in brain and retina. Thus, based on immunoreactivity and nucleotide sequencing, platelets and megakaryocytes contain substantial quantities of a protein identical to brain and retinal Gz alpha. The paucity of Gz alpha protein and RNA in the megakaryoblastic cell lines suggests that either there has been a selective loss of the ability to synthesize Gz alpha from these cells or that Gz alpha appears at a later stage in megakaryocyte development than does Gi alpha.

Base Sequence↗
PubMed1991

Receptor and G protein-mediated responses to thrombin in HEL cells.

Thrombin is believed to activate platelets via cell surface receptors coupled to G proteins. In order to better understand this process, we have examined the interaction of thrombin with HEL cells, a leukemic cell line that has served as a useful model for studies of platelet structure and function. In HEL cells, as in platelets, thrombin stimulated inositol trisphosphate (IP3) formation and suppressed cAMP synthesis. Both events were inhibited by pertussis toxin with 50% inhibition occurring at a toxin concentration that ADP-ribosylated 50% of the Gi alpha subunits present in HEL cells. IP3 formation was also stimulated by a second serine protease, trypsin. The trypsin response was identical to the thrombin response in time course, magnitude, and pertussis toxin sensitivity, suggesting that a similar mechanism is involved. Agonist-induced changes in the cytosolic-free Ca2+ concentration were used to test this hypothesis. Both proteases caused a transient increase in intracellular calcium [Ca2+]i that could be inhibited with D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone thrombin. Exposure to either protease desensitized HEL cells against subsequent increases in [Ca2+]i and IP3 caused by the other, although responses to other agonists were retained. This loss of responsiveness persisted despite repeated washing of the cells and the addition of hirudin. Complete recovery occurred after 20 h and could be prevented with cycloheximide. These observations suggest that 1) HEL cell thrombin receptors, like those on platelets, are coupled to phospholipase C and adenylylcyclase by pertussis toxin-sensitive G proteins, 2) the G proteins involved are equally accessible to pertussis toxin in situ, 3) when access is limited to the outside of the cell the response mechanisms for thrombin and trypsin are similar, if not identical, despite the broader substrate specificity of trypsin, 4) both proteases cause persistent changes that may involve proteolysis of their receptors or associated proteins, and 5) desensitization of the thrombin response occurs at a step no later than the activation of phospholipase C and requires protein synthesis for recovery.

Adenosine Diphosphate Ribose↗
PubMed1991

Subcellular distribution of the alpha subunit(s) of Gi: visualization by immunofluorescent and immunogold labeling.

The subcellular distribution of the alpha subunit(s) of Gi has an obvious bearing on the ability of this protein to interact with receptors and targets and on its potential to serve in still unexplored capacities. In this study, we have examined the distribution of Gi alpha by means of light and electron microscopy. The cells employed were mouse 3T3 fibroblasts, normal rat kidney fibroblasts, rat C6 glioma cells, human umbilical vein endothelial cells, and human 293 kidney fibroblasts. By indirect immunofluorescence, two patterns of Gi alpha were evident. The more prominent was that associated with phase-dense, cytoplasmic structures exhibiting a tubule-like morphology. A similar distribution was noted for mitochondria, indicating attachment to a subset of microtubules. The second pattern appeared as a diffuse, particulate fluorescence associated with the plasma membrane. By immunogold labeling and electron microscopy, two populations of Gi alpha were again evident. In this instance, labeling of the plasma membrane was the more prominent. Gold particles were most often evenly distributed along the plasma membrane and were concentrated along microspikes. The second, less abundant population of Gi alpha represented the subunit (or fragments) within lysosomes. Specificity in immunolabeling was confirmed in all instances by immunotransfer blotting, the use of antibodies differing in specificities for epitopes within Gi alpha, the absence of labeling with preimmune sera, and the decrease in labeling after preincubation of antisera with appropriate peptides. These results support the proposal that several populations of Gi alpha exist: those evident within the cytoplasm by immunofluorescence, those present at the plasma membrane, and those evident within lysosomes by immunogold labeling.

Amino Acid Sequence↗
PubMed1991

Beta-adrenergic receptor-G protein-adenylate cyclase complex in experimental canine congestive heart failure produced by rapid ventricular pacing.

Changes in the beta-adrenergic receptor-G protein-adenylate cyclase complex were investigated in an experimental canine model of low-output heart failure produced by chronic rapid ventricular pacing. The contractile response occurring after exposure to the beta-adrenergic agonist dobutamine, measured as peak left ventricular + dP/dt, was decreased after 3 weeks of pacing. To further characterize the diminished functional responsiveness to beta-adrenergic receptor stimulation, beta-adrenergic receptor-adenylate cyclase coupling was investigated using membranes prepared from both control and paced animals. The density of beta-adrenergic receptors was decreased by 40% with a selective downregulation of the beta 1-subtype. The affinity of the receptor for the antagonist radioligand [125I]iodocyanopindolol remained unchanged. A defect in coupling was suggested by a decreased ability of isoproterenol, fluoride, and forskolin to stimulate adenylate cyclase in membranes prepared from failing hearts. Determination of the levels of Gi alpha (the alpha-subunit of Gi) by immunoblotting and pertussis toxin labeling revealed modest increases of approximately 30%. Furthermore, Mn2+ and purified Gs failed to stimulate adenylate cyclase in membranes prepared from failing hearts, indicating an impairment in the catalytic moiety of adenylate cyclase itself or in the ability of adenylate cyclase to couple to Gs. In contrast, complementation assay did not reveal differences in the functional activity of Gs alpha (the alpha-subunit of Gs). Taken together, these data demonstrate a selective decrease in the beta 1-subtype of adrenergic receptors and an increase in a 40-kd G1-like protein in the failing heart. Similar changes have been described in human idiopathic dilated cardiomyopathy. In addition to these changes, we identified a possible defect at the level of the catalytic subunit of adenylate cyclase.

Adenylate Cyclase Toxin↗
PubMed1991

Tissue-dependent association of muscarinic acetylcholine receptors with guanine nucleotide-binding regulatory proteins.

The muscarinic acetylcholine receptors in heart and cerebellum form a stable association with guanine nucleotide-binding regulatory proteins (G proteins) in the presence of receptor agonists. This has been confirmed by purification of the muscarinic receptor-G protein complexes using an immunoprecipitation protocol. The isolated complexes were subjected to Western blotting to identify the G protein subunits present in the complexes. At saturating concentrations of carbachol, the muscarinic receptors in atrial membranes co-purified exclusively with Go, whereas in cerebellar and ventricular membranes an association with both Gi and Go was demonstrated. Further characterization of the G protein subunits allowed identification of the species of Gi alpha subunits present in the complexes of muscarinic receptor and G protein; in ventricle Gi alpha 2 was the only subtype present, whereas in cerebellum both Gi alpha 1 and Gi alpha 2 were present. These results demonstrate that a single muscarinic receptor subtype, depending on the tissue studied, is capable of interacting with more than one G protein subtype. The concentrations of agonist required to promote receptor-G protein association in atrial and ventricular membranes correlated with the high affinity component of receptor occupancy by agonist, as measured in equilibrium binding assays. Furthermore, incubation of cardiac membranes with saturating concentrations of pilocarpine or McN A343 resulted in reduced amounts of receptor-G protein complexes, compared with carbachol. Overall, our results suggest that the specificity of cellular effects of muscarinic agonists may relate, in part, to the selective interaction of receptor with G proteins.

(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethy↗
PubMed1990

Identification of the pertussis toxin-sensitive G proteins in platelets, megakaryocytes, and human erythroleukemia cells.

Guanine nucleotide-binding regulatory proteins, or G proteins, mediate the interaction of agonist receptors on the platelet surface with phospholipase C and adenylyl cyclase. To better understand this process, we have used several approaches to identify which G proteins are present in platelets, normal human megakaryocytes, and human erythroleukemia (HEL) cells, a leukemic cell line with megakaryocytic features. Because platelet and HEL cell responses to thrombin are inhibited by pertussis toxin, we have focused upon the members of the Gi family, whose alpha subunits can be ADP-ribosylated by that toxin. Western blots with antisera specific for Gi alpha demonstrated the presence in both platelets and HEL cells of the three best-described forms of this protein: Gi alpha 1, Gi alpha 2, and Gi alpha 3. Based upon immunoprecipitation studies with [35S]-methionine-labeled HEL cells, their relative abundance appears to be Gi alpha 2 much greater than Gi alpha 3 greater than Gi alpha 1. A HEL cell cDNA library screened with the Gi alpha antisera produced clones encoding Gi alpha 2 and Gi alpha 3 that had sequences similar to those reported from other sources. Gi alpha-specific probes created from these cDNA clones confirmed the presence of mRNA encoding Gi alpha 2 and Gi alpha 3 in both platelets (by Northern blotting) and megakaryocytes (by in situ hybridization). Thus the pertussis toxin substrates that have previously been detected in platelets and HEL cells are shown to be members of the Gi alpha family, all of which are candidates for interaction with receptors for thrombin and other agonists.

Adenylate Cyclase Toxin↗
PubMed1990

Transforming growth factor beta enhances parathyroid hormone stimulation of adenylate cyclase in clonal osteoblast-like cells.

The effects of transforming growth factor beta (TGF beta) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Purified TGF beta incubated with UMR-106 cells for 48 hr produced a concentration-dependent increase in PTH stimulation of adenylate cyclase, with maximal increase in PTH response (37%) occurring at 1 ng/ml TGF beta. TGF beta also enhanced receptor-mediated activation of adenylate cyclase by isoproterenol and prostaglandin E2 (PGE2) and nonreceptor-mediated enzyme activation by cholera toxin and forskolin. In cells in which PTH-stimulated adenylate cyclase activity was augmented by treatment with pertussis toxin, the incremental increase in PTH response produced by TGF beta was reduced by 33%. However, TGF beta neither mimicked nor altered the ability of pertussis toxin to catalyze the ADP-ribosylation of a 41,000-Da protein, presumably the alpha subunit of the inhibitory guanine nucleotide-binding regulatory component (Gi) of adenylate cyclase, in cholate-extracted UMR-106 cell membranes. TGF beta also had no effect on the levels of alpha or beta subunits of Gi, as assessed by immunotransfer blotting. In time course studies, brief (less than or equal to 30 min) exposure of cells to TGF beta during early culture was sufficient to increase PTH response but only after exposed cells were subsequently allowed to grow for prolonged periods. TGF beta enhancement of PTH and isoproterenol responses was blocked by prior treatment of cells with cycloheximide but not indomethacin. The results suggest that TGF beta enhances PTH response in osteoblast-like cells by action(s) exerted at nonreceptor components of adenylate cyclase. The effect of TGF beta may involve Gi, although in a manner unrelated to either pertussis toxin-catalyzed ADP-ribosylation of the alpha subunit of Gi or changes in levels of Gi subunits. The regulatory action of TGF beta on adenylate cyclase is likely to be mediated by the rapid generation of cellular signals excluding prostaglandins, followed by a prolonged sequence of events involving protein synthesis. These observations suggest a mechanism by which TGF beta may regulate osteoblast responses to systemic hormones.

Adenylyl Cyclases↗
PubMed1990

G protein complement of SV40-transformed ciliary epithelial cells.

Guanine nucleotide-binding proteins (G proteins) are a family of receptor-coupled signal-transducing proteins that regulate a variety of second-messenger systems and ion channels. The complement of G proteins in SV40-transformed pigmented and nonpigmented ciliary epithelial cells was determined by Western blot analysis utilizing peptide and holoprotein derived antisera to known G protein alpha and beta subunits and cholera toxin catalyzed ADP-ribosylation. The complement of alpha subunits found in both SV40-transformed NPE and PE cells includes Gs alpha and all three members of the Gi alpha family. Neither cell type contains Go alpha or Gz alpha. Both cell lines contain beta 35 and beta 36. Future studies will examine the functional involvement of these G proteins in the regulation of aqueous humor stimulus-secretion coupling.

Amino Acid Sequence↗
PubMed1990

Ontogeny of cortical muscarinic receptor subtypes and muscarinic receptor-mediated responses in rat.

To determine the ontogenetic relationship of muscarinic receptor and effector systems in the central nervous system, the developmental time courses for binding sites with high (M1) and low (M2) affinity for pirenzepine as well as muscarinic receptor-mediated stimulation of phosphoinositide breakdown and inhibition of cyclic AMP accumulation were examined in rat cortex. M1 sites were 30% of adult levels at 1 week, 70% at 2 weeks, 90% at 3 weeks and equal to adult levels at 4 weeks postpartum. M2 sites, on the other hand, did not show a significant change between the ages of 1 and 6 weeks. Acetylcholine-stimulated phosphoinositide breakdown was detected at all ages tested (1, 2, 3, 4 and 6 weeks). The percentage of conversion of [3H] phosphoinositides to [3H]inositol phosphates, stimulated by 1 mM acetylcholine, increased with age until 3 weeks after birth and then decreased slightly at ages 4 and 6 weeks. Carbachol inhibition of [3H]cyclic AMP accumulation, on the other hand, was undetectable in tissues from 1- and 2-week-old rats, whereas in tissues from 3- and 4-week-old rats, the responses were at 6-week level. Thus, for carbachol inhibition of cyclic AMP accumulation, a time in development existed (2 weeks after birth) at which receptors appeared to be present but response to stimulation was absent. To examine indirectly the coupling of binding sites to second messenger systems via guanine nucleotide-binding regulatory proteins, the density of binding sites for the muscarinic receptor agonist, [3H]oxotremorine-M, was measured.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed1989

Pharmacological and biochemical characterization of complexes of muscarinic acetylcholine receptor and guanine nucleotide-binding protein.

Complexes of agonist-bound muscarinic acetylcholine receptor (mAChR) and guanine nucleotide-binding protein (G protein) were solubilized and isolated from rat heart. Heart membranes were incubated with mAChR agonists or antagonists, solubilized using digitonin and cholate, and subjected to chromatography over wheat germ agglutinin-Affi-Gel. Eluted fractions were precipitated using a cardiac-selective anti-mAChR antibody (Luetje, C. W., Brumwell, C., Norman, M. G., Peterson, G. L., Schimerlik, M. I., and Nathanson, N. M. (1987) Biochemistry 26, 6892-6898). Using samples obtained from membranes initially incubated with carbachol (10 nM, 100 nM, or 1 mM), G alpha immunoreactivity was detected on Western blots probed using antibodies with specificity for G alpha subunits. The G alpha immunoreactivity was not detected when atropine alone (10 nM or 1 microM) or when excess atropine (1 microM) plus carbachol (100 nM) was used during the membrane preincubation. G beta immunoreactivity, when detectable on Western blots, was present in substoichiometric amounts relative to that of G alpha. The G alpha immunoreactivity was not present if GTP was included during incubation of membranes with agonist and following membrane solubilization. Further results indicate that although agonist binding to receptors is rapidly reversed by GTP or GDP (t1/2 less than 10 min), the mAChR-G protein complex is reversed more slowly or not at all. It was also shown that at high agonist concentrations, the cardiac mAChR interacts with both Go and Gi-like proteins. Together, these results demonstrate the utility of an immunoaffinity approach to the purification and biochemical study of receptor-G protein interactions.

Animals↗
PubMed1989

Thrombin and phorbol esters cause the selective phosphorylation of a G protein other than Gi in human platelets.

Preincubation of human platelets with activators of protein kinase C such as phorbol 12-myristate 13-acetate (PMA) has been shown previously to attenuate the ability of agonists both to suppress formation of cAMP and to stimulate hydrolysis of phosphoinositides. In the present study, we have examined whether the attenuation caused by PMA can be attributed to the phosphorylation of the alpha subunit(s) of Gi, a GTP-binding regulatory protein implicated in several pathways of signal transduction. PMA was found to promote the phosphorylation of several proteins within saponin-permeabilized and intact platelets incubated with [gamma-32P]ATP and [32P]H3PO4, respectively. None of the phosphoproteins, however, was precipitated by either of two antisera containing antibodies differing in specificities for epitopes within Gi alpha, despite precipitation of a substantial fraction of the subunit itself. In contrast, other antisera, containing antibodies specific for the recently described Gz alpha or both Gz alpha and Gi alpha, precipitated a 40-kDa phosphoprotein. Phosphorylation of this protein occurred not only in response to PMA, but to thrombin and the thromboxane A2 analog U46619. These data suggest that activators of protein kinase C lead to the phosphorylation within platelets of a select population of G alpha subunits. The identified phosphoprotein is not Gi alpha, but is similar or identical to Gz alpha. Because Gz alpha does not contain the consensus site for ADP-ribosylation by the Bordetella pertussis toxin islet-activating protein, the data also suggest that effects of PMA on processes otherwise sensitive to this toxin are not exerted at the level of G proteins responsible for transduction.

Amino Acid Sequence↗
PubMed1989

Chemoattractant-stimulated GTPase activity is decreased on membranes from polymorphonuclear leukocytes incubated in chemoattractant.

Polymorphonuclear leukocytes, PMNs, incubated in a chemoattractant undergo a time-dependent decrease in responsiveness to the chemoattractant; i.e. they desensitize or adapt. We have examined the role of ligand-induced changes at early steps in signal transduction for adaptation of PMNs to chemoattractants. The chemoattractant stimulation of a pertussis toxin-sensitive GTPase activity on PMN membranes was used as an assay of signal transduction. We find a decreased basal GTPase activity and a decrease in the ability of N-formylnorleucylleucylphenylalanine (FN-LLP) to stimulate this activity on membranes prepared from PMNs incubated with the chemotactic peptide FNLLP. The basal GTPase activity is decreased by up to 70% and the peptide-stimulated GTPase activity by up to 95% on membranes from PMNs incubated for 20 min at 37 degrees C in 10(-7) M FNLLP. The decrease in peptide-stimulated GTPase activity cannot be accounted for by the decreased number of FNLLP receptors on the membranes. Rather, receptors that remain available for binding stimulate the GTPase activity with a decreased efficiency. The ligand-induced change in GTPase activity is not stimulus specific. GTPase activity stimulated by both C5a and LTB4 was decreased on membranes from PMNs incubated in FNLLP. The decrease in chemoattractant-stimulated GTPase activity is partially reversed if cells are subsequently incubated at 37 degrees C in the absence of peptide prior to membrane preparation. We detected no quantitative or qualitative change in either pertussis toxin substrates or immunoreactive G proteins when membranes from control and FNLLP-treated cells were compared.

Animals↗
PubMed1988

Interactions in platelets between G proteins and the agonists that stimulate phospholipase C and inhibit adenylyl cyclase.

Platelet responses to agonists are believed to be mediated by at least two pertussis toxin-sensitive guanine nucleotide-binding (G) proteins: Gi which inhibits adenylyl cyclase and Gp, which stimulates phospholipase C. The present studies compare the properties of Gi and Gp and examine their interactions with the receptors for various platelet agonists. In permeabilized platelets and platelet membranes, pertussis toxin [32P]ADP-ribosylated a protein(s) (alpha 41) which migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis fractionally below rabbit and bovine alpha i (Mr = 41,000). Prior exposure of the platelets to an agonist inhibited the [32P]ADP-ribosylation of alpha 41 to an extent which correlated with the pattern of responses to that agonist. Thrombin, which elicited responses that were mediated by both Gi and Gp, decreased radiolabeling by greater than 90%. Epinephrine, which was functionally coupled only to Gi, decreased radiolabeling by 50%, as did vasopressin and platelet-activating factor (PAF), which were coupled only to Gp. U46619, a thromboxane analog which neither inhibited cAMP formation nor caused pertussis toxin-sensitive phosphoinositide hydrolysis, had no effect on 32P-ADP-ribosylation. These results suggest that either G alpha 41 regulates more than one enzyme or that alpha subunits from more than one G protein comigrate within alpha 41. Two-dimensional electrophoresis was used to test the latter possibility. Upon isoelectric focusing, alpha 41 resolved into two distinct subspecies. However, these appear to be minor variants rather than functionally distinct alpha subunits since: 1) both proteins produced the same proteolytic fragments after digestion with chymotrypsin or Staphylococcus aureus V8 protease and 2) preincubation of the platelets with agonists, including those which appear to interact in intact platelets solely with Gp (PAF and vasopressin) or solely with Gi (epinephrine), inhibited the [32P]ADP-ribosylation of both proteins to the same extent. The pattern of functional responses produced by some of the agonists was found to depend upon the conditions used for the assay. Although unable to inhibit cAMP formation in intact platelets, both PAF and vasopressin caused pertussis toxin-sensitive inhibition of adenylyl cyclase in isolated membranes. Collectively, these observations suggest that 1) in platelets a single pertussis toxin-sensitive, alpha 41-containing G protein may be involved in the regulation of both adenylyl cyclase and phospholipase C and 2) additional constraints which are altered during membrane isolation may help to determine which enzyme is coupled to which agonist.

Adenosine Diphosphate Ribose↗