Biomedical subjects

T Wieland

Publications and source records attributed to T Wieland.

At least 55 records · Page 3Linked to original sources

PubMed1994

Transfected muscarinic acetylcholine receptors selectively couple to Gi-type G proteins and Gq/11.

Regulation of effector functions by muscarinic acetylcholine receptor subtypes is mediated by pertussis toxin-sensitive and -insensitive G proteins. In membranes from human embryonic kidney 293 cells transfected with m1, m2, and m3 muscarinic acetycholine receptors, we detected the pertussis toxin-sensitive G proteins Gi1, Gi2, and Gi3 and the pertussis toxin-insensitive G proteins Gq/11 and Gs. Subtype-specific immunoprecipitation of G protein alpha subunits photolabeled with [alpha-32P] GTP azidoanilide, in the absence and presence of carbachol, revealed the selective coupling of activated muscarinic receptors to G protein subtypes. Gq/11 was activated via m1 and m3 receptors and Gi2 was activated via m2 receptors. All three receptors subtypes mediated the activation of Gi1 and Gi3. Effective activation of Gi1 and Gi3 via m1 and m3 receptors occurred only at high carbachol concentrations (EC50 about 10-20 microM), whereas carbachol with higher potency (EC50 about 1 microM) induced activation of all G1 subtypes via m2 receptors. Thus, coupling of muscarinic receptors and G protein subtypes was principally selective; however, activation of distinct G protein subtypes by different muscarinic receptors occurred with different efficacies.

Affinity Labels↗

PubMed1993

Guanine nucleotide-specific phosphate transfer by guanine nucleotide-binding regulatory protein beta-subunits. Characterization of the phosphorylated amino acid.

One major substrate protein was phosphorylated with [gamma-32P]GTP in membranes of human leukemia (HL-60) cells. The phosphoprotein comigrated with beta-subunits of heterotrimeric GTP-binding proteins (G proteins) in different gel systems. Upon solubilization of the phosphorylated membranes, the phosphoprotein could be immunoprecipitated by a G protein beta-subunit-specific antiserum. The beta-subunit phosphorylation was transient and was found to be specific for GTP and its analog, guanosine 5'-O-(gamma-thio)triphosphate. When phosphorylated membranes were incubated with various nucleotides, the bound phosphate was specifically removed by GDP, suggesting that the phosphate can be retransferred onto GDP. Divalent cations, preferentially Mg2+ and Mn2+, were required for both phosphorylation and dephosphorylation. The phosphorylation was stable against treatment with NaOH but sensitive to treatment with heat, HCl, and hydroxylamine. Moreover, treatment of the membranes with the histidine-modifying agent, diethyl pyrocarbonate, resulted in a loss in phosphate incorporation. The data suggest that G protein beta-subunits are involved in a guanine nucleotide-specific enzymatic activity transferring the gamma-phosphate from GTP to GDP, presumably at G protein alpha-subunits, via a phosphohistidine intermediate.

Biological Transport↗

PubMed1993

Receptor-stimulated dissociation of GTP[S] from Gi-proteins in membranes of HL-60 cells.

Formyl peptides stimulate binding of the stable GTP analogue, guanosine 5'-O-[gamma-thio]triphosphate (GTP[S]), to G-proteins in membranes of myeloid differentiated human leukaemia (HL-60) cells. On the other hand, agonist-activated formyl peptide receptors can also cause rapid and substantial release of GTP[S] bound to HL-60 membrane G-proteins. For fMet-Leu-Phe-stimulated dissociation of labelled GTP[S], an additional guanine nucleotide, in the potency order, unlabelled GTP[S] >> GTP >> guanosine 5'-[beta,gamma-imino]triphosphate > or = guanosine 5'-O-[beta-thio]diphosphate > or = GDP > GMP = ATP (no effects at 1 mM), was absolutely necessary. While with unlabelled GTP[S] and GTP similar concentrations were required for control and fMet-Leu-Phe-stimulated release, about 50-100-fold higher concentrations of the other nucleotides were necessary for agonist-stimulated than for basal release of bound GTP[S]. The receptor action appeared to be catalytic, required Mg2+ and was pertussis toxin sensitive. The data indicate that binding of GTP[S] to HL-60 membrane G-proteins is reversible and that agonist-activated formyl peptide receptors can interact, either directly or indirectly, with GTP[S]-liganded Gi-proteins, resulting in release of bound GTP[S].

Binding Sites↗

PubMed1992

Stimulation and inhibition of human platelet adenylylcyclase by thiophosphorylated transducin beta gamma-subunits.

The effect of beta gamma-dimers isolated from the retinal guanine nucleotide-binding protein (G protein) transducin eluted from illuminated bovine rod outer segment membranes with GTP, guanosine 5'-O-(beta, gamma-imino)triphosphate (Gpp(NH)p), or guanosine 5'-O-(gamma-thio)triphosphate (GTP gamma S) on basal and forskolin-stimulated adenylylcyclase activities in membranes of human platelets was studied. beta gamma-Subunits isolated from transducin eluted with GTP gamma S (TD beta gamma GTP gamma S) had a concentration-dependent stimulatory effect on basal adenylylcyclase activity. The stimulatory agonist prostaglandin E1 increased the potency and the maximum extent of stimulation due to TD beta gamma GTP gamma S). With a similar concentration dependence, TD beta gamma GTP gamma S exerted an inhibitory influence on forskolin-stimulated adenylylcyclase activity. At the same concentrations, beta gamma-dimers isolated with either GTP or Gpp(NH)p did not alter enzyme activities. The observed effects of TD beta gamma GTP gamma S were similar to those of directly added GTP gamma S with regard to maximum levels, time dependence, and persistence; however, TD beta gamma GTP gamma S was approximately 10-fold more potent than GTP gamma S. Treatment of TD beta gamma GTP gamma S, but not of free GTP gamma S, with hydroxylamine caused a loss of adenylylcyclase regulation by TD beta gamma GTP gamma S. The data presented indicated that TD beta gamma GTP gamma S potently and efficiently activates the stimulatory and inhibitory G proteins of adenylylcyclase in human platelet membranes. Furthermore, evidence is provided suggesting that the observed effects of TD beta gamma GTP gamma S, which can be thiophosphorylated by GTP gamma S at the beta-subunit (Wieland, T., Ulibarri, I., Gierschik, P., and Jakobs, K. H. (1991) Eur. J. Biochem. 196, 707-716), are due to formation of GTP gamma S at the G proteins.

Adenylyl Cyclase Inhibitors↗

PubMed1992

ADP receptor-induced activation of guanine-nucleotide-binding proteins in human platelet membranes.

ADP receptor-regulated binding of the labeled GTP analog, guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTP[gamma S]), to guanine-nucleotide-binding proteins (G proteins) was studied in human platelet membranes. The potent ADP receptor agonist, 2-methyl-thio-adenosine 5'-diphosphate (2MeSADP), a non-hydrolyzable analog of ADP, increased the binding of [35S]GTP[gamma S] without apparent lag phase. Under optimal conditions, i.e. in the presence of GDP (1-10 microM), 2MeSADP increased the binding up to about threefold, with half-maximal and maximal increase observed at 10 nM and 1 microM 2MeSADP, respectively. ADP itself increased the binding of [35S]GTP[gamma S] by maximally about twofold, with half-maximal increase occurring at 0.1 microM ADP. The agonist-induced stimulation was competitively antagonized by the ADP receptor(s) antagonist, (1S)-adenosine 5'-O-(1-thiotriphosphate) [(Sp)-ATP[alpha S]]. Other platelet receptor agonists known to act through receptors coupled to G proteins also increased binding of [35S]GTP[gamma S] in human platelet membranes, but without being inhibited by (Sp)-ATP[alpha S]. The data presented indicate that the platelet ADP receptor(s) can interact with and efficiently activate G proteins, the nature of which remains to be identified.

6-Phytase↗

PubMed1992

Role of GDP in formyl-peptide-receptor-induced activation of guanine-nucleotide-binding proteins in membranes of HL 60 cells.

Membranes of myeloid differentiated human leukemia (HL 60) cells contain receptors for the chemotactic peptide, fMet-Leu-Phe (fMet, N-formylmethionine), interacting with pertussis-toxin-sensitive guanine-nucleotide-binding proteins (G proteins). Agonist activation of the receptors increases binding of the GTP analog, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), to membrane G proteins, at 30 degrees C only in the presence of exogenous GDP. In contrast, at 0 degrees C fMet-Leu-Phe stimulated binding of GTP[S] to G proteins maximally without addition of GDP. Under conditions resulting in marked degradation of membrane-bound GDP, control binding of GTP[S] measured at 0 degrees C was significantly increased, whereas the extent of agonist-stimulated binding was reduced. Furthermore, there was a rapid spontaneous release of membrane-bound GDP at 30 degrees C, but not at 0 degrees C. The data suggest that in intact membranes of HL 60 cells G proteins are initially in a GDP-liganded form, which state allows the receptor-induced exchange of bound GDP for GTP[S] at low temperature. In contrast, at or near physiological temperature, bound GDP is rapidly released (and degraded), resulting in unligated G proteins to which GTP[S] will bind independently of agonist-activated receptors.

Alkaline Phosphatase↗

PubMed1992

Dissociation of guanosine 5'-[gamma-thio]triphosphate from guanine-nucleotide-binding regulatory proteins in native cardiac membranes. Regulation by nucleotides and muscarinic acetylcholine receptors.

Binding of the poorly hydrolyzable GTP analog, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), to purified guanine-nucleotide-binding regulatory proteins (G proteins) has been shown to be nonreversible in the presence of millimolar concentrations of Mg2+. In porcine atrial membranes, binding of [35S]GTP[S] to G proteins was stable in the presence of 1 mM Mg2+. However, either large dilution or, even more strongly, addition of unlabelled guanine nucleotides, in the potency order, GTP[S] greater than GTP greater than or equal to guanosine 5'-[beta,gamma-imino]triphosphate greater than GDP greater than or equal to guanosine 5'-[beta-thio]diphosphate greater than GMP, markedly enhanced the observed dissociation, with 20-30% of bound [35S]GTP[S] being released by unlabelled guanine nucleotide within 20 min at 25 degrees C. Most interestingly, dissociation of [35S]GTP[S] was rapidly and markedly stimulated by agonist (carbachol) activation of cardiac muscarinic acetylcholine receptors. Carbachol-stimulated release of [35S]GTP[S] was strictly dependent on the presence of Mg2+ and an unlabelled guanine nucleotide. Although having different potency and efficiency in releasing [35S]GTP[S] from the membranes by themselves, the guanine nucleoside triphosphates and diphosphates studied, at maximally effective concentrations, promoted the carbachol-induced dissociation to the same extent, while GMP and ATP were ineffective. GTP[S]-binding-saturation experiments indicated that one agonist-activated muscarinic acetylcholine receptor can cause release of bound GTP[S] from three to four G proteins. The data presented indicate that binding of GTP[S] to G proteins in intact membranes, in contrast to purified G proteins, is reversible, and that agonist-activated receptors can even, either directly or indirectly, interact with GTP[S]-bound G proteins, resulting in release of bound guanine nucleoside triphosphate.

Animals↗

PubMed1992

G protein-mediated receptor-receptor interaction: studies with chemotactic receptors in membranes of human leukemia (HL 60) cells.

Differentiated human leukemia (HL 60) cells contain high numbers of receptors for the chemotactic factors, N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) and complement component 5a (C5a), both coupled to pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins (G proteins). Agonist activation of either receptor stimulated binding of the GTP analog, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), to membrane G proteins and by a similar extent in a non-additive manner. The possible interaction of the two receptors was studied by measuring agonist binding to one receptor in the presence of the other receptor agonist. fMet-Leu-Phe and C5a had no effects on [125I]C5a and fMet-Leu-[3H]Phe receptor binding, respectively, when studied in the absence of regulatory ligands. Similarly, the inhibitory effects of NaCl and GDP on agonist receptor binding were not altered in the presence of the other receptor agonist. In contrast, in the presence of the GTP analogs, GTP[S] and guanosine 5'-[beta,gamma-imino] triphosphate, fMet-Leu-Phe and C5a reduced the binding of [125I]C5a and fMet-Leu-[3H]Phe, respectively, in a concentration-dependent manner. The potencies of the GTP analogs to inhibit binding of [125I]C5a and fMet-Leu-[3H]Phe was increased about 3-fold by fMet-Leu-Phe and C5a, respectively. The data presented suggest that fMet-Leu-Phe and C5a receptors share the same G protein pool in membranes of HL 60 cells and that activation of these G proteins by one of the two receptors decreases the availability of G proteins for the other receptor.

Cell Membrane↗

PubMed1992

Structure-toxicity relationships in the amatoxin series. Structural variations of side chain 3 and inhibition of RNA polymerase II.

The amatoxins, highly toxic components of death cap Amanita mushrooms, bind strongly to RNA polymerase II (or B) in cell nuclei thus preventing the transcription of DNAs to hn-RNAs (Pre-mRNAs), the precursors of messenger RNAs. Three of the binding sites of the bicyclic octapeptides have been identified: an isoleucine side chain in position 6, a trans-4-hydroxyl group at proline in position 2 and a hydroxylated L-isoleucine side chain in position 3. No information exists about the stereochemical conditions at the beta-C-atom (C-atom 3) of this side chain. We have now synthesized the diastereomeric S-deoxo-amaninamides (Fig. 1) containing, in position 3, L-allo-isoleucine (analog 1), (2S, 3R)-2-amino-4-hydroxy-3-methyl butyric acid (analog 2), the diastereomer (2S, 3S)-2-amino-4-hydroxy-3-methylbutyric acid (analog 3) and D-isoleucine (analog 4). In the last synthesis, besides the "normal" bicyclic octapeptide 4, an isomeric Iso-4 was formed. The affinities for Drosophila RNA polymerase II were 100 times weaker as compared to gamma-amanitin for 1, 10 times weaker for 2, 200 times weaker for 3, 100 times weaker for 4, and more than 1000 times weaker for Iso-4. The results point to the importance of a methyl group in (R)-configuration at the beta-C atom of side chain 3.

Amanitins↗

PubMed1992

Evidence for nucleoside diphosphokinase-dependent channeling of guanosine 5'-(gamma-thio)triphosphate to guanine nucleotide-binding proteins.

Agonist binding to guanine nucleotide-binding protein (G protein)-coupled receptors in membranes of myeloid differentiated human leukemia (HL-60) cells is inhibited by guanine nucleotides, most potently by the GTP analog guanosine 5'-(gamma-thio)triphosphate (GTP gamma S). In order to study whether GTP gamma S formed locally from adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) and GDP by nucleoside diphosphokinase has any advantage over exogenously added GTP gamma S in binding to and activating G proteins, regulation of complement component 5a (C5a) binding to its receptors, as well as formation of GTP gamma S, was studied in membranes of HL-60 cells. GTP gamma S added to HL-60 membranes potently inhibited binding of 125I-C5a (IC50 about 3 nM), an effect not influenced by addition of either GDP or ATP gamma S. When HL-60 membranes were incubated with the combination of ATP gamma S and GDP, a marked potentiation (up to 300-fold) of the inhibition caused by either GDP or ATP gamma S alone was observed. By measuring nucleoside diphosphokinase-catalyzed formation of GTP gamma S and inhibition of 125I-C5a binding in the presence of GDP and ATP gamma S under identical assay conditions, it was found that formed GTP gamma S inhibited binding of 125I-C5a with an IC50 value of about 0.3 nM, thus being about 10-fold more potent than exogenously added GTP gamma S. These data suggest that the GTP gamma S-forming nucleoside diphosphokinase is closely associated with the C5a receptor-G protein complex and channels the formed GTP gamma S into the G protein.

Adenosine Triphosphate↗

PubMed1991

Fifty years of amanitin.

Pharmacokinetic studies have provided new insights into human Amanita poisoning, but it appears to be impossible to treat this intoxication by immunotherapy. New synthetic analogs have revealed structure-activity relationships that were unknown so far. The main toxin, alpha-amanitin, is in constant use as a tool in molecular biology and in biological research. First experiments have been reported in which amanitin bound to polymers could be internalized into tumor cells via a receptor-mediated endocytosis.

Amanitins↗

PubMed1991

Contribution of nucleoside diphosphokinase to guanine nucleotide regulation of agonist binding to formyl peptide receptors.

High-affinity agonist binding to formyl peptide receptors in membranes of myeloid differentiated human leukemia (HL 60) cells is known to be regulated by guanine nucleotides, most potently by the GTP analog, guanosine-5'-O-(3-thiotriphosphate) (GTP[S]). Here we analyzed whether nucleoside diphosphokinase present in these membranes and capable of forming GTP[S] from GDP and adenosine-5'-O-(3-thiotriphosphate) (ATP[S]) can contribute to nucleotide regulation of agonist receptor binding. Using GDP and ATP[S] at concentrations causing by themselves only small reductions in receptor binding of the labelled formyl peptide, N-formyl-methionyl-leucyl-phenylalanine ([3H]FMLP), a marked potentiation (up to 30-fold) was observed when both nucleotides were combined. Under conditions in which the combination of GDP and ATP[S] induced 70-90% of maximal inhibition of [3H]FMLP binding, a total concentration of about 7 nM GTP[S] formed was measured. The synergistic effect of GDP and ATP[S] on [3H]FMLP binding was not seen in the presence of UDP (1 mM), which blocked formation of GTP[S] from GDP and ATP[S]. Furthermore, no potentiation was observed when instead of GDP and ATP[S], guanosine-5'-O-(2-thiodiphosphate) and adenylyl-5'-imidodiphosphate, respectively, were used. Finally, regulation of [3H]FMLP binding by ATP[S] plus GDP (or GTP) was a time-dependent process, reaching maximal inhibition after 20-30 min of incubation at 25 degrees C. The data indicate that nucleoside diphosphokinase present in membranes of HL 60 cells can transfer the thiophosphate group of ATP[S] to GDP leading to formation of GTP[S] and that the GTP[S] thus formed efficiently binds to G proteins interacting with formyl peptide receptors and thereby regulates their agonist binding affinity.

Adenosine Triphosphate↗

PubMed1991

Activation of signal-transducing guanine-nucleotide-binding regulatory proteins by guanosine 5'-[gamma-thio]triphosphate. Information transfer by intermediately thiophosphorylated beta gamma subunits.

Signal-transducing guanine-nucleotide-binding regulatory proteins (G proteins) are heterotrimers, composed of the nucleotide-binding alpha subunit and a beta gamma dimer. The influence of beta gamma dimer preparations of the retinal G protein transducin (TD) was studied on formylpeptide-receptor--G-protein interactions in membranes of differentiated HL 60 cells. For this, TD was prepared from bovine rod outer segment (ROS) membranes with either GTP or its analogs, guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta gamma-imino]triphosphate (Gpp[NH]p). After removal of free nucleotides, TD beta gamma was separated from TD alpha and its function analyzed. Addition of TD beta gamma isolated from TD prepared with GTP[S] (TD beta gamma GTP[S]) to HL 60 membranes abolished high-affinity binding of fMet-Leu-[3H]Phe (fMet, N-formylmethionine) to its receptor. In contrast, TD beta gamma isolated from TD prepared with GTP (TD beta gamma GTP), boiled TD beta gamma GTP[S] and TD alpha prepared with GTP[S] had no or only slight effects. The inhibitory effect of TD beta gamma GTP[S] on fMet-Leu-[3H]Phe receptor binding was potentiated by GDP at low concentrations but not by GTP[S]. Furthermore, TD beta gamma GTP[S], but not TD beta gamma GTP or TD beta gamma isolated from TD prepared with Gpp[NH]p (TD beta gamma Gpp[NH]p), prevented fMet-Leu-Phe-stimulated binding of [35S]GTP[S] to G proteins in HL 60 membranes, measured in the presence of GDP. When TD beta gamma GTP was incubated with GTP [S] and TD-depleted illuminated ROS membranes, and subsequently separated from the membranes and free GTP[S], this TD beta gamma GTP, similar to TD beta gamma GTP[S], abolished high-affinity binding of fMet-Leu-[3H]Phe to its receptor, fMet-Leu-Phe-stimulated binding of [35S]GTP[S], and fMet-Leu-Phe-stimulated GTP hydrolysis in HL 60 membranes. Inhibition of [35S]GTP[S] binding by TD beta gamma was not seen in the presence of the metabolically stable GDP analog, guanosine 5'-[beta-thio]diphosphate. In order to obtain an insight into the modification of TD beta gamma apparently caused by GTP[S], and into its mechanism of action in HL 60 membranes, TD, TD alpha and TD beta gamma, all prepared in the presence of GTP, were incubated with [35S]GTP[S] and TD-depleted illuminated ROS membranes. Fluorographic analysis of the supernatant proteins revealed 35S labelling of the beta band of the G protein. When apparently thiophosphorylated TD beta gamma was incubated with [3H]GDP in the presence of HL 60 membranes, [3H]GTP[S] was rapidly formed.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Interaction of recombinant rho A GTP-binding proteins with photoexcited rhodopsin.

The small molecular mass GTP-binding proteins rho A, B and C are targets for ADP-ribosyltransferase activity of the botulinum exoenzyme C3. The possible interaction of recombinant rho A proteins expressed in E. coli with photoexcited rhodopsin was studied by reconstitution with bovine rod outer segment (ROS) membranes depleted of endogenous GTP-binding proteins by treatment with urea. As reported for C3 substrates present in untreated ROS membranes, ADP-ribosylation of recombinant rho A proteins, both normal and Val-14 mutant, by C3 was inhibited when reconstituted with illuminated compared to dark-adapted ROS membranes pretreated with urea. GDP reduced the light-induced inhibition, while GTP[S] and light inhibited ADP-ribosylation of rho A proteins in a synergistic manner.

Adenosine Diphosphate Ribose↗

PubMed1990

Interaction of small G proteins with photoexcited rhodopsin.

Bovine rod outer segment (ROS) membranes contain in addition to the heterotrimeric G protein transducin, several small GTP-binding proteins (23-27 kDa). Furthermore, these membranes contain two substrate proteins (about 22 and 24 kDa) for botulinum C3 ADP-ribosyltransferase known to ADP-ribosylate small G proteins in any mammalian cell type studied so far. Most interestingly, [32P]ADP-ribosylation of ROS membrane small G proteins by C3 is regulated by light and guanine nucleotides in a manner similar to pertussis toxin-catalyzed [32P]ADP-ribosylation of the alpha-subunit of transducin. These findings suggest that not only the heterotrimeric G protein transducin but also the C3 substrate small G proteins present in ROS membranes interact with photoexcited rhodopsin and thus contribute to its signalling action.

ADP Ribose Transferases↗

PubMed1990

S-deoxo-Abu1,Ile3-amaninamide, an inactive amatoxin analogue.

The title compound 3, an amatoxin analogue containing L-alpha-aminobutyric acid instead of L-asparagine in position 1, as in natural toad stool peptides, has been synthesized. It does not inhibit the eukaryotic DNA-dependent RNA polymerase form II (or B) in concentrations up to 10(-4)M, whereas 50% inhibition is exerted in 10(-6)M solution by the corresponding Asn-analogue S-deoxo-Ile3-amaninamide 2. The striking difference seems to be due to a relatively small variation of the conformation recognized by sensitive NMR spectroscopic methods.

Amanitins↗

PubMed1989

Evidence for receptor-regulated phosphotransfer reactions involved in activation of the adenylate cyclase inhibitory G protein in human platelet membranes.

The activity of the adenylate cyclase inhibitory guanine-nucleotide-binding regulatory protein (Gi), measured as inhibition of forskolin-stimulated cyclic AMP formation, and its regulation by various nucleotides and the inhibitory alpha 2-adrenoreceptor agonist epinephrine was studied in membranes of human platelets. When adenylate cyclase activity was measured with ATP as substrate and in the absence of a nucleoside-triphosphate-regenerating system, GTP (0.1-10 microM) by itself potently and efficiently inhibited the enzyme. GDP was almost as potent and as effective as GTP. In the additional presence of epinephrine, the potencies of both GTP and GDP were increased about threefold, while maximal inhibition by these nucleotides was only slightly increased by the receptor agonist. In contrast to GTP and GDP, the metabolically stable GDP analog, guanosine 5'-[beta-thio]diphosphate, had only a very small effect, suggesting that GDP but not its stable analog is converted to the active GTP. Addition of UDP (1 mM), used to block the GDP to GTP conversion reaction, completely suppressed the inhibitory effect of GDP, while that caused by GTP was not affected. Most important, the inhibitory receptor agonist epinephrine counteracted the suppressive effect of UDP on GDP's action, suggesting that, while UDP inhibits the formation of GTP from GDP, the activated receptor stimulates this conversion reaction. In the presence of a complete nucleoside-triphosphate-regenerating system, which by itself had no influence on control forskolin-stimulated adenylate cyclase activity, GTP alone, at concentrations up to 10 microM, did not decrease enzyme activity, but required the presence of an inhibitory receptor agonist (epinephrine) to activate the Gi protein. Addition of the regenerating system creatine phosphate plus creatine kinase not only abolished adenylate cyclase inhibition by GTP alone, but also largely reduced both the potency and efficiency of epinephrine to activate the Gi protein in the presence of GTP. Furthermore, the nucleoside-triphosphate-regenerating system also largely delayed the onset of adenylate cyclase inhibition by the GTP analog, guanosine-5'-[beta-thio]triphosphate (10 nM), which was accelerated by epinephrine, and it also decreased the final enzyme inhibition caused by this GTP analog.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylyl Cyclase Inhibitors↗

PubMed1989

Receptor-regulated formation of GTP[gamma S] with subsequent persistent Gs-protein activation in membranes of human platelets.

Preincubation of human platelet membranes with the ATP analog ATP[gamma S] led to persistent adenylate cyclase activation. This stimulation was increased by copreincubation with PGE1 and obliterated by removing endogenous GDP by the NTP-regenerating system, creatine phosphate plus creatine kinase. PGE1 partially reversed the action of the regenerating system. Control formation of GTP[gamma S] from ATP[gamma S] and GDP in platelet membranes was apparently not stimulated by PGE1. In contrast, in the presence of creatine phosphate plus creatine kinase, which prevented formation of GTP[gamma S], PGE1 stimulated formation of this GTP analog, by partially reversing the action of the NTP-regenerating system. The data indicate that GTP[gamma S] can be formed by a membrane-associated nucleoside diphosphokinase from ATP[gamma S] and GDP, resulting in persistent Gs-protein activation, and that this process can be stimulated by an agonist-activated receptor.

Adenosine Triphosphate↗