Forskolin stimulation of adenylate cyclase in human thyroid membranes

Abstract. Forskolin stimulates adenylate cyclase in human thyroid membranes approximately 7-fold with halfmaximal stimulation occurring at 5–10 μm. Guanine nucleotides are not required for stimulation of the enzyme by forskolin. Forskolin-stimulation is additive or greater than additive with that of TSH or Gpp(NH)p-(above 1 μm). Different from TSH- or Gpp(NH)p-stimulation of adenylate cyclase, uncoupling of the guanine nucleotide-binding regulatory component by increasing concentrations of MnCl2 did not result in uncoupling of forskolin stimulation. The finding indicates that forskolin may mainly act on the catalytic component of adenylate cyclase. From the present study, it is suggested that the diterpene forskolin stimulates adenylate cyclase in human thyroid membranes by a novel mechanism that differs from TSH- or Gpp(NH)p-stimulation, and that the diterpene may be a useful tool to investigate the metabolism of thyroid and its regulation in normal and pathological situations.

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Detection and analysis of agonist-induced formation of the complex of the stimulatory guanine nucleotide-binding protein with adenylate cyclase in intact wild-type and β2-adrenoceptor-expressing NG108-15 cells

Biochemical Journal ◽

10.1042/bj3080275 ◽

1995 ◽

Vol 308 (1) ◽

pp. 275-281 ◽

Cited By ~ 11

Author(s):

G D Kim ◽

I C Carr ◽

G Milligan

Keyword(s):

Adenylate Cyclase ◽

Nucleotide Binding ◽

Dose Effect ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Guanine Nucleotide ◽

Guanine Nucleotide Binding Protein ◽

Guanine Nucleotide Binding ◽

Dose Dependent ◽

Stimulation Of

Neuroblastoma x glioma hybrid, NG108-15, cells appear to express the alpha-subunit of the guanine nucleotide-binding protein Gs in a substantial molar excess over its effector adenylate cyclase [Kim, Adie and Milligan (1994) Eur. J. Biochem. 219, 135-143]. Addition of the IP prostanoid receptor agonist iloprost to intact NG108-15 cells resulted in a dose-dependent increase in formation of the complex between Gs alpha and adenylate cyclase (GSAC) as measured by specific high-affinity binding of [3H]forskolin. NG108-15 cells transfected to express either relatively high (clone beta N22) or low (clone beta N17) levels of beta 2-adrenoceptor both showed dose-dependent increases in specific [3H]forskolin binding in response to the beta-adrenoceptor agonist isoprenaline, and maximally effective concentrations of isoprenaline resulted in the generation of similar numbers of GSAC complexes in both clones. The dose-effect curve for clone beta N22, however, was some 15-fold to the left of that for clone beta N17, which is similar to that noted for isoprenaline-mediated stimulation of adenylate cyclase activity [Adie and Milligan (1994) Biochem. J. 303, 803-808]. In contrast, dose-effect curves for iloprost stimulation of [3H]forskolin binding were not different in clones beta N22 and beta N17. Basal specific [3H]forskolin binding in the absence of agonist was significantly greater in cells of clone beta N22 than clone beta N17. This was not a reflection of higher immunological levels of adenylate cyclase, indicating that the higher basal formation of GSAC probably reflects empty-receptor activation of Gs. This higher basal specific [3H]forskolin binding was partially reversed by propranolol. The addition of the opioid peptide D-Ala-D-Leu-enkephalin to NG108-15 cells did not reduce iloprost-stimulated [3H]forskolin binding even though this peptide inhibits stimulated adenylate cyclase activity by activation of a delta opioid receptor.

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Evidence for two GTPases activated by thrombin in membranes of human platelets

Biochemical Journal ◽

10.1042/bj2370669 ◽

1986 ◽

Vol 237 (3) ◽

pp. 669-674 ◽

Cited By ~ 35

Author(s):

R Grandt ◽

K Aktories ◽

K H Jakobs

Keyword(s):

Adenylate Cyclase ◽

Binding Protein ◽

Nucleotide Binding ◽

Guanine Nucleotide ◽

Guanine Nucleotide Binding Protein ◽

Gtp Hydrolysis ◽

Platelet Membranes ◽

Guanine Nucleotide Binding ◽

Stimulation Of ◽

Nucleotide Binding Protein

Thrombin inhibits adenylate cyclase and stimulates GTP hydrolysis by high-affinity GTPase(s) in membranes of human platelets at almost identical concentrations. Both of these thrombin actions are similar to those observed with agonist-activated alpha 2-adrenoceptors coupling to the inhibitory guanine nucleotide-binding protein N1. However, stimulation of GTP hydrolysis caused by adrenaline (alpha 2-adrenoceptor agonist) and by thrombin at maximally effective concentrations was partially additive, whereas with regard to adenylate cyclase inhibition no additive response was observed. Furthermore, treatment of platelet membranes with pertussis toxin, which inactivates Ni and largely abolishes thrombin- and adrenaline-induced adenylate cyclase inhibition and adrenaline-induced GTPase stimulation, decreased the thrombin-induced stimulation of GTP hydrolysis by only about 30%. Additionally, the thiol reagent N-ethylmalemide (NEM) at rather low concentrations abolished thrombin- and adrenaline-induced stimulation of GTP hydrolysis was decreased by only 30-40% by treatment of platelet membranes with even high concentrations of NEM. Treatment with cholera toxin, which inhibits GTPase activity of the Ns (stimulatory guanine nucleotide-binding) protein, has no effect on thrombin-stimulated GTP hydrolysis. The data suggest that thrombin interaction with its receptor sites in platelet membranes leads to stimulation of two GTP-hydrolysing enzymes. One of these enzymes is apparently Ni and is also activated by agonist-activated alpha 2-adrenoceptors and is inactivated by pertussis toxin and NEM treatment. The other GTP-hydrolysing enzyme activated by thrombin may represent a guanine nucleotide-binding protein apparently involved in the coupling of thrombin receptors to the phosphoinositide phosphodiesterase.

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Effects of tiazofurin on guanine nucleotide binding regulatory proteins in HL-60 cells

Blood ◽

10.1182/blood.v75.3.583.583 ◽

1990 ◽

Vol 75 (3) ◽

pp. 583-588 ◽

Cited By ~ 23

Author(s):

SM Kharbanda ◽

ML Sherman ◽

DW Kufe

Keyword(s):

Adenylate Cyclase ◽

Phospholipase C ◽

G Protein ◽

G Proteins ◽

Binding Sites ◽

Nucleotide Binding ◽

Membrane Receptors ◽

Guanine Nucleotide ◽

Guanine Nucleotide Binding ◽

Stimulation Of

Abstract Guanine nucleotide binding proteins (G proteins) are regulatory molecules that couple membrane receptors to effector systems such as adenylate cyclase and phospholipase C. The alpha subunits of G proteins bind to guanosine 5′-diphosphate (GDP) in the unstimulated state and guanosine 5′ triphosphate (GTP) in the active state. Tiazofurin (2-beta- D-ribofuranosylthiazole-4-carboxamide), a specific inhibitor of inosine monophosphate (IMP) dehydrogenase, decreases guanylate synthesis from IMP in HL-60 promyelocytic leukemia cells and depletes intracellular guanine nucleotide pools. This study demonstrates that treatment of HL- 60 cells with tiazofurin is associated with a fourfold increase in membrane binding sites for the nonhydrolyzable analogue GDP beta S. This increase in binding sites was associated with a 3.2-fold decrease in GDP beta S binding affinity. Similar findings were obtained with GTP gamma S. These effects of tiazofurin treatment on guanine nucleotide binding were also associated with decreased adenosine diphosphate- ribosylation of specific G protein substrates by cholera and pertussis toxin. The results further demonstrate that tiazofurin treatment results in inhibition of G protein-mediated transmembrane signaling mechanisms. In this regard, stimulation of adenylate cyclase by prostaglandin E2 was inhibited by over 50% in tiazofurin-treated cells. Furthermore, tiazofurin treatment resulted in inhibition of N- formylmethionylleucylphenylalanine-induced stimulation of phospholipase C. Taken together, these results indicate that tiazofurin acts at least in part by inhibiting the ability of G proteins to function as transducers of intracellular signals.

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Effects of tiazofurin on guanine nucleotide binding regulatory proteins in HL-60 cells

Blood ◽

10.1182/blood.v75.3.583.bloodjournal753583 ◽

1990 ◽

Vol 75 (3) ◽

pp. 583-588

Author(s):

SM Kharbanda ◽

ML Sherman ◽

DW Kufe

Keyword(s):

Adenylate Cyclase ◽

Phospholipase C ◽

G Protein ◽

G Proteins ◽

Binding Sites ◽

Nucleotide Binding ◽

Membrane Receptors ◽

Guanine Nucleotide ◽

Guanine Nucleotide Binding ◽

Stimulation Of

Guanine nucleotide binding proteins (G proteins) are regulatory molecules that couple membrane receptors to effector systems such as adenylate cyclase and phospholipase C. The alpha subunits of G proteins bind to guanosine 5′-diphosphate (GDP) in the unstimulated state and guanosine 5′ triphosphate (GTP) in the active state. Tiazofurin (2-beta- D-ribofuranosylthiazole-4-carboxamide), a specific inhibitor of inosine monophosphate (IMP) dehydrogenase, decreases guanylate synthesis from IMP in HL-60 promyelocytic leukemia cells and depletes intracellular guanine nucleotide pools. This study demonstrates that treatment of HL- 60 cells with tiazofurin is associated with a fourfold increase in membrane binding sites for the nonhydrolyzable analogue GDP beta S. This increase in binding sites was associated with a 3.2-fold decrease in GDP beta S binding affinity. Similar findings were obtained with GTP gamma S. These effects of tiazofurin treatment on guanine nucleotide binding were also associated with decreased adenosine diphosphate- ribosylation of specific G protein substrates by cholera and pertussis toxin. The results further demonstrate that tiazofurin treatment results in inhibition of G protein-mediated transmembrane signaling mechanisms. In this regard, stimulation of adenylate cyclase by prostaglandin E2 was inhibited by over 50% in tiazofurin-treated cells. Furthermore, tiazofurin treatment resulted in inhibition of N- formylmethionylleucylphenylalanine-induced stimulation of phospholipase C. Taken together, these results indicate that tiazofurin acts at least in part by inhibiting the ability of G proteins to function as transducers of intracellular signals.

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