scholarly journals Persistent activation of Gsα through limited proteolysis by calpain

2000 ◽  
Vol 347 (3) ◽  
pp. 733-740 ◽  
Author(s):  
Kaori SATO-KUSUBATA ◽  
Yukiko YAJIMA ◽  
Seiichi KAWASHIMA
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Cell Membranes ◽  
Active Form ◽  
Limited Proteolysis ◽  
Specific Inhibitor ◽  
Activation Mechanism ◽  
Calpain Inhibitor ◽  
Pituitary Cells ◽  
Rat Pituitary

Treatment of rat pituitary GH4C1 cell membranes with calpain, a calcium-activated cysteine protease, increased adenylate cyclase activity, and this activity was inhibited by a calpain inhibitor, leupeptin. Calpain treatment potentiated the activity of guanosine 5ʹ-[γ-thio]triphosphate (GTP[S]), but did not attenuate MnCl2 action on adenylate cyclase, suggesting that calpain acted at the G-protein level, rather than directly on adenylate cyclase. This calpain stimulation of adenylate cyclase was inhibited by an antibody raised against the C-terminal portion of Gsα, but not by anti-Gi2α or anti-Gβ antibodies. Furthermore, it was shown that Gsα is more susceptible to calpain-mediated proteolysis than Gi2α or Gβ. Therefore the stimulatory effect of calpain on adenylate cyclase is due to the cleavage of Gsα in GH4C1 cell membranes. Proteolysis of Gsα by μ-calpain involved sequential cleavages at two sites, resulting in the generation of a 39 kDa fragment first, and then a 20 kDa fragment, from the C-terminus. Treatment of GH4C1 cell membranes with cholera toxin increased the rate of cleavage. Cholera toxin treatment of intact GH4C1 cells induced the translocation of calpain from the cytosol to the membranes, a hallmark of calpain activation. In addition, treatment of intact GH4C1 cells with a calpain-specific inhibitor, benzyloxycarbonyl-Leu-leucinal, blocked the increased cAMP production and the down-regulation of Gsα, which were produced by cholera toxin or pituitary adenylate cyclase-activating polypeptide. These results suggest that calpain sustains adenylate cyclase in an active form through the cleavage of Gsα to an active Gsα fragment. This is a novel calpain-dependent activation mechanism of Gsα and, thus, of adenylate cyclase in rat pituitary cells.

HDL promotes adiponectin gene expression via the CAMKK/CAMKIV pathway

2021 ◽  
Author(s):  
Toshihiro Kobayashi ◽  
Hitomi Imachi ◽  
Kensaku Fukunaga ◽  
Jingya Lyu ◽  
Seisuke Sato ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signaling Pathways ◽  
Promoter Activity ◽  
Active Form ◽  
Specific Inhibitor ◽  
Density Lipoprotein ◽  
Dominant Negative ◽  
Activation Mechanism ◽  
Dominant Negative Mutant ◽  
Type I

Adiponectin (APN) is an adipokine that protects against diabetes and atherosclerosis. High-density lipoprotein (HDL) mediates reverse cholesterol transport, which also protects against atherosclerosis. In this process, the human homolog of the B class type I scavenger receptor (SR-BI/CLA-1) facilitates the cellular uptake of cholesterol from HDL. The level of circulating adiponectin is positively correlated with the serum level of HDL-cholesterol. In this study, we investigated whether HDL stimulates the gene expression of adiponectin through the Ca²+/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) cascade. Adiponectin expression was examined using real-time PCR and western blot analysis in 3T3-L1 cells incubated with HDL. CaMKIV activity was assessed by detection of activation loop phosphorylation (at Thr196 residue), and the effect of the constitutively active form, CaMKIVc, on adiponectin promoter activity was investigated. Our results showed that HDL stimulated APN gene expression via hSR-BI/CLA-1. Furthermore, we explored the signaling pathways by which HDL stimulated APN expression in 3T3-L1 cells. The stimulation of APN gene expression by HDL appears to be mediated by CaMKK, as STO-609, a specific inhibitor of CaMKK2, prevents this effect. We revealed that CaMKIVc increased APN gene transcriptional activity, and the CaMKIV dominant negative mutant blocked the effect of HDL on APN promoter activity. Finally, knockdown of hSR-BI/CLA-1 also cancelled the effect of HDL on APN gene expression. These results suggest that HDL has important role to improve the function of adipocytes by activating hSR-BI/CLA-1 and CaMKK/CaMKIV pathway is conceivable as one of the signaling pathways of this activation mechanism.

scholarly journals Short communications. Subunit A from cholera toxin is an activator of adenylate cyclase in pigeon erythrocytes

1975 ◽  
Vol 146 (1) ◽  
pp. 269-271 ◽  
Author(s):  
S Van Heyningen ◽  
C A King
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Cell Membranes ◽  
Erythrocyte Membranes ◽  
Subunit A ◽  
Subunit B

Intact cholera toxin and its purified subunit A both activate the adenylate cyclase of pigeon erythrocyte membranes, but subunit B does not. The activation by subunit A is unaffected by treatments that inhibit whole toxin by interfering with the binding of subunit B to cell membranes.

Signal transduction alterations in GH12C1 rat pituitary tumour cells following treatment with 5-azacytidine

1991 ◽  
Vol 6 (3) ◽  
pp. 257-268
Author(s):  
V. C. Parrow ◽  
J. O. Gordeladze ◽  
E. J. Paulssen ◽  
P. Aleström ◽  
K. M. Gautvik
Keyword(s):  
Drug Treatment ◽  
Adenylyl Cyclase ◽  
Cholera Toxin ◽  
Pertussis Toxin ◽  
Pituitary Tumour ◽  
Tumour Cells ◽  
Temporary Increase ◽  
Pituitary Cells ◽  
Intact Cells ◽  
Rat Pituitary

ABSTRACT In GH12C1 rat pituitary cells treated with 5-azacytidine, the stimulatory effects exerted by vasoactive intestinal peptide (VIP), the GTP analogue guanyl-5′-yl imidodiphosphate (Gpp(NH)p), 12-O-tetradecanoyl phorbol 13-acetate, cholera toxin and pertussis toxin on the membrane-bound adenylyl cyclase were almost completely abolished. The corresponding inhibitory effect of somatostatin was increased. Alterations in adenylyl cyclase responsiveness began at the end of the drug treatment, and were most pronounced on day 5 after removal of 5-azacytidine. The cells subsequently and completely recovered after 10 days in the absence of the drug. Measurements of cholera toxin- and VIP-enhanced cyclic AMP levels in intact cells confirmed these results, and VIP appeared to have no stimulatory effect on GH secretion after 5-azacytidine treatment. Down-regulation of Gαs RNA also occurred on day 5 after cessation of drug treatment. ADP-ribosylation subsequent to stimulation with pertussis toxin was markedly increased, indicating an enhancement of Gαi and/or GGαo. Furthermore, both basal and Gpp(NH)p-stimulated phospholipase C activities were augmented by pre-exposure to 5-azacytidine. Treatment of GH12C1 rat pituitary tumour cells with 5-azacytidine therefore causes a marked but temporary increase in the ratio of Gαi/Gαs protein levels.

scholarly journals The stimulatory guanine-nucleotide regulatory unit of adenylate cyclase from bovine cerebral cortex ADP-ribosylation and purification

1987 ◽  
Vol 241 (2) ◽  
pp. 325-336 ◽  
Author(s):  
E J Neer ◽  
L G Wolf ◽  
D M Gill
Keyword(s):  
Cerebral Cortex ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Polyacrylamide Gel Electrophoresis ◽  
Regulatory Protein ◽  
Active Form ◽  
Acid Hydrazide ◽  
Guanine Nucleotide ◽  
Adp Ribosylation ◽  
Catalytic Unit

Hormonal stimulation of adenylate cyclase from bovine cerebral cortex is mediated by a guanine-nucleotide regulatory protein (Gs). This protein contains at least three polypeptides: a guanine nucleotide-binding alpha s component and a beta X gamma component, which modulates the function of alpha s. The alpha s component from many tissues can be ADP-ribosylated with cholera toxin, but has been unusually difficult to modify in brain. We have improved incorporation of ADP-ribose by including isonicotinic acid hydrazide to inhibit the potent NAD glycohydrolase activity of brain. ADP-ribosylation is further improved by addition of detergent to render the substrates accessible and 20 mM-EDTA to chelate metal ions. Although Mg2+ is absolutely required for activation of adenylate cyclase by the GTP analogue guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG), it is not obligatory for p[NH]ppG-stimulated ADP-ribosylation by cholera toxin. Under these conditions, the ADP-ribosylation of brain membranes is not enhanced by a cytosolic protein. We find that there are two major sizes of brain alpha s, which we have named ‘alpha sL’, with an apparent Mr of 42,000-45,000, and ‘alpha sH’ with an apparent Mr of 46,000-51,000 depending on the gel-electrophoretic system used. The alpha sL and alpha sH components can incorporate different amounts of ADP-ribose depending on the reaction conditions, so that one or the other may appear to predominate. Thus we show that incomplete ADP-ribosylation by cholera toxin is not a good indication of the relative amounts of alpha s units. Functionally, however, both forms of alpha s appear to be similar. Both forms associate with the catalytic unit of adenylate cyclase, but neither of them does so preferentially. There is an excess of each of them over the amount associated with catalytic unit. We have now substantially purified Gs from brain by a modification of the method of Sternweis et al. [(1981) J. Biol. Chem. 256, 11517-11526] as well as by a new, simplified, procedure. On SDS/polyacrylamide-gel electrophoresis, the purified brain Gs contains both the 45 and 51 kDa alpha s polypeptides revealed by ADP-ribosylation and a beta X gamma component. Activation of purified alpha s by guanine nucleotides or fluoride can be reversed by addition of purified beta X gamma component. The activated form of purified brain Gs has an Mr of 49,000 as determined by hydrodynamic measurements, which is consistent with the idea that the active form of brain Gs is the dissociated one.

Effect of Pituitary Adenylate Cyclase-Activating Polypeptide on GH Gene Expression in Rat Pituitary Cells

2006 ◽  
Vol 805 (1) ◽  
pp. 684-691 ◽  
Author(s):  
YUJIN SHUTO ◽  
ANIKÓ SOMOGYVÁRI-VIGH ◽  
SÁNDOR VIGH ◽  
ICHIJI WAKABAYASHI ◽  
AKIRA ARIMURA
Keyword(s):  
Gene Expression ◽  
Adenylate Cyclase ◽  
Pituitary Cells ◽  
Pituitary Adenylate Cyclase ◽  
Rat Pituitary ◽  
Gh Gene ◽  
Rat Pituitary Cells

scholarly journals Gsα is a substrate for mono(ADP-ribosyl)transferase of NG108-15 cells. ADP-ribosylation regulates Gsα activity and abundance

1992 ◽  
Vol 288 (1) ◽  
pp. 331-336 ◽  
Author(s):  
L E Donnelly ◽  
R S Boyd ◽  
J MacDermot
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Cell Membranes ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Major Protein ◽  
Adp Ribosylation ◽  
Short Term Exposure ◽  
Significant Difference ◽  
Gs Alpha

NG108-15 neuroblastoma x glioma somatic hybrid cells were permeabilized in the presence of [32P]NAD+ and then cultured for 18 h. Resolution of the cell proteins on polyacrylamide gels revealed [32P]ADP-ribosylation of five major protein species with molecular mass values of 52 kDa, 44 kDa, 35 kDa, 30 kDa and 25 kDa. A similar pattern of labelling was also seen when NG108-15 cell membranes were incubated with [32P]NAD+ and hydrolysis of the product revealed mono(ADP-ribosyl)ation. Immunoprecipitation of these products with anti-Gs alpha antiserum revealed a single band identical to cholera toxin substrate. Culture of [32P]NAD(+)-loaded cells for 18 h in the presence of 50 mM-nicotinamide inhibited the eukaryotic mono(ADP-ribosyl)transferase activity. Inhibition of the eukaryotic enzyme was also accompanied by an increase in the abundance of Gs alpha, whether measured by Western blotting with anti-Gs alpha antibody (two separate antisera) or by cholera toxin-dependent [32P]ADP-ribosylation. There was no accompanying change in the abundance of G beta. The increase in Gs alpha abundance in nicotinamide-treated NG108-15 cells was accompanied by a 2-fold increase in basal adenylate cyclase activity (measured in the presence of GTP), and by a smaller but significant increase in iloprost-dependent activation of adenylate cyclase. Receptor number or affinity was not affected by nicotinamide, since this treatment did not alter the binding parameters of [3H]iloprost to NG108-15 cell membranes. Short-term exposure of cells to nicotinamide for 1 h revealed no significant difference in either basal or agonist-stimulated adenylate cyclase activity. These results reveal that mono(ADP-ribosyl)ation of Gs alpha by eukaryotic ADP-ribosyltransferase modifies the abundance and activity of Gs alpha in NG108-15 cells, and hence may play a role in the hormonal regulation of cell function.

scholarly journals Proteasome inhibitors induce apoptosis in growth hormone- and prolactin-secreting rat pituitary tumor cells

2002 ◽  
Vol 174 (3) ◽  
pp. 379-386 ◽  
Author(s):  
R Yu ◽  
SG Ren ◽  
S Melmed
Keyword(s):  
Tumor Cells ◽  
Pituitary Tumor ◽  
Proteasome Inhibitors ◽  
Prolactin Secretion ◽  
Gh3 Cells ◽  
Calpain Inhibitor ◽  
Pituitary Cells ◽  
Caspase Inhibitor ◽  
Rat Pituitary ◽  
Rat Pituitary Tumor Cells

Proteasome inhibitors induce apoptosis in some malignant cells, and we show here that these inhibitors induce apoptosis in rat pituitary MMQ and GH3 tumor cells but not in normal pituitary cells. Three proteasome inhibitors, PSI, MG-132, and lactacystin, but not the calpain inhibitor, ALLM, dose- and time-dependently caused apoptosis in these cells, and 10 microM PSI caused apoptosis in 70% of MMQ cells and in 25% of GH3 cells within 24 h. A lower PSI dose (10 nM) inhibited GH3 cell growth without causing significant apoptosis or affecting prolactin secretion. Primary rat pituitary cells were resistant to both PSI and MG-132 and did not undergo apoptosis. In MMQ cells, DNA synthesis was slowed (approximately 30%) after 6 h of 10 microM PSI treatment and a partial cell cycle block at G2/M was evident after 8 h. Colorimetric caspase substrate assay and Western blotting of caspase substrates showed that caspases 2 and 3 are activated by PSI while caspases 6 and 8 remained inactive. A broad-range caspase inhibitor, caspase inhibitor III, prevented apoptosis induced by PSI. The results show that proteasome inhibitors induce apoptosis in rat pituitary tumor cells by specific caspase activation. This novel group of drugs may potentially be used in treatment of aggressive pituitary tumors, especially as their action appears relative for tumor cells.

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