Identification of adenylyl cyclase isoenzymes in CHO and B82 cells

Varga, Éva; Stropova, Dagmar; Rubenzik, Marc; Wang, Man; Landsman, Robert S.; Roeske, William R.; Yamamura, Henry I.

doi:10.1016/s0014-2999(98)00258-1

Cited by 40 publications

(30 citation statements)

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“…Heterologous sensitization of adenylyl cyclase occurs after the long-term activation of G␣ i/o -coupled receptors, and results in a subsequent increase in adenylyl cyclase activity (Watts, 2002). Heterologous sensitization after prolonged ␦-opioid receptor activation in CHO cells correlates with an increase in phosphorylation of the predominant endogenous adenylyl cyclase isoform AC6 (Varga et al, 1998(Varga et al, , 1999. The Raf1 inhibitor GW5074 or a combination of tyrosine kinase and PKC inhibitors have recently been demonstrated to markedly attenuate the magnitude of heterologous sensitization in CHO cells after prolonged ␦-opioid receptor activation (Varga et al, 2002(Varga et al, , 2003.…”

Section: Discussionmentioning

confidence: 99%

“…PKC increases AC5 activity (Kawabe et al, 1994(Kawabe et al, , 1996; however, PKC phosphorylation inhibits AC6 activation in insect or mammalian cell membranes (Lai et al, 1997(Lai et al, , 1999Lin et al, 2002;Wu et al, 2001). Although PMA activation of PKC is reported to have no significant effect on drug-stimulated AC6 activity in transiently transfected HEK293 cells (Jacobowitz et al, 1993;Yoshimura and Cooper, 1993), a recent study suggested that PKC may be involved in ␦-opioid receptor-induced heterolo-gous sensitization of endogenous AC6 in CHO cells (Varga et al, 1998(Varga et al, , 2003. The same CHO cell model has also been used to demonstrate that the Raf1 inhibitor GW5074 partially attenuated heterologous sensitization (Varga et al, 2002).…”

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confidence: 99%

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Protein Kinase C and Epidermal Growth Factor Stimulation of Raf1 Potentiates Adenylyl Cyclase Type 6 Activation in Intact Cells

Beazely¹,

Alan²,

Watts³

2004

Mol Pharmacol

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Adenylyl cyclase type 6 (AC6) activity is inhibited by protein kinase C (PKC) in vitro; however, in intact cells, PKC activation does not inhibit the activity of transiently expressed AC6. To investigate the effects of PKC activation on AC6 activity in intact cells, we constructed human embryonic kidney (HEK) 293 cells that stably express wild-type AC6 (AC6-WT) or an AC6 mutant lacking a PKC and cyclic AMP-dependent protein kinase (PKA) phosphorylation site, Ser674 (AC6-S674A). In contrast to in vitro observations, we observed a PKC-mediated enhancement of forskolin-and isoproterenol-stimulated cyclic AMP accumulation in HEK-AC6 cells. Phorbol 12-myristate 13-acetate also potentiated cyclic AMP accumulation in cells expressing endogenous AC6, including Chinese hamster ovary cells and differentiated Cath.a differentiated cells. In HEK-AC6-S674A cells, the potentiation of AC6 stimulation was significantly greater than in cells expressing AC6-WT. The positive effect of PKC activation on AC6 activity seemed to involve Raf1 kinase because the Raf1 inhibitor 3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one (GW5074) inhibited the PKC potentiation of AC6 activity. Furthermore, the forskolin-stimulated activity of a recombinant AC6 in which the putative Raf1 regulatory sites have been eliminated was not potentiated by activation of PKC. The ability of Raf1 to regulate AC6 may involve a direct interaction because AC6 and a constitutively active Raf1 construct were coimmunoprecipitated. In addition, we report that epidermal growth factor receptor activation also enhances AC6 signaling in a Raf1-dependent manner. These data suggest that Raf1 potentiates drug-stimulated cyclic AMP accumulation in cells expressing AC6 after activation of multiple signaling pathways.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Protein Kinase C and Epidermal Growth Factor Stimulation of Raf1 Potentiates Adenylyl Cyclase Type 6 Activation in Intact Cells

Beazely¹,

Alan²,

Watts³

2004

Mol Pharmacol

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“…One possible explanation of our results is that the latter types of adenylyl cyclase might be involved in the LPA-induced cAMP accumulation in p2y 9 /GPR23-expressing CHO cells. Indeed, type VI and type VII adenylyl cyclases are expressed in CHO cells (33).…”

Section: Screening Of Candidate Ligands-phylogenetic Analysismentioning

confidence: 99%

Identification of p2y9/GPR23 as a Novel G Protein-coupled Receptor for Lysophosphatidic Acid, Structurally Distant from the Edg Family

Noguchi

Ishii

Shimizu

2003

Journal of Biological Chemistry

519

425

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Lysophosphatidic acid (LPA) is a bioactive lipid mediator with diverse physiological and pathological actions on many types of cells. LPA has been widely considered to elicit its biological functions through three types of G protein-coupled receptors, Edg-2 (endothelial cell differentiation gene-2)/LPA 1 /vzg-1 (ventricular zone gene-1), Edg-4/LPA 2 , and Edg-7/LPA 3 . We identified an orphan G protein-coupled receptor, p2y 9 /GPR23, as the fourth LPA receptor (LPA 4 ). Membrane fractions of RH7777 cells transiently expressing p2y 9 /GPR23 displayed a specific binding for 1-oleoyl-LPA with a K d value of around 45 nM. Competition binding and reporter gene assays showed that p2y 9 /GPR23 preferred structural analogs of LPA with a rank order of 1-oleoyl-> 1-stearoyl-> 1-palmitoyl-> 1-myristoyl-> 1-alkyl-> 1-alkenyl-LPA. In Chinese hamster ovary cells expressing p2y 9 /GPR23, 1-oleoyl-LPA induced an increase in intracellular Ca 2؉ concentration and stimulated adenylyl cyclase activity. Quantitative real-time PCR demonstrated that mRNA of p2y 9 /GPR23 was significantly abundant in ovary compared with other tissues. Interestingly, p2y 9 /GPR23 shares only 20 -24% amino acid identities with Edg-2/LPA 1 , Edg-4/LPA 2 , and Edg-7/LPA 3 , and phylogenetic analysis also shows that p2y 9 /GPR23 is far distant from the Edg family. These facts suggest that p2y 9 /GPR23 has evolved from different ancestor sequences from the Edg family.

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“…, 1997). We have previously determined that AC VI is a major native isoenzyme in CHO cells (Varga et al, 1998). Thus, we hypothesized that AC VI is that major isoenzyme involved in forskolin-stimulated cAMP overshoot upon chronic ␦-opioid agonist treatment in the hDOR/CHO cells.…”

Section: Resultsmentioning

confidence: 99%

“…It has also been shown previously that adenylyl cyclase superactivation is isoenzyme-specific (Thomas and Hoffman, 1996;Avidor-Reiss et al, 1997, Cumbay andWatts, 2001). Using a reverse transcription-polymerase chain reaction method, we previously found that AC VI and AC VII are the major AC isoenzymes expressed in CHO cells (Varga et al, 1998). It was demonstrated previously that AC VI is one of the few AC isoenzymes that becomes sensitized to forskolin after chronic opioid receptor stimulation and subsequent withdrawal (Avidor-Reiss et al, 1997).…”

Section: Discussionmentioning

confidence: 96%

Converging Protein Kinase Pathways Mediate Adenylyl Cyclase Superactivation upon Chronic δ-Opioid Agonist Treatment

Varga

Rubenzik

Stropova

et al. 2003

J Pharmacol Exp Ther

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Adenylyl cyclase (AC) superactivation is thought to play an important role in opioid tolerance, dependence, and withdrawal. In the present study, we investigated the involvement of protein kinases in chronic ␦-opioid agonist-mediated AC superactivation in Chinese hamster ovary (CHO) cells stably expressing the human ␦-opioid receptor (hDOR/CHO). Maximal forskolin-stimulated cAMP formation in hDOR/CHO cells increased by 472 Ϯ 91, 399 Ϯ 2, and 433 Ϯ 73% after chronic treatment with the ␦-opioid-enkephalin, and deltorphin II, respectively. Concurrently, chronic SNC 80 (1 M, 4-h) treatment augmented 32 P incorporation into a 200-kDa protein immunoreactive with the ACV/VI antibody by 300 Ϯ 60% in hDOR/CHO cell lysates. The calmodulin antagonist calmidazolium significantly attenuated chronic deltorphin II-mediated AC superactivation. Tyrosine kinase (genistein) and protein kinase C (chelerythrine) inhibitors individually had minimal effect on chronic ␦-opioid agonist-mediated AC superactivation. Conversely, simultaneous treatment with both genistein and chelerythrine significantly attenuated AC superactivation. Because we showed previously that the Raf-1 inhibitor 3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one (GW5074) attenuates AC superactivation, we hypothesize that parallel calmidazolium-, chelerythrine-, and genistein-sensitive pathways converge at Raf-1 to mediate AC superactivation by phosphorylating AC VI in hDOR/CHO cells.Chronic opioid receptor activation frequently leads to the sensitization of adenylyl cyclase to stimulators after the inhibitory agonist has been removed (AC superactivation). AC superactivation after chronic opioid agonist exposure is thought to contribute to the development of opioid tolerance, dependence, and withdrawal (Williams et al., 2001). A better understanding of the molecular mechanisms of chronic ␦-opioid agonist treatment-mediated AC superactivation should aid in the development of longer acting analgesics with fewer side effects.We have reported previously that in Chinese hamster ovary (CHO) cells stably expressing the human ␦-opioid receptor (hDOR/CHO), chronic ␦-opioid agonist treatment gives rise to AC superactivation (Malatynska et al., 1996). In addition, we also demonstrated that chronic ␦-opioid agonist treatment of the hDOR/CHO cells augments 32 P incorporation into proteins immunoreactive with an AC V/VI-specific antibody (Varga et al., 1999). The identity of protein kinase(s) involved in chronic ␦-opioid agonist-mediated phosphorylation in hDOR/CHO cells, and the role of the phosphorylation in AC superactivation, however, have not been investigated previously. In the present work, we studied the effect of protein kinase inhibitors on chronic ␦-opioid agonistmediated AC superactivation.Recent data (Tan et al., 2001) indicate the important role of the protein kinase p74Raf-1 in phosphorylation of adenylyl cyclase VI in transfected human embryonic kidney 293 cells. Moreover, Raf-1-mediated phosphorylation led to the sensitization of AC VI to s...

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Identification of adenylyl cyclase isoenzymes in CHO and B82 cells

Cited by 40 publications

References 6 publications

Protein Kinase C and Epidermal Growth Factor Stimulation of Raf1 Potentiates Adenylyl Cyclase Type 6 Activation in Intact Cells

Protein Kinase C and Epidermal Growth Factor Stimulation of Raf1 Potentiates Adenylyl Cyclase Type 6 Activation in Intact Cells

Identification of p2y9/GPR23 as a Novel G Protein-coupled Receptor for Lysophosphatidic Acid, Structurally Distant from the Edg Family

Converging Protein Kinase Pathways Mediate Adenylyl Cyclase Superactivation upon Chronic δ-Opioid Agonist Treatment

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