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Published on: June 9, 2017
Negative regulation of alpha2-adrenergic receptor-mediated Gi signalling by a novel pathway
A Takesono1, J Zahner, K J Blumer
1Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
Abstract:
Chinese hamster ovary (CHO) cells stably expressing alpha(2) adrenergic receptor (alpha(2)AR) were pretreated with cholera toxin (CTX) and then treated with or without PMA. The alpha(2A)AR-mediated inhibition of forskolin-stimulated cAMP accumulation was completely ablated by CTX pretreatment only after additional treatment with PMA. Although the addition of cycloheximide (protein synthesis inhibitor) and H-89 (cAMP dependent protein kinase inhibitor) did not completely counteract the negative regulation, the elevation of cAMP was a primary factor for negative regulation by treatment with CTX and PMA. In contrast with the cAMP response, the inhibition of membrane adenylate cyclase activity and the agonist competition curve were not influenced by treatment with CTX or PMA, suggesting that a cytosolic factor was involved in this negative regulation. The m2-muscarinic-acetylcholine-receptor-mediated inhibition of the forskolin-stimulated accumulation of cAMP was also attenuated by treatment with CTX and PMA. The ablation of alpha(2A)AR-mediated inhibition was not observed when alpha(2A)AR was expressed in Rat2 fibroblast cells, suggesting that this negative regulation is not dependent on the receptor type but is instead a phenomenon common to G(i)-coupled receptors in CHO cells. Reverse-transcriptase-mediated PCR and Northern blot analysis showed that the expression of GOS8/RGS2 mRNA, which is a member of the regulator of G-protein signalling (RGS) group of proteins, was considerably increased by pretreatment with CTX. These results indicate a novel regulatory pathway, whereby a cytosolic factor induced by the elevation of cellular cAMP levels negatively regulates G(i) signalling in a protein-kinase-C-dependent manner.
Insights
Cholera toxin and PMA treatment in Chinese hamster ovary cells revealed a novel pathway where elevated cAMP levels induce a cytosolic factor that negatively regulates G(i) signaling, impacting alpha(2A) adrenergic receptor function.
Area of Science:
- Cellular and Molecular Pharmacology
- Signal Transduction Pathways
- G Protein-Coupled Receptor Regulation
Background:
- Alpha(2) adrenergic receptors (α(2)AR) mediate cellular responses through G(i) protein signaling.
- Cholera toxin (CTX) activates G(s) proteins, increasing cyclic AMP (cAMP) levels.
- Phorbol 12-myristate 13-acetate (PMA) activates protein kinase C (PKC).
Purpose of the Study:
- To investigate the novel regulatory pathway of G(i)-coupled receptor signaling in Chinese hamster ovary (CHO) cells.
- To elucidate the role of cAMP and protein kinase C in modulating α(2A)AR-mediated signaling.
- To identify potential cytosolic factors involved in negative regulation.
Main Methods:
- Stable expression of α(2A)AR in CHO cells.
- Treatment with CTX, PMA, cycloheximide, and H-89.
- Measurement of forskolin-stimulated cAMP accumulation.
- Analysis of adenylate cyclase activity and receptor binding.
- Reverse-transcriptase-mediated PCR and Northern blot analysis for GOS8/RGS2 mRNA expression.
Main Results:
- CTX pretreatment followed by PMA treatment ablated α(2A)AR-mediated inhibition of cAMP accumulation.
- Elevated cAMP levels were crucial for this negative regulation.
- Cytosolic factors, not membrane-bound components, were implicated.
- GOS8/RGS2 mRNA expression significantly increased after CTX pretreatment.
- Similar attenuation was observed for m2-muscarinic-acetylcholine-receptor-mediated inhibition.
- The phenomenon was specific to CHO cells and G(i)-coupled receptors.
Conclusions:
- A novel cAMP-induced cytosolic factor negatively regulates G(i) signaling in a PKC-dependent manner in CHO cells.
- This regulation is independent of receptor type but dependent on the cell type and G(i)-coupling.
- The induction of GOS8/RGS2 (a regulator of G-protein signaling) is a key component of this pathway.
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