Negative regulation of alpha2-adrenergic receptor-mediated Gi signalling by a novel pathway.
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.
Takesono
A
A
Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
Zahner
J
J
Blumer
K J
KJ
Nagao
T
T
Kurose
H
H
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002772:Cholera Toxin / Q000494:pharmacology; D006224:Cricetinae; D017931:DNA Primers; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism*; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D017931:DNA Primers; D018341:Receptors, Adrenergic, alpha-2; D002772:Cholera Toxin; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate