Negative regulation of alpha(2)-adrenergic receptor-mediated G(i) signalling by a novel pathway

Chinese hamster ovary (CHO) cells stably expressing alpha(2) adrenergic rec eptor (alpha(2)AR) were pretreated with cholera toxin (CTX) and then treate d with or without PMA. The alpha(2A)AR-mediated inhibition of forskolin-sti mulated cAMP accumulation was completely ablated by CTX pretreatment only a fter additional treatment with PMA. Although the addition of cycloheximide (protein synthesis inhibitor) and H-89 (cAMP dependent protein kinase inhib itor) 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 ade nylate cyclase activity and the agonist competition curve were not influenc ed by treatment with CTX or PMA, suggesting that a cytosolic factor was inv olved in this negative regulation. The m2-muscarinic-acetylcholine-receptor -mediated inhibition of the forskolin-stimulated accumulation of cAMP was a lso attenuated by treatment with CTX and PMA. The ablation of alpha(2A)AR-m ediated 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 re ceptors 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 conside rably increased by pretreatment with CTX. These results indicate a novel re gulatory pathway, whereby a cytosolic factor induced by the elevation of ce llular cAMP levels negatively regulates G(i) signalling in a protein-kinase -C-dependent manner.