POSTTRANSLATIONAL REGULATION OF NEURONAL ACETYLCHOLINE-RECEPTORS STABLY EXPRESSED IN A MOUSE FIBROBLAST CELL-LINE

Second messenger regulation of neuronal acetylcholine receptors (AChRs ) was investigated in a mouse fibroblast cell line, M10, stably transf ected with chicken alpha 4 and beta 2 cDNAs. Both forskolin and 8-brom o-cyclic adenosine 3',5'-monophosphate (cAMP) induced large increases in the numbers of AChRs. The increases were due in part to increased t ranscription and translation of the alpha 4 and beta 2 genes. Blockade of protein synthesis with cycloheximide, however, revealed that forsk olin also exerts a post-translational effect, increasing the number of surface receptors by twofold. Immunoblot analysis of sucrose gradient fractions confirmed that the cells had a large fraction of unassemble d subunits potentially available for receptor assembly. The post-trans lational effect of forskolin was blocked by H-89, an inhibitor of cAMP -dependent protein kinase, and by okadaic acid, an inhibitor of phosph atases 1 and 2A. Nicotine also acted posttranslationally to induce a t wofold increase in the number of surface receptors, but the mechanism differed from that utilized by forskolin, since the effects of the two agents were additive and were differentially affected by okadaic acid . The results suggest that protein phosphorylation-dephosphorylation m echanisms act post-translationally to increase the number of neuronal AChRs maintained on the cell surface. This could be achieved by increa sing the efficiency of receptor assembly, transport, or stabilization on the cell surface. (C) 1996 John Wiley & Sons, Inc.