The transcription factor CREB (cyclic AMP response-element binding protein) is primarily regulated by the phosphorylation of Ser-133 by multiple protein kinases (Shaywitz & Greenberg, 1999). These protein kinase activities are regulated by a variety of receptor systems, including G protein-coupled receptors, which regulate the extent and time-course of CREB phosphorylation/activation. Here, we have examined the abilities of ß₂-adrenoceptors and M₃ muscarinic acetylcholine (mACh) receptors, stably co-expressed in a Chinese hamster ovary (CHO) cell background, either individually or interactively to regulate CREB phosphorylation.

CHO-ß2m3 cells (expression levels: ß₂, 332±41; M₃, 1175±94 fmol mg^-1 protein) were grown under standard cell culture conditions. Agonist stimulations were performed on confluent cells in Krebs-Henseleit buffer. Lysates were prepared by addition of 125mM Tris-HCl, 4%(w/v) SDS, 20% glycerol, 50mM dithiothreitol, 0.01% bromophenol blue, pH 6.8, followed by sonication, incubation at 100°C, and separated by SDS-PAGE (10% gel) before transfer to nitrocellulose. Total CREB and Ser-133 phosphorylated CREB were assessed using western blotting and quantified (mean±SEM, n3). For each experiment data were normalized to the response to forskolin (Fk; 10 µM).

Methacholine (MCh) and isoprenaline (ISO) each stimulated time-dependent increases in the phosphorylation of ^Ser133CREB (pCREB) that were maximal at 5 min and sustained for at least 15 min. Total CREB immunoreactivity did not change over this time-course. Concentration-dependencies could be established for both ISO- (pEC₅₀, 8.46 ± 0.34) and MCh- (pEC₅₀, 7.73 ± 0.43) stimulated pCREB at 15min. In comparison, ISO stimulated an approx. 100 fold increase in cAMP accumulation (pEC₅₀, 8.21 ± 0.05), while MCh caused only a modest 4 fold response (pEC₅₀, 4.18 ± 0.07). Reversibility of CREB phosphorylation was assessed by incubation with agonists for 5 min followed by respective addition of timolol or atropine. Each antagonist rapidly decreased CREB phosphorylation with full reversal by 10 min. Under similar conditions, ISO-stimulated cAMP accumulation was reversed by timolol with a t_1/2 value of 2.1 min. Co-addition of MCh with ISO markedly enhanced the cAMP response (ISO, 964 ± 66; ISO+MCh, 1812 ± 136 pmol mg^-1 protein), while co-addition of maximal concentrations of ISO and MCh produced an increase in pCREB that was increased, but less than additive. Preliminary experiments demonstrated that the responses observed at the level of pCREB were accurately reflected by CRE-luciferase reporter gene assays.

These data demonstrate that both M₃ mACh receptors and ß₂-adrenoceptors link to the regulation of CREB. While the ß₂-adrenoceptor-CREB linkage is likely to be mediated by cAMP, the M₃ mACh receptor-CREB pathway(s) and mechanisms of crosstalk with the ß₂-adrenoceptor remain to be delineated.

Shaywitz, A.J. & Greenberg, M.E. (1999) Ann Rev Biochem 68, 821-861.

We gratefully acknowledge the support of the Wellcome Trust.