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Regulation of cyclic AMP response-element binding-protein (CREB) by Gq/11-protein-coupled receptors in human SH-SY5Y neuroblastoma cells

By Elizabeth M. Rosethorne, Stefan R. Nahorski and R.A. John Challiss

Abstract

This paper was published as Biochemical Pharmacology, 2008, 75 (4), pp. 942-955. It is available from http://www.sciencedirect.com/science/journal/00062952. Doi: 10.1016/j.bcp.2007.10.015Metadata only entryHuman SH-SY5Y neuroblastoma cells have been used to investigate mechanisms involved in CREB phosphorylation after activation of two endogenously expressed Gq/11-protein-coupled receptors, the M3 muscarinic acetylcholine (mACh) and B2 bradykinin receptors. Stimulation with either methacholine or bradykinin resulted in maximal increases in CREB phosphorylation within 1 min, with either a rapid subsequent decrease (bradykinin) to basal levels, or a sustained response (methacholine). Inhibitor studies were performed to assess the involvement of a number of potential kinases in signalling to CREB phosphorylation. Removal of extracellular Ca2+, inhibition of Ca2+/calmodulin-dependent protein kinase II and down-regulation of protein kinase C (PKC) resulted in reduced CREB phosphorylation after both M3 mACh and B2 bradykinin receptor activation. In contrast, inhibition of MEK1/2 by U0126 resulted in significantly reduced CREB phosphorylation levels after B2 bradykinin, but not M3 mACh receptor activation. In addition, we demonstrate that maintained phosphorylation of CREB is necessary for CRE-dependent gene transcription as the M3 mACh, but not the B2 bradykinin receptor activates both a recombinant CRE-dependent reporter gene, and the endogenous c-Fos gene. These data highlight the involvement of multiple, overlapping signalling pathways linking these endogenous Gq/11-coupled metabotropic receptors to CREB and emphasize the importance of the duration of signalling pathway activation in converting a CREB phosphorylation event into a significant change in transcriptional activity

Publisher: Elsevier
Year: 2008
DOI identifier: 10.1016/j.bcp.2007.10.015
OAI identifier: oai:lra.le.ac.uk:2381/8077
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