Regulation of cyclic AMP response-element binding-protein (CREB) by Gq/11-protein-coupled receptors in human SH-SY5Y neuroblastoma cells.
Human 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.
Department of Cell Physiology and Pharmacology, University of Leicester, Henry Wellcome Building, Lancaster Road, Leicester LE1 9HN, UK.
Elizabeth M
EM
Rosethorne
Stefan R
SR
Nahorski
R A John
RA
Challiss
D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism / Q000502:physiology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D016210:Methacholine Chloride / Q000494:pharmacology; D009447:Neuroblastoma; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D043782:Receptor, Bradykinin B2 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism / Q000502:physiology*; D015290:Second Messenger Systems / Q000187:drug effects / Q000502:physiology
D017362:Cyclic AMP Response Element-Binding Protein; D043782:Receptor, Bradykinin B2; D043562:Receptors, G-Protein-Coupled; D016210:Methacholine Chloride; D019869:Phosphatidylinositol 3-Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D001920:Bradykinin; D002118:Calcium