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

Elizabeth M Rosethorne1, Stefan R Nahorski, R A John Challiss

  • 1Department of Cell Physiology and Pharmacology, University of Leicester, Henry Wellcome Building, Lancaster Road, Leicester LE1 9HN, UK.

Biochemical Pharmacology
|November 27, 2007
PubMed

Insights

Gq/11-protein-coupled receptors, M3 muscarinic acetylcholine (mACh) and B2 bradykinin receptors, activate CREB phosphorylation through distinct signaling pathways. Sustained signaling is crucial for CRE-dependent gene transcription.

Area of Science:

  • Neuroscience
  • Cellular Signaling
  • Molecular Biology

Background:

  • Investigates signaling mechanisms of Gq/11-protein-coupled receptors.
  • Focuses on CREB phosphorylation and its role in gene transcription.
  • Utilizes human SH-SY5Y neuroblastoma cells.

Purpose of the Study:

  • To elucidate the signaling pathways downstream of M3 muscarinic acetylcholine (mACh) and B2 bradykinin receptors.
  • To determine the role of CREB phosphorylation in CRE-dependent gene transcription.
  • To compare the signaling dynamics and transcriptional outcomes of mACh and bradykinin receptor activation.

Main Methods:

  • Stimulation of SH-SY5Y cells with methacholine and bradykinin.
  • Assessment of CREB phosphorylation using inhibitor studies targeting various kinases (Ca2+/calmodulin-dependent protein kinase II, PKC, MEK1/2).
  • Analysis of CRE-dependent reporter gene and c-Fos gene expression.

Main Results:

  • Both M3 mACh and B2 bradykinin receptors rapidly increased CREB phosphorylation.
  • Methacholine induced sustained CREB phosphorylation, while bradykinin caused a transient response.
  • Ca2+, Ca2+/calmodulin-dependent protein kinase II, and PKC were involved in both receptor pathways.
  • MEK1/2 inhibition affected B2 bradykinin but not M3 mACh receptor signaling.
  • M3 mACh receptor activation, but not B2 bradykinin, led to sustained CREB phosphorylation and subsequent CRE-dependent gene transcription (c-Fos).

Conclusions:

  • Multiple, overlapping signaling pathways link Gq/11-coupled receptors to CREB.
  • The duration of signaling pathway activation is critical for translating CREB phosphorylation into transcriptional changes.
  • Distinct downstream signaling and transcriptional outcomes arise from M3 mACh and B2 bradykinin receptor activation despite shared upstream components.

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