Regulation of the cAMP-response element-binding (CREB) protein by the group I metabotropic glutamate receptors Helen K. Warwick, Stefan R. Nahorski and R.A. John Challiss Dept. of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester, LE1 9HN. UK.

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While the group I metabotropic glutamate receptors, mGlu1a and mGlu5a, are closely related in terms of their structure and pharmacology, differences are seen with respect to their regulation of intracellular signalling pathways (Hermans & Challiss, 2001). The aim of the current study was to investigate the regulation of cAMP-response element-binding protein (CREB) phosphorylation by mGlu1a and mGlu5a receptors.

Chinese hamster ovary (CHO) cells stably expressing either human mGlu1a or mGlu5a receptors under the control of the LacSwitch II inducible expression system were studied. Assay conditions were adapted from Thandi et al., (2002). Total CREB and Ser-133 phosphorylated CREB immunoreactivity were assessed using western blotting and quantified (mean ± SEM, n=3-5) using the BioRad Imaging Densitometry system. Statistical differences between data sets were determined using a 1-way ANOVA followed by Bonferroni's multiple comparison test at P<0.05.

Time-courses of CREB phosphorylation in CHO-mGlu1a and -mGlu5a cell lines in response to quisqualate (10 µM) showed a maximal increase within 2 min that was remained for at least 30min. No changes in total CREB expression were observed. The magnitude of the response was 6.6 ± 1.3 and 2.9 ± 0.3 fold-over-basal for mGlu1a- and mGlu5a-expressing cells, respectively. Analysis of concentration-dependencies of CREB phosphorylation in response to quisqualate revealed pEC₅₀ values of 6.63 ± 0.50 and 6.82 ± 0.23 for CHO-mGlu1a and -mGlu5a cells. The involvement of Ca²⁺ in receptor-mediated CREB phosphorylation was assessed by removal of extracellular and/or intracellular Ca²⁺. Stimulation of cells in Ca²⁺-free medium containing EGTA (100 µM) revealed 76 ± 16% and 64 ± 16% decreases in agonist-mediated responses in mGlu1a- and mGlu5a-expressing cells. Additional depletion of intracellular stores (by pre-addition of 2 µM thapsigargin) did not significantly increase the observed inhibitory effects. To examine the possible role of protein kinase C (PKC), cells were pre-treated with phorbol dibutyrate (1 µM, 24 h). This caused 54 ± 16% and 71 ± 23% suppressions of agonist-mediated responses in mGlu1a and mGlu5a cell-lines. Previous studies have identified ERK as a possible downstream effector involved in PKC-dependent CREB phosphorylation (Schinelli et al., 2001). However, pre-incubation of cells with MEK inhibitor, U0126 (10 µM, 30 min) did not affect quisqualate-stimulated CREB phosphorylation.

These data suggest that mGlu1a and mGlu5a receptor activation can regulate CREB phosphorylation and thus influence transcriptional activity. Both receptors appear to employ a Ca²⁺ - and PKC-dependent pathway similar to that recently described for ₁-adrenoceptor-mediated CREB phosphorylation in brown adipocytes (Thonberg et al., 2002).

We gratefully acknowledge the support of the Wellcome Trust.

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Schinelli, S. et al. (2001) J. Neurosci. 21, 8842-8853.
Thandi, S. et al. (2002) J. Neurochem. 83, 1139-1153.
Thonberg, H. et al. (2002) Biochem. J. 364, 73-79.