Korean J Physiol Pharmacol.  2011 Apr;15(2):95-100. 10.4196/kjpp.2011.15.2.95.

Regulation of DREAM Expression by Group I mGluR

• Lee J
• Kim I
• Oh SR
• Ko SJ
• Lim MK
• Kim DG
• Kim CH

• KMID: 2285413
• DOI: http://doi.org/10.4196/kjpp.2011.15.2.95

Abstract

DREAM (downstream regulatory element antagonistic modulator) is a calcium-binding protein that regulates dynorphin expression, promotes potassium channel surface expression, and enhances presenilin processing in an expression level-dependent manner. However, no molecular mechanism has yet explained how protein levels of DREAM are regulated. Here we identified group I mGluR (mGluR1/5) as a positive regulator of DREAM protein expression. Overexpression of mGluR1/5 increased the cellular level of DREAM. Up-regulation of DREAM resulted in increased DREAM protein in both the nucleus and cytoplasm, where the protein acts as a transcriptional repressor and a modulator of its interacting proteins, respectively. DHPG (3,5-dihydroxyphenylglycine), a group I mGluR agonist, also up-regulated DREAM expression in cortical neurons. These results suggest that group I mGluR is the first identified receptor that may regulate DREAM activity in neurons.

Keyword

Calcium; DREAM; Group I mGluR; mGluR5; mGluR1; Pain

MeSH Terms

Calcium
Cytoplasm
Dynorphins
Methoxyhydroxyphenylglycol
Neurons
Potassium Channels
Presenilins
Proteins
Receptors, Metabotropic Glutamate
Up-Regulation
Calcium
Dynorphins
Methoxyhydroxyphenylglycol
Potassium Channels
Presenilins
Proteins
Receptors, Metabotropic Glutamate

Figure

• 

  Fig. 1. Group I mGluR increases DREAM protein expression. (A, C, and D∼F) HEK293 cells were co-transfected with 500 ng of each plasmid expressing DREAM or hippocalcin and 50 ng of pRK5 empty vector, pRK5-mGluR subtypes, or pRK5-mGluR5 F767S (Gαq binding site mutant of mGluR5). Cell lysates were immunoblotted with rabbit anti-DREAM or anti-hippocalcin antibody. (B) pRK5 or pRK5-mGluR5 (200 ng) was transfected into SK-N-MC cells for 36 h before cell lysis. Immunoblotting using mouse anti-DREAM antibody was performed. (E) Cells were treated with MPEP for 15 h before lysis. Lysis was performed by boiling with denaturing lysis buffer after 36 h of cultivation. WT, wild-type.

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  Fig. 2. mGluR5 stabilizes DREAM protein. (A) pCBA-DREAM was co-transfected with pRK5 or pRK5-mGluR5 into HEK293 cells. Cells were lysed after 48 h of incubation. Cells were treated with cycloheximide (CHX) at 100 μg/ml for 0, 2, 4, 6, or 8 h before cell lysis. DREAM expression was analyzed by immunoblotting with rabbit anti-DREAM antibody. (B) HEK293 cells were transfected as indicated above. EDTA-AM (100 nM) was added to culture for 12 h to chelate intracellular Ca2+. Immunoblotting was performed using rabbit anti-DREAM antibody. (C) Lysates from cells expressing mGluR5 and DREAM (E186Q/E234Q) double mutant or DREAM (D150N/E186Q/E234Q) triple mutant were subjected to Western blotting.

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  Fig. 3. Subcellular localization of increased DREAM by mGluR5. (A) pCBA-DREAM and/or pRK5-myc-mGluR5 were transfected into HeLa cells. Following 36 h incubation, cells were fixed with 4% paraformaldehyde, blocked with goat serum, and incubated with rabbit anti-DREAM and anti-myc antibody. After incubation, fixed cells were washed and treated again with Alexa 488 anti-rabbit and Alexa 568 anti-mouse antibodies. Images were obtained under a Zeiss LSM 710 confocal microscope. (B) HEK293 cells were cotransfected with pRK5-mGluR5 and pCBA-DREAM. Cytoplasmic and nuclear extracts were prepared from transfected cells. Western blotting was performed to determine the amount of DREAM in each fraction.

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  Fig. 4. DHPG increases DREAM levels in primary cultured neurons. Primary cultures of rat cortical neurons were prepared from 17-day-old embryonic brains. At day 12 in vitro, neurons were treated with 200 μM DHPG for 12 h. Changes in DREAM level were measured using Western blot analysis.

Reference

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