Regulation of DREAM Expression by Group I mGluR

Jinu Lee; Insook Kim; So Ra Oh; Suk Jin Ko; Mi Kyung Lim; Dong Goo Kim; Chul Hoon Kim

doi:10.4196/kjpp.2011.15.2.95

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Lee, Kim, Oh, Ko, Lim, Kim, and Kim: Regulation of DREAM Expression by Group I mGluR

Original Article

Korean J Physiol Pharmacol 2011;15(2):95-100.

Published online: 18 February 2011

DOI: https://doi.org/10.4196/kjpp.2011.15.2.95

Regulation of DREAM Expression by Group I mGluR

Jinu Lee¹, Insook Kim³, So Ra Oh¹, Suk Jin Ko^1,², Mi Kyung Lim^1,², Dong Goo Kim^1,², Chul Hoon Kim^1,^2,^4,

¹Department of Pharmacology, Yonsei University College of Medicine, Seoul 120-752, Korea

²Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Korea

³Division of Metabolic Disease, Department of Biomedical Science, National Institutes of Health, Osong 363-951, Korea

⁴Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 120-752, Korea

Corresponding to: Chul Hoon Kim, Department of Pharmacology, Yonsei University College of Medicine, 134, Shinchon-dong, Seodaemun-gu, Seoul 120-752, Korea. (Tel) 82-2-2228-1783, (Fax) 82-2-313-1894, (E-mail) kimhoon@yuhs.ac

Received 23 February 2011 Revised 18 March 2011 Accepted 23 March 2011

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Keywords: Calcium, DREAM, Group I mGluR, mGluR5, mGluR1, Pain

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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 (G_αq 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.

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.

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.

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.

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