Analysis of RXR/THR and RXR/PPARG2 Heterodimerization by Bioluminescence Resonance Energy Transfer (BRET)

被引:9
作者
Mulero, Miquel [1 ,3 ]
Perroy, Julie [4 ,5 ,6 ,7 ]
Federici, Carole [1 ]
Cabello, Gerard [1 ,2 ]
Ollendorff, Vincent [1 ,2 ]
机构
[1] INRA, Dynam Musculaire & Metab UMR866, F-34060 Montpellier, France
[2] Univ Montpellier I, Montpellier, France
[3] Univ Rovira & Virgili, Nutrigen Res Grp, Dept Biochem & Biotechnol, E-43007 Tarragona, Spain
[4] CNRS, UMR 5203, Inst Genom Fonct, Montpellier, France
[5] INSERM, U661, Montpellier, France
[6] Univ Montpellier I, UMR 5203, Montpellier, France
[7] Univ Montpellier 2, UMR 5203, Montpellier, France
关键词
PROTEIN-PROTEIN INTERACTIONS; PPAR-GAMMA-RXR; PEROXISOME PROLIFERATOR; DNA-BINDING; PRIMARY TARGET; IDENTIFICATION; MOBILITY; AGONISTS; COMPLEX; GENES;
D O I
10.1371/journal.pone.0084569
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Background: Nuclear receptors (NR) regulate transcription of genes involved in many biological processes such as development, cell proliferation, differentiation and cell death. Amongst them, PPARG2 and THR control tissue glucose and lipid homeostasis which are deregulated in severe pathophysiological conditions such as metabolic syndromes. Methodology/Principal Findings: Here, we describe a real time BRET approach to monitor heterodimerization between RXR and PPARG2 or THR in vitro or in living cells. The presence of a specific DNA target was required to induce in vitro a BRET shift reflecting heterodimerization of RXR/PPARG2 or RXR/THR. As in electrophoretic mobility shift assay (EMSA), the stringency and specificity of the BRET shift assay depended upon assay condition optimization including MgCl2 concentration. For the nuclear receptors, we found by mutagenesis analysis that each heterodimer partner must harbor an intact DNA binding domain to induce BRET and heterodimerization on a DNA target. Moreover the interaction between the PPARG2 ligand binding domain and the RXR DNA binding domain stabilized the heterodimer on its DNA target. BRET microscopy in living cells highlighted the heterodimerization of RXR/PPARG2 within the nucleus clustered in discrete foci that may represent active target gene transcription regulation regions. BRET imaging also suggested that heterodimerization between RXR and PPARG2 required the DNA binding of PPARG2. Conclusions/Significance: The BRET approach described here allowed us to study the dynamic interactions which exist between NR in vitro or in living cells and can provide important information on heterodimerization modes, affinity with a given RE and subcellular localization of the heterodimers. This method could be used to study real time changes of NR heterodimers occurring on DNA depending upon cell activation, chromatin state and help to define the mechanisms of ligands or drug action designed to target NRs.
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页数:15
相关论文
共 36 条
[1]   Non-DNA binding, dominant-negative, human PPARγ mutations cause lipodystrophic insulin resistance [J].
Agostini, Maura ;
Schoenmakers, Erik ;
Mitchell, Catherine ;
Szatmari, Istvan ;
Savage, David ;
Smith, Aaron ;
Rajanayagam, Odelia ;
Semple, Robert ;
Luan, Jian'an ;
Bath, Louise ;
Zalin, Anthony ;
Labib, Mourad ;
Kumar, Sudhesh ;
Simpson, Helen ;
Blom, Dirk ;
Marais, David ;
Schwabe, John ;
Barroso, Ines ;
Trembath, Richard ;
Wareham, Nicholas ;
Nagy, Laszlo ;
Gurnell, Mark ;
O'Rahilly, Stephen ;
Chatterjee, Krishna .
CELL METABOLISM, 2006, 4 (04) :303-311
[2]   PPAR ligands: Potential therapies for metabolic syndrome [J].
Akiyama T.E. ;
Meinke P.T. ;
Berger J.P. .
Current Diabetes Reports, 2005, 5 (1) :45-52
[3]   Selective intranuclear redistribution of PPAR isoforms by RXRα [J].
Akiyama, TE ;
Baumann, CT ;
Sakai, S ;
Hager, GL ;
Gonzalez, FJ .
MOLECULAR ENDOCRINOLOGY, 2002, 16 (04) :707-721
[4]  
Aparicio Oscar, 2004, Curr Protoc Cell Biol, VChapter 17, DOI 10.1002/0471143030.cb1707s23
[5]   Manipulation of reciprocal salt bridges at the heterodimerization interface alters the dimerization properties of mouse RXRα and PPARγ1 [J].
Chan, Lap Shu ;
Wells, Richard A. .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2007, 358 (04) :1080-1085
[6]   Structure of the intact PPAR-γ-RXR-α nuclear receptor complex on DNA [J].
Chandra, Vikas ;
Huang, Pengxiang ;
Hamuro, Yoshitomo ;
Raghuram, Srilatha ;
Wang, Yongjun ;
Burris, Thomas P. ;
Rastinejad, Fraydoon .
NATURE, 2008, 456 (7220) :350-U33
[7]   THYROID-HORMONE (T-3) INHIBITS CIPROFIBRATE-INDUCED TRANSCRIPTION OF GENES ENCODING BETA-OXIDATION ENZYMES - CROSS-TALK BETWEEN PEROXISOME PROLIFERATOR AND T-3 SIGNALING PATHWAYS [J].
CHU, RY ;
MADISON, LD ;
LIN, YL ;
KOPP, P ;
RAO, MS ;
JAMESON, JL ;
REDDY, JK .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1995, 92 (25) :11593-11597
[8]   Subcellular imaging of dynamic protein interactions by bioluminescence resonance energy transfer [J].
Coulon, Vincent ;
Audet, Martin ;
Homburger, Vincent ;
Bockaert, Joel ;
Fagni, Laurent ;
Bouvier, Michel ;
Perroy, Julie .
BIOPHYSICAL JOURNAL, 2008, 94 (03) :1001-1009
[9]   The insulin-like growth factor-binding protein 1 gene is a primary target of peroxisome proliferator-activated receptors [J].
Degenhardt, Tatjana ;
Matilainen, Merja ;
Herzig, Karl-Heinz ;
Dunlop, Thomas W. ;
Carlberg, Carsten .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2006, 281 (51) :39607-39619
[10]   PPARγ agonists partially restores hyperglycemia induced aggravation of vascular dysfunction to angiotensin II in thoracic aorta isolated from rats with insulin resistance [J].
Gaikwad, Anil Bhanudas ;
Viswanad, Bhoomi ;
Ramarao, Poduri .
PHARMACOLOGICAL RESEARCH, 2007, 55 (05) :400-407