The role of hinge domain in heterodimerization and specific DNA recognition by nuclear receptors.
Four structural domains are characteristic of the members of the nuclear receptor superfamily. The hinge (D) domain which is located between the DNA binding (C) domain and the ligand binding (EF) domain, is less conserved among the nuclear receptors. In this study, we investigated the effects of the D domain on receptor function with regard to ligand binding, protein-protein interaction and DNA recognition. We found that EF domain of TR lacked T3 binding activity and additional D domain was required for its ligand binding. Using pull down assays and two-hybrid assays, we also demonstrated that the EF domain of TR did not dimerize with TR or RXR in solution, while the DEF domain was able to homo-and heterodimerize with RXR. In contrast, the RXR EF domain alone was able to heterodimerize with TR. The D domain of TR is required but that of RXR is not necessary for the interaction. We further demonstrated that the D domain was required for receptor specific DNA recognition. The ABC domain of vitamin D receptor (VDR) and TR(DEF) chimeric receptor could not bind to VDR response element (VDRE). Addition of own D domain of VDR to the ABC domain enables the chimeric receptor to bind VDRE and transactivate. The D domain of TR cannot substitute for that of VDR in context of specific DNA recognition. These data suggest that the D domain is important to maintain the integrity of the functional structure of the nuclear receptors.
Miyamoto
T
T
Department of Aging Medicine and Geriatrics, Shinshu University School of Medicine, 3-1-1 Asahi, 390-8621, Matsumoto, Japan. mitamoto@hsp.md.shinshu-u.ac.jp
Kakizawa
T
T
Ichikawa
K
K
Nishio
S
S
Takeda
T
T
Suzuki
S
S
Kaneko
A
A
Kumagai
M
M
Mori
J
J
Yamashita
K
K
Sakuma
T
T
Hashizume
K
K
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D008024:Ligands; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018167:Receptors, Calcitriol / Q000737:chemistry / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000737:chemistry / Q000378:metabolism; D011988:Receptors, Thyroid Hormone / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D047488:Retinoid X Receptors; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014284:Triiodothyronine / Q000378:metabolism
D008024:Ligands; D018167:Receptors, Calcitriol; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D011988:Receptors, Thyroid Hormone; D011993:Recombinant Fusion Proteins; D047488:Retinoid X Receptors; D014157:Transcription Factors; D014284:Triiodothyronine; D004247:DNA