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Activation Function 1 of Glucocorticoid Receptor Binds TATA-Binding Protein in Vitro and in Vivo
- Source :
- Molecular Endocrinology. 20:1218-1230
- Publication Year :
- 2006
- Publisher :
- The Endocrine Society, 2006.
-
Abstract
- The mechanism through which the glucocorticoid receptor (GR) stimulates transcription is still unclear, although it is clear that the GR affects assembly of the transcriptional machinery. The binding of the TATA-binding protein (TBP) to the TATA-box is accepted as essential in this process. It is known that the GR can interact in vitro with TBP, but the direct interaction of TBP with GR has not been previously characterized quantitatively and has not been appreciated as an important step in assembling the transcriptional complex. Herein, we demonstrate that the TBP-GR interaction is functionally significant by characterizing the association of TBP and GR in vitro by a combination of techniques and confirming the role of this interaction in vivo. Combined analysis, using native gel electrophoresis, sedimentation equilibrium, and isothermal microcalorimetry titrations, characterize the stoichiometry, affinity, and thermodynamics of the TBP-GR interaction. TBP binds recombinant GR activation function 1 (AF1) with a 1:2 stoichiometry and a dissociation constant in the nanomolar range. In vivo fluorescence resonance energy transfer experiments, using fluorescently labeled TBP and various GR constructs, transiently transfected into CV-1 cells, show GR-TBP interactions, dependent on AF1. AF1-deletion variants showed fluorescence resonance energy transfer efficiencies on the level of coexpressed cyan fluorescent protein and yellow fluorescent protein, indicating that the interaction is dependent on AF1 domain. To demonstrate the functional role of the in vivo GR-TBP interaction, increased amounts of TBP expressed in vivo stimulated expression of GR-driven reporters and endogenous genes, and the effect was also specifically dependent on AF1.
- Subjects :
- Yellow fluorescent protein
Transcription, Genetic
genetic processes
macromolecular substances
Plasma protein binding
In Vitro Techniques
Biology
Transfection
Models, Biological
Cell Line
Receptors, Glucocorticoid
Endocrinology
Glucocorticoid receptor
Transcription (biology)
In vivo
Animals
Humans
Promoter Regions, Genetic
Molecular Biology
TATA-Box Binding Protein
General Medicine
Recombinant Proteins
Protein Structure, Tertiary
Kinetics
Förster resonance energy transfer
Gene Expression Regulation
Biochemistry
Multiprotein Complexes
health occupations
Biophysics
biology.protein
Thermodynamics
TATA-binding protein
Protein Binding
Subjects
Details
- ISSN :
- 19449917 and 08888809
- Volume :
- 20
- Database :
- OpenAIRE
- Journal :
- Molecular Endocrinology
- Accession number :
- edsair.doi.dedup.....bac78544d59ac76ee8a231440935d260