The N-terminal Regions of Estrogen Receptor α and β Are Unstructured in Vitro and Show Different TBP Binding Properties*

The N-terminal regions of the estrogen receptor α (ERα-N) and β (ERβ-N) were expressed and purified to homogeneity. Using NMR and circular dichroism spectroscopy, we conclude that both ERα-N and ERβ-N are unstructured in solution. The TATA box-binding protein (TBP) has been shown previously to interact with ERα-N in vitro and to potentiate ER-activated transcription. We used surface plasmon resonance and circular dichroism spectroscopy to confirm and further characterize the ER-N-TBP interaction. Our results show that the intrinsically unstructured ERα-N interacts with TBP, and suggest that structural changes are induced in ERα-N upon TBP interaction. Conformational changes upon target factor interaction have not previously been demonstrated for any N-terminal region of nuclear receptors. In addition, no binding of ERβ-N to TBP was detected. This difference in TBP binding could imply differential recruitment of target proteins by ERα-N and ERβ-N. The affinity of the ERα-N-TBP interaction was determined to be in the micromolar range (K D = 10−6 to 10−5 m). Our results support models of TBP as a target protein for the N-terminal activation domain of ERα. Further, our results suggest that target proteins can induce and/or stabilize ordered structure in N-terminal regions of nuclear receptors upon interaction.

[1]  K. Dahlman-Wright,et al.  Structural characterization of a minimal functional transactivation domain from the human glucocorticoid receptor. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Y. Sadovsky,et al.  Transcriptional activators differ in their responses to overexpression of TATA-box-binding protein , 1995, Molecular and cellular biology.

[3]  J. Gustafsson,et al.  Differential Recruitment of the Mammalian Mediator Subunit TRAP220 by Estrogen Receptors ERα and ERβ* , 2001, The Journal of Biological Chemistry.

[4]  D. Edwards,et al.  The Steroid Receptor Coactivator-1 Contains Multiple Receptor Interacting and Activation Domains That Cooperatively Enhance the Activation Function 1 (AF1) and AF2 Domains of Steroid Receptors* , 1998, The Journal of Biological Chemistry.

[5]  K. Berndt,et al.  How Transcriptional Activators Bind Target Proteins* , 2001, The Journal of Biological Chemistry.

[6]  K. Umesono,et al.  The nuclear receptor superfamily: The second decade , 1995, Cell.

[7]  R. Cole,et al.  Alternative O-glycosylation/O-phosphorylation of the murine estrogen receptor beta. , 2000, Biochemistry.

[8]  B. O’Malley,et al.  Assessment of structural similarities in chick oviduct progesterone receptor subunits by partial proteolysis of photoaffinity-labeled proteins. , 1983, The Journal of biological chemistry.

[9]  J. Gustafsson,et al.  Cloning of a novel receptor expressed in rat prostate and ovary. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[10]  A. Levine,et al.  Induced α Helix in the VP16 Activation Domain upon Binding to a Human TAF , 1997 .

[11]  P. Chambon,et al.  Functional domains of the human estrogen receptor , 1987, Cell.

[12]  C. Ingles,et al.  Direct interaction between the transcriptional activation domain of human p53 and the TATA box-binding protein. , 1993, The Journal of biological chemistry.

[13]  K. Horwitz,et al.  The N-terminal Region of the Human Progesterone A-receptor , 2000, The Journal of Biological Chemistry.

[14]  C. Ingles,et al.  Direct and selective binding of an acidic transcriptional activation domain to the TATA-box factor TFIID , 1990, Nature.

[15]  J. Shine,et al.  Sequence and expression of human estrogen receptor complementary DNA. , 1986, Science.

[16]  Simak Ali,et al.  Human Estrogen Receptor β Binds DNA in a Manner Similar to and Dimerizes with Estrogen Receptor α* , 1997, The Journal of Biological Chemistry.

[17]  P. O’Hare,et al.  Structural studies of the acidic transactivation domain of the Vmw65 protein of herpes simplex virus using 1H NMR. , 1992, Biochemistry.

[18]  N. Webster,et al.  The human estrogen receptor has two independent nonacidic transcriptional activation functions , 1989, Cell.

[19]  M. Czisch,et al.  Structural and functional analysis of the NF-kappa B p65 C terminus. An acidic and modular transactivation domain with the potential to adopt an alpha-helical conformation. , 1994, The Journal of biological chemistry.

[20]  M. Schmitz,et al.  Basal transcription factors TBP and TFIIB and the viral coactivator E1A 13S bind with distinct affinities and kinetics to the transactivation domain of NF-kappaB p65. , 1997, Nucleic acids research.

[21]  P. Chambon,et al.  Role of the two activating domains of the oestrogen receptor in the cell‐type and promoter‐context dependent agonistic activity of the anti‐oestrogen 4‐hydroxytamoxifen. , 1990, The EMBO journal.

[22]  K. Dahlman-Wright,et al.  Functional interaction of the c-Myc transactivation domain with the TATA binding protein: evidence for an induced fit model of transactivation domain folding. , 1996, Biochemistry.

[23]  J. Knutson,et al.  Transcriptional Activation Domain of the Herpesvirus Protein VP16 Becomes Conformationally Constrained upon Interaction with Basal Transcription Factors (*) , 1996, The Journal of Biological Chemistry.

[24]  P. Chambon,et al.  Stimulation of RARα Activation Function AF-1 through Binding to the General Transcription Factor TFIIH and Phosphorylation by CDK7 , 1997, Cell.

[25]  T. Kodadek,et al.  The acidic activation domains of the GCN4 and GAL4 proteins are not α helical but form β sheets , 1993, Cell.

[26]  J. Capone,et al.  Purification and characterization of the carboxyl-terminal transactivation domain of Vmw65 from herpes simplex virus type 1. , 1992, The Journal of biological chemistry.

[27]  R. Dickerson,et al.  How proteins recognize the TATA box. , 1996, Journal of molecular biology.

[28]  P. Chambon,et al.  Characterization of the Amino-terminal Transcriptional Activation Function of the Human Estrogen Receptor in Animal and Yeast Cells (*) , 1995, The Journal of Biological Chemistry.

[29]  V. Giguère,et al.  Ligand-independent recruitment of SRC-1 to estrogen receptor beta through phosphorylation of activation function AF-1. , 1999, Molecular cell.

[30]  B. Komm,et al.  A novel human estrogen receptor β: identification and functional analysis of additional N-terminal amino acids , 1998, The Journal of Steroid Biochemistry and Molecular Biology.

[31]  A. Levine,et al.  Structure of the MDM2 Oncoprotein Bound to the p53 Tumor Suppressor Transactivation Domain , 1996, Science.

[32]  Gene regulation by steroid hormones , 1989, Cell.

[33]  R. Evans,et al.  Multiple and cooperative trans-activation domains of the human glucocorticoid receptor , 1988, Cell.

[34]  Miguel Beato,et al.  Steroid hormone receptors: Many Actors in search of a plot , 1995, Cell.

[35]  H. Gronemeyer,et al.  Activation Function 2 in the Human Androgen Receptor Ligand Binding Domain Mediates Interdomain Communication with the NH2-terminal Domain* , 1999, The Journal of Biological Chemistry.

[36]  F. Claessens,et al.  The Androgen Receptor Amino-Terminal Domain Plays a Key Role in p160 Coactivator-Stimulated Gene Transcription , 1999, Molecular and Cellular Biology.

[37]  J. Lee,et al.  Interdomain Signaling in a Two-domain Fragment of the Human Glucocorticoid Receptor* , 1999, The Journal of Biological Chemistry.

[38]  Peter E Wright,et al.  Solution Structure of the KIX Domain of CBP Bound to the Transactivation Domain of CREB: A Model for Activator:Coactivator Interactions , 1997, Cell.

[39]  J. Gustafsson,et al.  Functional Differences between the Amino-Terminal Domains of Estrogen Receptors α and β , 2000 .

[40]  S. Cowley,et al.  Estrogen Receptors α and β Form Heterodimers on DNA* , 1997, The Journal of Biological Chemistry.