The long term goal of this study is to understand the role of estrogen in regulating activation function 2 (AF-2) of the estrogen receptor (ER). AF- 2 is located in the ligand binding domain of the receptor and is engaged by hormone (but not by antihormone) binding. It is the major activation function that allows ER to stimulate transcription from its estrogen response element (ERE) in the promoter region of target gene. Our pilot studies have shown that the ER AF-2 domain binds three basal transcription factors, and overexpression of one of these (the TATA box binding protein) potentiates AF-2 action in transfected cells. Our pilot studies have also revealed that a putative co-activator, cloned RIP140, binds ER only in the presence of estrogen and not antiestrogen. We plan to employ biochemical binding, directed mutation and transfection studies to analyze the contribution of these interactions with target proteins to AF-2.
The aims are: 1) To map the sites of interaction between the ER AF-2 domain and the three basal transcription factors to which ER binds - TATA box binding protein (TBP), TFIIB, and the 30kD subunit of TFIIF. The mapping information will then be used to construct mutations in the ER that specifically disrupt the binding to one or the other target protein. 2) To test the ability of the ER mutants and also of isolated subdomains of the ER ligand binding domain to activate transcription of transfected ERE reporter genes. This will test us whether the binding interaction disrupted by the mutation contributes to AF-2 function. 3) To test the hypothesis that estrogen binding to ER removes an inhibitory function that in the absence of hormone blocks binding of RIP140, an important co- activator, and hence AF-2 function. 4) To test whether competition for RIP140 or related proteins underlies the ability of steroid/thyroid receptors to squelch each other. That such competition for target proteins underlies squelching is an old hypothesis. It can now be tested.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK051083-03
Application #
2749594
Study Section
Biochemical Endocrinology Study Section (BCE)
Program Officer
Margolis, Ronald N
Project Start
1996-08-01
Project End
2000-07-31
Budget Start
1998-08-11
Budget End
1999-07-31
Support Year
3
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Webb, Paul; Nguyen, Phuong; Kushner, Peter J (2003) Differential SERM effects on corepressor binding dictate ERalpha activity in vivo. J Biol Chem 278:6912-20
Lopez, G N; Turck, C W; Schaufele, F et al. (2001) Growth factors signal to steroid receptors through mitogen-activated protein kinase regulation of p160 coactivator activity. J Biol Chem 276:22177-82
Feng, W; Webb, P; Nguyen, P et al. (2001) Potentiation of estrogen receptor activation function 1 (AF-1) by Src/JNK through a serine 118-independent pathway. Mol Endocrinol 15:32-45
Webb, P; Nguyen, P; Valentine, C et al. (2000) An antiestrogen-responsive estrogen receptor-alpha mutant (D351Y) shows weak AF-2 activity in the presence of tamoxifen. J Biol Chem 275:37552-8
Pissios, P; Tzameli, I; Kushner, P et al. (2000) Dynamic stabilization of nuclear receptor ligand binding domains by hormone or corepressor binding. Mol Cell 6:245-53
Lopez, G N; Webb, P; Shinsako, J H et al. (1999) Titration by estrogen receptor activation function-2 of targets that are downstream from coactivators. Mol Endocrinol 13:897-909
Webb, P; Nguyen, P; Valentine, C et al. (1999) The estrogen receptor enhances AP-1 activity by two distinct mechanisms with different requirements for receptor transactivation functions. Mol Endocrinol 13:1672-85
Ma, H; Hong, H; Huang, S M et al. (1999) Multiple signal input and output domains of the 160-kilodalton nuclear receptor coactivator proteins. Mol Cell Biol 19:6164-73
An, J; Ribeiro, R C; Webb, P et al. (1999) Estradiol repression of tumor necrosis factor-alpha transcription requires estrogen receptor activation function-2 and is enhanced by coactivators. Proc Natl Acad Sci U S A 96:15161-6
Ding, X F; Anderson, C M; Ma, H et al. (1998) Nuclear receptor-binding sites of coactivators glucocorticoid receptor interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC-1): multiple motifs with different binding specificities. Mol Endocrinol 12:302-13

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