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.
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