Mechanistic analysis of estrogen receptor action using dominant negative mutants

Zhuang, Yao

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https://hdl.handle.net/2142/19086 Copy

Description

Title

Mechanistic analysis of estrogen receptor action using dominant negative mutants

Author(s)

Zhuang, Yao

Issue Date

1995

Doctoral Committee Chair(s)

Shapiro, David J.

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Biology, Molecular
• Biology, Cell
• Chemistry, Biochemistry

Language

eng

Abstract

We have generated and characterized dominant negative mutants of human estrogen receptor, which are mutant forms of ER that are capable of suppressing the activity of wild-type receptor when they are co-expressed in the same cells. These mutants can be divided into two different categories: (1) The non-hormone binding mutants, NHB1 and NHB7, in which the hormone binding region has been mutated making them unable to bind estrogen; (2) the transactivation mutants S554Fs, L540Q and ER1-530, in which amino acids directly involved in ligand-dependent transcriptional activation have been mutated or deleted. Although they are still able to bind estrogen, these ER mutants are no longer able to activate gene transcription. While the non-hormone binding mutants NHB1 and NHB7 are less effective than the transactivation domain mutants in inhibiting the ability of wild-type ER to activate transcription, studies of these mutants have led us to a better understanding of the role of hormone binding in estrogen receptor action. These mutants are able to dimerize and to bind to estrogen response element DNA both in vitro and in vivo, indicating that hormone binding is not a prerequisite for receptor dimerization or DNA binding. We propose that the primary function of the hormone is to convert the receptor to a state in which it is able to activate gene transcription. The three transactivation mutants, S554Fs, L540Q and ER1-530, are the most powerful dominant negative mutants that we have generated. Characterization of these mutants revealed that dimerization with the wild-type ER may play an important role in their ability to act as dominant negative mutants. We demonstrated that all three mutants are able to form heterodimers with the wild-type ER when they are co-expressed in COS cells. Consequently, these heterodimers may be transcriptionally inactive. When the powerful dominant negative mutant, S554Fs, is coexpressed with the wild-type ER, it is able to inhibit the binding of wild-type ER to the ERE in gel mobility shift assays. Introducing into these dominant negative mutants a mutation which reduces the ability of the ER to dimerize, reduces their ability to block the activity of the wild-type ER. Taken together these facts indicate that heterodimerization between the mutants and wild-type ER plays a major role in their dominant negative inhibition of wild-type ER. Studies on the dimerization of the human estrogen receptor showed that in CHO cells, the dimerization deficient mutant is still able to bind to DNA and dimerize in vivo, and consequently is able to activate gene transcription. In contrast, this mutant has little ability to activate transcription in the human breast cancer cell line, 231 cells. This suggests that one or more cell-specific proteins may play a role in ER dimerization. Furthermore, it indicates that under some circumstances an amino acid thought to be critical for dimerization can be mutated without abolishing the ability of the ER to dimerize. This suggests that regions of the ER other than the sequence in the ligand binding domain initially identified as important in dimerization may be important in intracellular dimerization of the hER.

Type of Resource

text

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http://hdl.handle.net/2142/19086

Copyright and License Information

Copyright 1995 Zhuang, Yao

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