Random mutagenesis of human estrogen receptor ligand binding domain identifies mutations that decrease sensitivity to...

Area of Science:

• Molecular biology
• Endocrinology
• Biochemistry

Background:

• The human estrogen receptor (ER) plays a critical role in various physiological processes.
• Understanding the structure-function relationship of the ER ligand-binding domain (LBD) is crucial for drug development and gene regulation.
• Existing ER modulators have limitations, necessitating the development of novel variants with altered specificities.

Purpose of the Study:

• To generate and characterize mutations in the human estrogen receptor ligand-binding domain (LBD).
• To identify specific mutations that alter the receptor's responsiveness to estradiol and synthetic compounds.
• To develop an ER mutant with reversed specificity for potential use in mammalian gene expression systems.

Main Methods:

• Low fidelity polymerase chain reaction (PCR) amplification was employed to introduce random mutations into the ER LBD.
• Mutagenic libraries were screened in yeast to identify phenotypic changes in receptor activity.
• Positional cloning and sequencing were used to identify the specific mutations responsible for altered phenotypes.

Main Results:

• Mutagenesis yielded ER variants with altered ligand binding and functional responses.
• Specific mutations were mapped to discrete regions within the LBD, crucial for ER function.
• A mutant ER was generated with reversed specificity, showing higher responsiveness to a diphenol indene-ol than to estradiol.

Conclusions:

• Targeted mutagenesis of the ER LBD can effectively alter receptor specificity.
• The identified mutations provide insights into the functional regions of the ER LBD.
• The reversed-specificity ER mutant holds promise for developing regulated gene expression systems in mammalian cells.