Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 279, Issue 32, Pages 33855-33864Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M402118200
Keywords
-
Categories
Funding
- NCI NIH HHS [CA18119] Funding Source: Medline
- NIDDK NIH HHS [DK15556] Funding Source: Medline
Ask authors/readers for more resources
Human estrogen receptor alpha (hERalpha) and human androgen receptor exhibit exquisite ligand specificity, which underlies their remarkable ability to effect ligand-regulated gene transcription in a highly distinctive and specific manner. Here we used a directed evolution approach to create hERalpha variants with enhanced androgen specificity and affinity with the goal to better understand the molecular basis of ER ligand specificity and the evolutionary mechanism of nuclear receptors. We developed a sensitive yeast two-hybrid system to screen for hERalpha variants with increased transactivation potency toward testosterone. After two rounds of directed evolution, we identified five hERalpha variants with dramatically improved transactivation potency toward testosterone in both yeast and mammalian cells. These variants showed up to 7,600-fold improvement in the binding affinity for testosterone and only slightly reduced affinity toward 17beta-estradiol. Detailed analysis of these evolved variants and a few site-directed mutants generated de novo led to several unexpected findings including the following. 1) Only two beneficial mutations were needed to create hERalpha variants with near nanomolar affinity for testosterone. 2) Some beneficial mutations were synergistic, context-dependent, or nonadditive. 3) Of the five identified beneficial mutations, four of them were not in the ER ligand binding pocket and yet exerted important action on ligand specificity. 4) The single ligand-contacting mutation E353Q plays a dominant role in discriminating androgens and estrogens. These results, viewed in conjunction with the ligand exploitation model of nuclear receptor evolution, suggest that the mutation E353Q may represent a key event in the evolution of androgen receptors from an ancestral estrogen receptor and that ligand promiscuity may play an important role in the creation of new nuclear receptors via divergent evolution.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available