Understanding the human estrogen receptor-alpha using targeted mutagenesis.
The estrogen receptor-alpha is a wonderfully complex protein important in normal biology, breast cancer, and as a target for anti-cancer agents. We are using the available structures of the hERalpha as well as secondary structure predictions to guide site-directed mutagenesis in order to test the importance of specific interactions and regions in the ligand-regulated activity of the protein. In one area of interest, we are investigating the role of the F domain in the ligand-stimulated activity of the hERalpha. Results from our laboratory and others suggest that the F domain modulates the activity of the hERalpha. In order to better understand the role of the F domain in the hERalpha, we have constructed mutants within this region. Mutations within a predicted alpha-helical region alter the response of the ER to estradiol (E2), eliminate or impair the agonist activity of 4-hydroxytamoxifen (4-OHT), and alter the ability of E2 to overcome 4-OHT's antagonist activity. Deleting the F domain increases the affinity of the receptor for E2; by contrast, mutating a residue in the middle of the predicted helix to a proline does not alter the affinity for E2, but does change the binding mechanism from a positive cooperative to a noncooperative interaction. These and other results show the F domain exhibits substantial functional complexity, and support the idea that this domain modulates the activity of the hERalpha. In a second area of interest, we are investigating the role of hydrophobic and hydrogen-bonding interactions at the start of helix 12 in the activity of the hERalpha. Leucine-536 (L536) has been proposed to participate in hydrophobic interactions that form part of a capping motif stabilizing the start of helix 12. When mutated, the resulting receptors exhibit a reduced response, or even an inverted response, to E2 and 4-OHT on both ERE-driven and AP-1-driven promoters. Interestingly, these mutated receptors also exhibit altered interactions with probes that recognize the agonist-bound and 4-OHT-bound conformations of the ERalpha. Thus, L536 couples the binding of ligand with the conformation of the receptor. Overall, these results show that combining structure-based hypotheses with functional tests of the ER's activity can identify regions and interactions that are important in the ligand-stimulated activity of the protein.
Skafar
Debra F
DF
Department of Physiology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201, USA. dskafar@med.wayne.edu
Koide
Shohei
S
D000595:Amino Acid Sequence; D047628:Estrogen Receptor alpha / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
D047628:Estrogen Receptor alpha