Molecular mechanisms of agonism and antagonism for the Estrogen Receptor

The nuclear receptor (NR) superfamily includes receptors for the known steroid hormones, thyroid hormone, retinoids, etc.. These ligand-inducible transcription factors control many complex events during development, growth, and homeostasis. For example, the estrogen receptor a (ERa) regulates the differentiation and maintenance of neural, skeletal, cardiovascular, and reproductive tissues. The regulation of ERa transcriptional activity plays a critical role in osteoporosis, cardiovascular disease, endometrial cancer and breast cancer. Transcriptional activation is mediated by two different activation functions, one of which is controlled by ligand binding (AF-2). A family of proteins called transcriptional coactivators interact with agonist-bound receptors to mediate transcription. This interaction occurs through one or more Nuclear Receptor interaction regions, or NR boxes, which contain the conserved LXXLL sequence motif. The p160 family of coactivators contain multiple NR boxes that recognize different NRs with varying affinities. For the steroid receptors, function is also controlled by the binding of a large chaperone complex that includes Hsp-90 to the ligand binding domain (LBD). Formation of this complex is apparently required for maintaining the receptor in a ligand binding-competent state. Upon ligand binding, the chaperones dissociate, allowing the receptor to bind DNA and regulate transcription.

The ERa LBD can bind to pure agonists such as the endogenous estrogen, 17b-estradiol (E2) or the synthetic estrogen diethylstilbesterol (DES) , pure antagonists such as ICI-164,384. Other compounds such as tamoxifen and raloxifene (RAL) act as antagonists in particular tissue and promoter contexts. This agonist/antagonist behavior is clinically important for treating cancer. Recently, breast cancer prevention trials with tamoxifen showed a 45% reduction in breast cancer incidence and a decreased occurrence of bone fractures. However, a significant increase in incidence of endometrial cancer was also reported.

We are particularly interested in understanding how ligands modulate transcriptional activity and the role of molecular chaperones in this process. This is a critical step in the rational optimization of compounds for the successful treatment and prevention of breast and other cancers. Furthermore, lessons learned from studies on the ER should be applicable to the broad family of NRs. As a first step in this process, we have recently solved the structures of the ERa LBD bound to either the antagonist 4-hydroxy tamoxifen (OHT; the active tamoxifen metabolite) or to the synthetic steroid DES and a peptide from the GRIP1 coactivator NR Box 2. This work is in collaboration with Dr. Geoffrey Greene, U. Chicago. These structures have been quite informative about the coupling between ligand binding and the functional state of AF-2.

Previous work had indicated that although E2 and RAL bind at the same site within the core of the ERa LBD, each of these ligands induces a different conformation of the last helix in the LBD, helix 12. With agonist bound, helix 12 packs against helices 3,5/6 and 11; by contrast with antagonist bound the position of helix 12 is quite different - now occupying a hydrophobic groove constructed from helices 3 and 5. Mutagenesis has shown that residues in this cleft and on helix 12 form part of the AF-2 recognition surface. In our DES complex, the NR box peptide is bound in an a-helical conformation by the hydrophobic groove formed from helices 3,4,5, and 12. In the OHT complex, instead of forming part of AF-2, helix 12 binds to, and occludes, the coactivator recognition box using an LXXML motif to mimic the LXXLL from the NR box. The positioning of helix 12 is directed by effects on the secondary and tertiary structure of the LBD programmed by ligand binding. Agonists stabilize secondary structural elements, extending the lengths of helices 3,8 and 11. This then shortens the loop between helices 11 and 12, which in turn does not allow helix 12 to fit in the hydrophobic coactivator pocket. The precise geometry of the ligand and its interactions with different regions of LBD lead to a differential stabilization of either the agonist or antagonist conformations.

Relevant Publications

Shiau, A.K., Barstad, D., Radek, J.T., Meyers, M.J., Katzenellenbogen, B.S., Katzenellenbogen, J.A., Agard, D.A., and Greene, G.L. (2002). Structural Characterization of a Subtype-Selective Ligand Reveals a Novel Mode of Estrogen Receptor Antagonism. Nature Structural Biology 9(5), 359-64 (html or pdf).

Kushner, Peter J.; Agard, David; Feng, Wei-Jun; Lopez, Gabriela; Schiau, Andrew; Uht, Rosalie; Webb, Paul; Greene, Geoffrey. Oestrogen receptor function at classical and alternative response elements. Novartis Foundation Symposium (2000), 230(Neuronal and Cognitive Effects of Oestrogens), 20-32. (pdf).

Kushner, P.J., Agard D.A., Greene G.L., Scanlan T.S., Shiau, A.K., Uht R.M., Webb P. (2000) Estrogen receptor pathways to AP-1. J Steroid Biochem Mol Biol. 74(5):311-7 (pdf).

Shiau, A., Barstad,D., Loria,P.M., Cheng,L., Kushner,P.J., Agard,D.A., and Greene, G.L. (1998) The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by taxoxifen. Cell 95(7):927-37 (pdf).