Transactivation Function-1-Mediated Partial Agonist Activity of Selective Estrogen Receptor Modulator Requires Homo-Dimerization of the Estrogen Receptor α Ligand Binding Domain

Abstract

The isolation of estrogen receptor alpha (ERα) cDNA was successful around 30 years ago. The characteristics of ERα protein have been examined from various aspects, primarily through in vitro cell culture studies, but more recently using in vivo experimental models. There remains, however, some uncharacterized ERα functionalities. In particular, the mechanism of partial agonist activity of selective estrogen receptor modulators (SERMs) that involves control of the N-terminal transcription function of ERα, termed AF-1, is still an unsolved ERα functionality. We review the possible mechanism of SERM-dependent regulation of ERα AF-1-mediated transcriptional activity, which includes the role of helix 12 of ERα ligand binding domain (LBD) for SERM-dependent AF-1 regulation. In addition, we describe a specific portion of the LBD that associates with blocking AF-1 activity with an additional role of the F-domain in mediating SERM activity.

Keywords: AF-1; F-domain; estrogen receptor alpha; homo-dimerization; ligand binding domain; selective estrogen receptor modulator.

Figure 1

Schematic structure of mouse estrogen receptor α (mERα). ERα consists of six domains named A to F. C-domain binds to an estrogen responsive DNA element. The ligands bind to E-domain (ligand binding domain; LBD). A/B-domain possesses the transactivation function 1 (AF1). E-domain possesses ligand-dependent transcription activation domain (i.e., AF-2). AF-2 is composed of static region (AF2-S) and flexible region (AF2-F). AF2-F corresponds to the helix 12 of LBD. The schematic structures of AF2-F mutant (AF2ER) and AF2-S mutant (mERα-I362D) are shown. Red bars indicate the position of mutated amino acids in the AF-2 mutants.

Figure 2

Crystallographic structure of E2-bound LBD dimer of ERα. The diagram was generated from the human ERα LBD with 17β-E2 (Protein Data Bank ID: 1ERE). The dimer form of pink and blue ERα LBD molecules is shown. The residues suggest the position corresponding to the I362, K366, L543 and E546 of mouse ERα. I362 and L543 are hydrophobic amino acids (yellow), K366 is a basic amino acid (blue) E546 is an acidic amino acid (red).

Figure 3

Schematic diagram of a hybrid reporter assay. The activity of reporter gene, which contains a Gal4-binding element (Gal4RE) juxtaposed to an ER binding element (ERE), coexpressed with ERα fragments was analyzed. In experiment, the activities were normalized by the activity of ERαCD and Gal4DBD expressing cells (Control; white). The cells that were coexpressed with ERαCD and Gal4DBD-ERαEF demonstrate AF-2 activity (pink). The cells that were coexpressed with ERαΑΒCD and Gal4DBD demonstrate AF-1 activity (blue). The cells that were coexpressed with ERαΑΒCD and Gal4DBD-ERαEF demonstrate AF-1 and AF-2 cooperative activity (green).

Figure 4

The amino acid sequence of mouse and human ERα F domains. The stars indicate identical amino acids between two species. The region of predicted β-strand is marked with red square. Mouse specific residues around the predicted β-strand are denoted as bold letters.