Dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX1) (NR0B1) and small heterodimer partner (SHP) (NR0B2) form homodimers individually, as well as DAX1-SHP heterodimers

Dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX1) (NR0B1), and small heterodimer partner (SHP) (NR0B2) are atypical nuclear receptor superfamily members that function primarily as corepressors through heterodimeric interactions with other nuclear receptors. Mutations in DAX1 cause adrenal hypoplasia congenita, and mutations in SHP lead to mild obesity and insulin resistance, but the mechanisms are unclear. We investigated the existence and subcellular localization of DAX1 and SHP homodimers and the dynamics of homodimerization. We demonstrated DAX1 homodimerization in the nucleus and cytoplasm, and dissociation of DAX1 homodimers upon heterodimerization with steroidogenic factor 1 (SF1) or ligand-activated estrogen receptor-alpha (ERalpha). DAX1 homodimerization involved an interaction between its amino and carboxy termini involving its LXXLL motifs and activation function (AF)-2 domain. We observed SHP homodimerization in the nucleus of mammalian cells and showed dissociation of SHP homodimers upon heterodimerization with ligand-activated ERalpha. We observed DAX1-SHP heterodimerization in the nucleus of mammalian cells and demonstrated the involvement of the LXXLL motifs and AF-2 domain of DAX1 in this interaction. We further demonstrate heterodimerization of DAX1 with its alternatively spliced isoform, DAX1A. This is the first evidence of homodimerization of individual members of the unusual NR0B nuclear receptor family and heterodimerization between its members. Our results suggest that DAX1 forms antiparallel homodimers through the LXXLL motifs and AF-2 domain. These homodimers may function as holding reservoirs in the absence of heterodimeric partners. The formation of DAX1 and SHP homodimers and DAX1-SHP and DAX1-DAX1A heterodimers suggests the possibility of novel functions independent of their coregulator roles, suggesting additional complexity in the molecular mechanisms of DAX1 and SHP action.     

Activation function-1 domain of estrogen receptor regulates the agonistic and antagonistic actions of tamoxifen

The antiestrogen tamoxifen has been widely used for decades as selective estrogen receptor (ER) modulator for ERalpha-positive breast tumors. Tamoxifen significantly reduces tumor recurrence by binding to the activation function-2 (AF-2) domain of the ER. Acquired resistance to tamoxifen in breast cancer patients is a serious therapeutic problem. Antiestrogen-resistant breast cancer often shows increased expression of the epidermal growth factor receptor (EGFR) family members, EGFR and ErbB2. In this report we now show that overexpression of EGFR or activated AKT-2 in MCF-7 cells leads to phosphorylation of Ser167 in the AF-1 domain of ERalpha, enhanced ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of tamoxifen, and resistance to tamoxifen. In contrast, transfection of activated MAPK kinase, an immediate upstream activator of MAPK (ERK 1 and 2) into MCF-7 cells leads to phosphorylation of Ser118 in the AF-1 domain of ERalpha, inhibition of ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of Tam, and maintenance of sensitivity to tamoxifen. Inhibition of AKT by short inhibitory RNA blocked Ser167 phosphorylation in ER and restored tamoxifen sensitivity. However, maximum sensitivity to tamoxifen was observed when both AKT and MAPK were inhibited. Taken together, these data demonstrate that different phosphorylation sites in the AF-1 domain of ERalpha regulate the agonistic and antagonistic actions of tamoxifen in human breast cancer cells.