FXXLF and WXXLF Sequences Mediate the NH2-terminal Interaction with the Ligand Binding Domain of the Androgen Receptor

Abstract

The nuclear receptor superfamily members of eukaryotic transcriptional regulators contain a highly conserved activation function 2 (AF2) in the hormone binding carboxyl-terminal domain and, for some, an additional activation function 1 in the NH 2 -terminal region which is not conserved. Recent biochemical and crystallographic studies revealed the molecular basis of AF2 is hormone-dependent recruitment of LXXLL motifcontaining coactivators, including the p160 family, to a hydrophobic cleft in the ligand binding domain. Our previous studies demonstrated that AF2 in the androgen receptor (AR) binds only weakly to LXXLL motif-containing coactivators and instead mediates an androgendependent interaction with the AR NH 2 -terminal domain required for its physiological function. Here we demonstrate in a mammalian two-hybrid assay, glutathione S-transferase fusion protein binding studies, and functional assays that two predicted ␣-helical regions that are similar, but functionally distinct from the p160 coactivator interaction sequence, mediate the androgendependent, NH 2 -and carboxyl-terminal interaction. Nuclear receptors facilitate ligand-dependent increases of gene transcription by direct interactions with nuclear coactivators. p160 coactivators have histone acetyltransferase activity (1) and interact with nuclear receptors through their ligand binding and NH 2 -terminal regions (2-4). Additional acetyltransferases p300/cAMP response element binding protein and p300/cAMP response element binding protein associated factor interact with the p160 coactivators (5-7). Nuclear receptors also interact with multiprotein complexes referred to as thyroid hormone receptor-associated proteins (8), activator-recruited cofactor (9), or vitamin D receptor-interacting protein complex (10). The p160 coactivators and at least one of the thyroid hormone receptor-associated proteins/activator-recruited cofactor/vitamin D receptor-interacting protein subunit 205 interact in a ligand-dependent manner with activation function 2 (AF2) 1 in the ligand binding domain (LBD) of nuclear receptors through the consensus sequence LXXLL, where L is leucine and X is any amino acid (11-16). Crystal structures of nuclear receptor LBDs have shown that a hydrophobic cleft within a multilayered ␣-helical structure serves as the LXXLL coactivator binding surface AF2 (17, 18). In the estrogen receptor, agonist binding positions helix 12 over the binding cavity to complete the AF2 surface (19,20), whereas binding of an antagonist such as 4-hydroxytamoxifen displaces helix 12 (21) causing an LXXLL-like sequence in helix 12 to mimic and thereby block coactivator binding (22).It has become apparent that the AF2 region overlaps with regions that serve as the binding site for a variety of LXXLLrelated sequences as recently shown for corepressor binding (23)(24)(25). Furthermore, we demonstrated that the AR AF2 region mediates an androgen-dependent NH 2 -terminal/carboxylterminal (N/C) interaction (26). Therefore we investigated the possibility that AF2 in the ...

Two Panels of Steroid Receptor Luciferase Reporter Cell Lines for Compound Profiling

“…When expressing the chimeric AR-LBD fused to Gal4-DBD in U2OS cells we were almost not able to detect any androgen-induced luciferase activity from the reporter vector. Since the ligand-induced activation of AR requires the interaction between AF-1 and AF-2 [28, 29], we speculated that the co-expression of the full-length AR would be sufficient for creating a functional AR-LBD containing transactivation complex able to induce transcription from the reporter vector containing Gal4 UAS sites. This hypothesis was confirmed in transient transfections and later by preparation of the stable reporter cell line U2OS-AR-LBD/9xGal4UAS by cotransfecting pBIND-AR construct together with pcDNA3-hAR vector.…”

Cyclin D3 action in androgen receptor regulation and prostate cancer

“…Given the capacity of cyclin D3 to bind AR, functional studies were performed. To determine if cyclin D3 alters the requisitive intramolecular interactions of AR that occur after ligand binding (N-Cterminal interaction) (He et al, 2000;Li et al, 2007), a well-characterized mammalian two-hybrid assay was utilized (Lim et al, 2000;Burd et al, 2005) where luciferase activity from the Gal4 promoter provided a readout of AR N-C-terminal interaction. Introduction of cyclin D3 resulted in approximately a 75% reduction in N-C-terminal interaction (Figure 3c).…”

Specificity and heregulin regulation of Ebp1 (ErbB3 binding protein 1) mediated repression of androgen receptor signalling

“…For example, ARIP3 enhances transcription from minimal AREs, yet represses the probasin promoter (Kotaja et al, 2000). Activation of both the PSA and probasin promoters requires interactions of the N and C terminal domains of AR, but this association is not required for activation of MMTV (He et al, 2000). Thus, it was important to examine the effects of Ebp1 on different native promoters.…”