Dual Function of an Amino-terminal Amphipatic Helix in Androgen Receptor-mediated Transactivation through Specific and Nonspecific Response Elements

Abstract

Steroid receptors are transcription factors that, upon binding to their response elements, regulate the expression of several target genes via direct protein interactions with transcriptional coactivators. For the androgen receptor, additional interactions between the amino-and carboxyl-terminal regions have been reported. The first amino acids of the amino-terminal domain are necessary for this amino/carboxyl-terminal interaction. Deletion of a FQNLF core sequence in this region blunts the interaction, as does a G21E mutation. We investigated the effect of the aforementioned mutations in the context of the full size androgen receptor on a series of selective and nonselective androgen response elements. Strikingly, the FQNLF deletion strongly reduced the androgen receptor capacity to transactivate through nonselective motifs but did not affect its activity on selective elements. Although the G21E mutation strongly impairs the amino/carboxyl-terminal interaction, it does not significantly influence androgen receptor activity on either selective or nonselective elements. Surprisingly, this mutation leads to an increased binding of the amino-terminal domain to the glutamine-rich region of the steroid receptor coactivator-1 of the p160 family. Taken together, these data suggest that the amino-terminal amino acids of the androgen receptor play a key role in determining its transcriptional activity by modulating the interaction with the ligand-binding domain as well as interaction with p160 coactivators.Androgens are involved in the differentiation of the male reproductive organs in the embryo and the development and maintenance of secondary sexual characteristics. The biological actions of androgens are mediated by the androgen receptor (AR).1 The AR is a ligand-activated transcription factor and belongs to the class I subgroup of the nuclear receptor superfamily. Two structural domains are well conserved among the nuclear receptors (NR): the DNA-binding domain (DBD), consisting of two zinc finger modules, and the ligand-binding domain (LBD), containing a transcription activation function (AF-2) (1, 2). The amino-terminal domain (NTD) and the hinge region are more divergent. Because of their highly conserved DBD, it is not surprising that the class I steroid receptors (AR, GR, progesterone receptor, and mineralocorticoid receptor) recognize the same palindromic (inverted) repeats of the 5Ј-TGT-TCT-3Ј core sequence, spaced by three nucleotides. The mechanisms that lead to the in vivo steroid specificity of gene regulation are still not completely understood (3-6). Selective DNA binding by the AR could be one possible mechanism for hormone-specific gene regulation. It has been reported that the AR-DBD does not only recognize response elements organized as inverted repeats of 5Ј-TGTTCT-3Ј sequences with a threenucleotide spacer but can also bind to direct repeats of 5Ј-TGTTCT-3Ј-like sequences, which is proposed to contribute to the AR specificity of transcriptional responses (7-14). The NTD of the AR is indispensable f...

Regulation of Androgen Receptor-dependent Transcription by Coactivator MED1 Is Mediated through a Newly Discovered Noncanonical Binding Motif

“…The pGEX-2TK-RBD, -RBD-LXXAA, -RBD1, and -RBD2 bacterial expression vectors were described previously (24). pSG5-FLAG-AR-wt, -⌬NTD, -⌬Tau5, -⌬Tau1, -⌬cTau1, -⌬F, and -G21E were described previously (16,19,37). Transfer vectors baculovirusexpressing for HA-MED1-wt, -C⌬454, -C⌬690, -C⌬918, -C⌬1215, -ERK mutant (T1032A and T1457A), and full-length FLAG-AR were generated by subcloning the corresponding epitope-tagged open reading frames into plasmids pAcSG2 or pVL1392 (BD Biosciences).…”

“…27 motif or mutation of glycine 21 to glutamic acid (G21E) has been shown to blunt the N/C interaction (37). To directly address whether the N/C interaction influences AR's interaction with MED1, we tested MED1 for interaction with AR containing either of these mutations.…”

An androgen receptor mutation in the MDA-MB-453 cell line model of molecular apocrine breast cancer compromises receptor activity

“…Acquisition of this hydrogen bond is likely to influence folding of the adjacent helix 12 over the ligand binding pocket in response to ligand binding, and as such the formation of the essential activation function-2 (AF-2). AF-2 is a three-dimensional ligand-induced LBD structure considered necessary for AR agonist activity; it acts as an interaction surface for specific LXXLL-like sequences in receptor coregulators and the structurally similar FQNLF peptide of the AR amino-terminal transactivation domain (NTD) in the AR N/C interaction (Ikonen et al 1997, He et al 2000, Callewaert et al 2003. Indeed, mammalian twohybrid assays were able to demonstrate that the DHTinduced N/C interaction for AR-Q865H was weaker than that observed for the wtAR, with a significant reduction observed at 10 and 100 nM DHT (Fig.…”

Identification and Characterization of MEL-3, a Novel AR Antagonist That Suppresses Prostate Cancer Cell Growth

“…All protocols and plasmids for assessing activity on mutant ARs and on other steroid receptors were described before (34). The expression plasmids for the VP16 activation domain fused with the hAR-NTD (1-529) and for the DBD-LBD of the hAR (538-919) used in the mammalian interaction assay were described before (35). The VP16 activation domain-hAR (1-919) expression construct was a kind gift of Prof. S. Balk (Department of Medicine, Harvard Medical School, Boston, MA).…”