Characterization of the Two Coactivator-interacting Surfaces of the Androgen Receptor and Their Relative Role in Transcriptional Control*

Abstract

The androgen receptor interacts with the p160 coactivators via two surfaces, one in the ligand binding domain and one in the amino-terminal domain. The ligand binding domain interacts with the nuclear receptor signature motifs, whereas the amino-terminal domain has a high affinity for a specific glutamine-rich region in the p160s. We here describe the implication of two conserved motifs in the latter interaction. The amino-terminal domain of the androgen receptor is a very strong activation domain constituent of Tau5, which is mainly active in the absence of the ligand binding domain, and Tau1, which is only active in the presence of the ligand binding domain. Both domains are, however, implicated in the recruitment of the p160s. Mutation analysis of the p160s has shown that the relative contribution of the two recruitment mechanisms via the signature motifs or via the glutamine-rich region depend on the nature of the enhancers tested. We propose, therefore, that the androgen receptor-coactivator complex has several alternative conformations, depending partially on the context of the enhancer. The androgen receptor (AR)1 is a member of the steroid receptor family of transcription factors. Steroid receptors are ligand-inducible sequence-specific transcription factors with highly conserved DNA binding domains (DBDs), moderately conserved ligand binding domains (LBDs), and divergent amino-terminal domains (NTD) (1-4). Two transactivating functions (AFs) have been characterized, AF1 in the NTD and AF2 in the LBD. For the AR, AF1 has strong constitutive activity, since deletion of the LBD results in a molecule that can activate a reporter gene to the same extent as the full-length receptor in the presence of ligand, whereas AF2 appears to be weak (5-8). This is in contrast to what occurs in most other nuclear receptors, for example for the estrogen receptor (ER), in which AF2 is the major activation domain (9). The precise residues and mechanisms that contribute to the AF1 activity of the AR have not been conclusively established. Almost the entire NTD is required for full transcriptional activity of the full-length receptor, whereas a core region located between residues 101 and 360 (Tau1) contributes 50% of activity (10). When a constitutively active AR mutant lacking an LBD is studied, the region necessary for transcriptional activation shifts to the region 370 -494 (Tau5) (10).Binding of the appropriate hormones to the steroid hormone receptors causes a translocation of the receptors to enhancer elements in the promoters of target genes. Transcriptional coactivators are recruited to the promotor through proteinprotein interaction with the receptor (11-14). Most known coactivators are complex proteins that harbor multiple activation domains and receptor-interacting domains (15-16). The best studied group of coactivators is the p160 family of 160-kDa proteins. Three family members have been identified. The first p160 coactivators cloned were the human steroid receptor coactivator 1 (SRC1) and the transcription inte...

Repression of Androgen Receptor Activity by HEYL, a Third Member of the Hairy/Enhancer-of-split-related Family of Notch Effectors

“…We found that HEYL can repress the activity of AF1 in isolation, and furthermore, GST-HEYL was able to interact with AR through the AF1 domain (although it is not yet clear whether this interaction is direct). A characteristic of AR-AF1 is the ability of this domain to interact with the coactivator SRC1, and this interaction may be more functionally relevant than well characterized interactions of coactivators with AF2 (via LXXLL interaction motifs) as described for other nuclear receptors (41). We previously showed that HEY1 interacts with the bHLH-PAS domain of SRC1 and hypothesized that HEY1 can repress AR activity by blocking SRC1e/AR interactions and/or sequestering SRC1e from functional transcriptional complexes (25).…”

The Hinge Region Regulates DNA Binding, Nuclear Translocation, and Transactivation of the Androgen Receptor

“…The DAF1-hinge mutants were created by exchanging HindIII/PsyI inserts. VP16-NTD and VP16-DFQNLF are pSNATCH-II constructs described earlier as hAR and hAR 1-529 DFQNLF (16,21). The DFQNLF plasmid was described before Figure 1.…”

“…Unlike other nuclear receptors, the AR AF2 is almost not active, probably because the AR AF2-coactivator interactions are weaker if compared with, for example, the estrogen receptor (16). Instead, AF1 is the major transactivation function of the AR, and the AR-NTD is the main recruiting surface for coactivators (16,17).…”

“…Instead, AF1 is the major transactivation function of the AR, and the AR-NTD is the main recruiting surface for coactivators (16,17). In addition, the AF2 coactivator recruitment surface of the AR is the primary interaction site for the NTD (18)(19)(20).…”

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).…”