Conformational Analysis of the Androgen Receptor Amino-terminal Domain Involved in Transactivation

Abstract

The androgen receptor (AR) is a member of the nuclear receptor superfamily. Sequences within the large amino-terminal domain of the receptor have been shown to be important for transactivation and proteinprotein interactions; however, little is known about the structure and folding of this region. In the present study we show that a 344-amino acid polypeptide representing the main determinants for transactivation has the propensity to form ␣-helical structure and that mutations which disrupt putative helical regions alter conformation. Folding of the AR was observed in the presence of the helix-stabilizing solvent trifluoroethanol and the natural osmolyte trimethylamine N-oxide (TMAO). TMAO resulted in the movement of two tryptophan residues to a less solvent-exposed environment and the formation of secondary/tertiary structure resistant to protease cleavage. Critically, binding to the RAP74 subunit of the general transcription factor TFIIF resulted in extensive protease resistance, consistent with induced folding of the receptor transactivation domain. These data indicate that this region of the AR is structurally flexible and folds into a stable conformation upon interactions with a component of the general transcription machinery.The actions of the male sex hormones testosterone and dihydrotestosterone are mediated by the intracellular androgen receptor (AR) 1 (reviewed in Refs. 1 and 2). The AR belongs to a large family of nuclear receptors, whose members mediate the actions of steroid and thyroid hormones, retinoic acid, vitamin D 3 , and fatty acid derivatives. The majority of these receptor proteins share a common domain organization (see Fig. 1A). The ligand binding domain (LBD) in the carboxyl terminus binds ligand and participates in dimerization and ligand-dependent transactivation. A hinge region connects the LBD to the DNA binding domain (DBD), a region responsible for recognition of and binding to DNA target sequences and receptor dimerization. The amino-terminal domain (NTD) of these proteins is the most variable in length and amino acid sequence but is often critical for transactivation. In the case of the AR, the major transactivation domain has been mapped to the amino-terminal domain (3-8). Sequences within this domain have been shown to mediate protein-protein interactions with the carboxyl-terminal LBD (9 -15), the general transcription factors TFIIF (16) and TFIIH (17), members of the p160 family of nuclear receptor coactivator proteins (18 -21), cyclin E (22), and an AR-associated protein 160 (23). This region of the receptor also contains homopolymer stretches of the amino acids glutamine (Gln), glycine (Gly), and proline (Pro). The largest stretch of glutamines is of particular interest because expansion of this sequence from on-average 22 to greater than 40 residues results in the neuromuscular degenerative condition spinal bulbar muscular atrophy or Kennedy's disease (reviewed in Refs. 24 and 25). In addition, polymorphisms in this polyglutamine stretch have been associated with prosta...

An Androgen Receptor NH2-terminal Conserved Motif Interacts with the COOH Terminus of the Hsp70-interacting Protein (CHIP)

“…1B), numbered according to the human AR sequence of Lubahn et al (22) and the AR mutation data base. 2 Sequence conservation of hydrophobic residues in the region was noted previously (14). Also notable is Ser 256 -Pro 257 since Ser 256 was shown previously to be phosphorylated in human AR (10).…”

“…The predicted amphipathic ␣-helices of the p160 coactivator LXXLL motifs were also confirmed by structural studies (69,70). Thus, although transcriptional activation domains of regulatory proteins are largely unstructured and not conserved, short segments within these regions are predicted to form amphipathic ␣-helices whose structure is likely stabilized by binding coregulatory proteins (2,71). We previously proposed a reversal of the acidic transcriptional activation domain-coactivator interaction model, implicating charge interactions that precede nonpolar contacts of hydrophobic residues in protein-protein interactions mediated by amphipathic ␣-helices (21).…”

Activated Cdc42-associated kinase Ack1 promotes prostate cancer progression via androgen receptor tyrosine phosphorylation

“…Both Ack1-dependent AR phosphorylation sites that we identified, Tyr-267 and Tyr-363, are located within this AF-1 domain. The AF-1 domain lacked stable secondary structure in aqueous solution, but may adopt a more structured and protease-resistant conformation on intra-and/or intermolecular protein-protein interactions (20). Because the N-terminal AF-1 domain has been shown to interact with the p160 family of coactivators, such as steroid receptor coactivator-1 (21,22), it is possible that Ack1-dependent phosphorylation sites in AR may regulate recruitment of coactivators and histone acetyl transferases, thereby regulating transcriptional activation.…”

Role of Regulatory F-domain in Hepatocyte Nuclear Factor-4α Ligand Specificity

“…Mutants: Circular Dichroism-TFE is known to induce helical secondary structure in proteins that contain unordered structures and have potential for developing helixes under a hydrophobic environment (41,42). Since the long F-domain in HNF-4␣ molecule is random coil-proline-rich with very low helix content (22,38), the potential of the three forms of HNF-4␣ to form helix structure in the presence of TFE and the influence of F-domain were examined (Fig.…”