Functional Characterization of the Native NH₂-Terminal Transactivation Domain of the Human Androgen Receptor: Binding Kinetics for Interactions with TFIIF and SRC-1a

Abstract

The androgen receptor (AR) is a ligand-activated transcription factor that mediates the actions of the steroid hormones testosterone and dihydrotestosterone at the level of gene transcription. The main transactivation function is modular in structure, maps to the N-terminal domain (NTD), and is termed AF1. This region of the AR is structurally flexible and functions in multiple protein-protein interactions with coregulatory proteins and components of the general transcription machinery. Using surface plasmon resonance, the binding kinetics for the interaction of AR-AF1 with the large subunit of the general transcription factor TFIIF, termed RAP74, and the coactivator SRC-1a were measured. AR-AF1 interacts with both the NTD and CTD of RAP74 and the CTD of SRC-1a. The dissociation constants ( Kd) for the binding of polypeptides derived from RAP74 are in the submicromolar range, while a peptide from SRC-1a bound with a Kd of 14 microM. Significantly, the individual NTD and CTD of RAP74 interacted with AR-AF1 with distinct binding kinetics, with the NTD exhibiting slower on and off rates. TFIIF is involved in transcription initiation and elongation, and the CTD of RAP74 binds to the RNA polymerase II enzyme, the general transcription factor TFIIB, and a CTD phosphatase, FCP1. We have mutated hydrophobic residues in the RAP74-CTD structure to disrupt secondary structure elements and show that binding of AR-AF1 depends upon helix 3 in the winged-helix domain of the RAP74-CTD polypeptide. Altogether, a model is suggested for AR-AF1-dependent transactivation of receptor-target genes.

Targeting the androgen receptor pathway in castration-resistant prostate cancer: progresses and prospects

“…The AR NTD is flexible with a high degree of intrinsic disorder 142,143 making it challenging to develop a therapeutic using rational structure-based drug design. One compound reported to target the AR NTD is EPI-001, isolated from a compound screen of a library of extracts from the marine sponge Geodia lindgreni .…”

Melanoma Antigen Gene Protein-A11 (MAGE-11) F-box Links the Androgen Receptor NH2-terminal Transactivation Domain to p160 Coactivators

“…AR AF2 binds LXXLL motifs of steroid receptor p160 coactivators (SRC), but interacts preferentially with the FXXLF motif in the AR NH 2 terminus (6,7). AR activation function 1 (AF1) in the unstructured NH 2 -terminal region interacts with multiple coregulatory proteins (8,9).…”

A sting in the tail: the N-terminal domain of the androgen receptor as a drug target

“…Phosphorylation of serine residues within the NTD is implicated in receptor function, with specific effects on gene expression, coregulatory protein binding, cellular localization, and receptor degradation 27. A growing number of coregulatory proteins have been described binding to the NTD, of particular note are members of the p160 coactivator family (SRC-1 and -2), the histone acetyltransferase protein CBP and the general transcription factor TFIIF, which all bind to the AR-AF1 domain 28. Also, of note is the “transcriptional hub” protein MAGE-A11, which binds to the FxxLF motif in the AR-NTD, interacts with CBP (p300) and p160 proteins, and represents a potential tissue-selective AR transcriptional coactivator 29.…”