GRIP1 mediates the interaction between the amino- and carboxyl-termini of the androgen receptor

The androgen receptor (AR) mediates transactivation of target genes by acting as a dimer in which its amino-terminal domain (AR-NTD) interacts with its carboxyl-terminal, ligand-binding domain (AR-LBD) (N/C interaction). Here we assessed if and how AR N/C interaction relates to AR transactivation activity and how the p160 coactivator GRIP1 participates in both processes. The concentration of dihydrotestosterone needed for half-maximal N/C interaction was approximately 10-fold higher than for half-maximal transactivation, indicating a disparity between the two processes. Although a mutation of an LXXLL-like motif, 23 FQNLF 27 --> 23 FQNAA 27 , in the AR-NTD abolished AR N/C interaction, it could be restored by the co-expression of the coactivator GRIP1. Co-expression of mutated forms of GRIP1, possessing alterations known to abolish either of the two AR interaction domains, could not restore AR N/C interaction, suggesting that wild-type GRIP1 normally bridges the two AR domains. Although AR transactivation activity can proceed without AR N/C interaction, we propose that part of the GRIP1 coactivation activity resides in its ability to bind both AR-NTD and -LBD, to stabilize the N/C complex and allow for secondary cofactors to be recruited more efficiently. Our results also indicate that AR N/C interaction enhances but is not necessary for AR transactivation activity.     

Structural characterization of the native NH2-terminal transactivation domain of the human androgen receptor: a collapsed disordered conformation underlies structural plasticity and protein-induced folding

The androgen receptor (AR) mediates the action of the steroid hormones testosterone and dihydrotestosterone. The protein contains two globular alpha-helical domains responsible for binding hormone and DNA. In contrast, the N-terminal domain is less well structurally defined and contains the main determinants for receptor-dependent transactivation, termed AF1. Previously, we have shown this region has the propensity to form alpha-helix structure. Significantly, the binding of specific protein targets or a natural osmolyte resulted in a more protease resistant conformation for the AF1 domain, consistent with an increase in conformational stability. Computational and experimental analyses were used to investigate the conformational properties of the native AF1 domain. This region of the receptor is predicted to contain significant regions of natural disordered structure, when analyzed by amino acid composition, PONDR (Predictor of Natural Disordered Regions), RONN (Regional Order Neural Network), and GlobPlot, but is grouped with ordered proteins on a charge-hydropathy plot. The binding of a hydrophobic fluorescence probe, 8-anilinonaphthalene-1-sulfonic acid (ANS), together with size-exclusion chromatography suggests that native AR-AF1 exists in a collapsed disordered conformation, distinct from extended disordered (random coil) and a stable globular fold. This state has also been described as premolten or molten globule-like. These findings are discussed in terms of the functional importance of the intrinsic plasticity of the AF1 domain.     

Electrostatic modulation in steroid receptor recruitment of LXXLL and FXXLF motifs

Coactivator recruitment by activation function 2 (AF2) in the steroid receptor ligand binding domain takes place through binding of an LXXLL amphipathic alpha-helical motif at the AF2 hydrophobic surface. The androgen receptor (AR) and certain AR coregulators are distinguished by an FXXLF motif that interacts selectively with the AR AF2 site. Here we show that LXXLL and FXXLF motif interactions with steroid receptors are modulated by oppositely charged residues flanking the motifs and charge clusters bordering AF2 in the ligand binding domain. An increased number of charged residues flanking AF2 in the ligand binding domain complement the two previously characterized charge clamp residues in coactivator recruitment. The data suggest a model whereby coactivator recruitment to the receptor AF2 surface is initiated by complementary charge interactions that reflect a reversal of the acidic activation domain-coactivator interaction model.