Abstract
Molecular chaperones such as Hsp40 and Hsp70 hold the androgen receptor (AR) in an inactive conformation. They are released in the presence of androgens, enabling transactivation and causing the receptor to become aggregation-prone. Here we show that these molecular chaperones recognize a region of the AR N-terminal domain (NTD), including a FQNLF motif, that interacts with the AR ligand-binding domain (LBD) upon activation. This suggests that competition between molecular chaperones and the LBD for the FQNLF motif regulates AR activation. We also show that, while the free NTD oligomerizes, binding to Hsp70 increases its solubility. Stabilizing the NTD-Hsp70 interaction with small molecules reduces AR aggregation and promotes its degradation in cellular and mouse models of the neuromuscular disorder spinal bulbar muscular atrophy. These results help resolve the mechanisms by which molecular chaperones regulate the balance between AR aggregation, activation and quality control.
Highlights
The steroid hormone receptors (SHRs) system provides an excellent model for understanding how molecular chaperones balance the needs of protein folding, stability, function and quality control
We found that Hsp[70] and Hsp[40] bind the Nterminal transactivation domain (NTD), with μM affinity and, strikingly, that the binding site contains the same FQNLF motif that interacts with activation function 2 (AF-2) in the ligand-binding domain (LBD) during activation
This observation suggests a tug-of-war model in which the FQNLF motif can be bound by either chaperone or the LBD, such that hormone binding shifts partitioning between these two possibilities. We found that this part of the NTD has the propensity to form oligomers that are stabilized by interactions involving residues of the FQNLF motif, leading to aggregation; its interaction with Hsp[70] keeps the protein in solution, explaining why chaperones are so well positioned to block the aggregation and misfolding of androgen receptor (AR)
Summary
Hsp[70] and Hsp[40] bind to the FQNLF motif in the AR NTD. We used solution NMR to determine whether Hsp[40] and Hsp[70] bind to the NTD of AR and, if so, where this interaction occurs. The 1H,15N-HSQC spectrum of NTD144-450 was not altered by the addition of either Hsp[40] or Hsp[70], indicating weak or no interaction (Supplementary Fig. 1) This result is important because NTD144-450 includes hydrophobic motifs that could represent non-specific sites for chaperone binding, yet none were observed to interact under these conditions. This value is in good agreement with the FP studies as well as with the NMR titrations because this dissociation constant should lead, in the slow exchange regime, to I/I0 ~ 0.3 when the NTD1-155 (at 33 μM) is in the presence of an equimolar amount of molecular chaperone (see Fig. 1b) Together, these results suggest that the region of sequence corresponding to the FQNLF motif of AR and its flanking regions represents a canonical substrate for.
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