Evidence for DNA-Binding Domain–Ligand-Binding Domain Communications in the Androgen Receptor

Abstract

DNA binding as well as ligand binding by nuclear receptors has been studied extensively. Both binding functions are attributed to isolated domains of which the structure is known. The crystal structure of a complete receptor in complex with its ligand and DNA-response element, however, has been solved only for the peroxisome proliferator-activated receptor ␥ (PPAR␥)-retinoid X receptor ␣ (RXR␣) heterodimer. This structure provided the first indication of direct interactions between the DNA-binding domain (DBD) and ligand-binding domain (LBD). In this study, we investigated whether there is a similar interface between the DNA-and ligand-binding domains for the androgen receptor (AR). Despite the structural differences between the AR-and PPAR␥-LBD, a combination of in silico modeling and docking pointed out a putative interface between AR-DBD and AR-LBD. The surfaces were subjected to a point mutation analysis, which was inspired by known AR mutations described in androgen insensitivity syndromes and prostate cancer. Surprisingly, AR-LBD mutations D695N, R710A, F754S, and P766A induced a decrease in DNA binding but left ligand binding unaffected, while the DBD-residing mutations K590A, K592A, and E621A lowered the ligand-binding but not the DNA-binding affinity. We therefore propose that these residues are involved in allosteric communications between the AR-DBD and AR-LBD. N uclear receptors (NRs) are involved in many physiological processes, diseases, and therapeutic applications. They are transcription factors that contain a DNA-binding domain (DBD) composed of 2 zinc fingers (40) and a ligand-binding domain (LBD) formed by 12 ␣ helices (60). The structures of the separate DNA-binding and ligand-binding domains of many receptors have already revealed a large amount of information. The structure of the LBD has especially led to a more focused search for new agonists and antagonists for many therapeutic indications where NRs are involved. The exact structure of full-size NRs bound to DNA and ligand will help us in understanding the basic mechanism of nuclear receptor signaling but will also provide new targets for therapeutic strategies. The coordinates of the peroxisome proliferator-activated receptor ␥ (PPAR␥)-retinoid X receptor ␣ (RXR␣) cocrystal bound as a heterodimer to the DNA have been reported (11) and revealed a contact between PPAR␥ and RXR␣ more intimate than expected. The existence of the previously unknown interface between PPAR␥-LBD and RXR␣-DBD was corroborated with a mutation analysis. Since this has not yet been confirmed by other techniques like small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) (49), it is debated whether such a communication exists. There is, however, strong evidence for allosteric communications, e.g., between the DBD and LBD, in nuclear receptors. A most remarkable observation was made for the glucocorticoid receptor (GR) when slightly different response elements were tested in gene reporter assays: small changes in the DNA sequence had an importa...

Androgen Induces a Switch from Cytoplasmic Retention to Nuclear Import of the Androgen Receptor

“…Since the LBD has a strong inhibitory effect on AR import, the data suggest that the T877A substitution weakens the repressive effect of the LBD on nuclear import of androgen-free AR. Thus, interdomain interactions, already known to be relevant to the transactivation function of AR (26,27,39) and detected by FRET in both the WT and T877A forms of AR (40), can play a role in regulating nuclear import as well. A combination of protein modeling and mutagenesis was used by the Claussens group to show that interdomain communication in AR also occurs between the LBD and DBD (39), and this type of interaction likely occurs in other nuclear receptors as well (41).…”

“…Thus, interdomain interactions, already known to be relevant to the transactivation function of AR (26,27,39) and detected by FRET in both the WT and T877A forms of AR (40), can play a role in regulating nuclear import as well. A combination of protein modeling and mutagenesis was used by the Claussens group to show that interdomain communication in AR also occurs between the LBD and DBD (39), and this type of interaction likely occurs in other nuclear receptors as well (41). Whether the T877A substitution stabilizes a conformation that is similar to an androgen-induced state in WT AR or whether this AR variant adopts a unique structure remain an open question.…”

Hydrogen Sulfide Represses Androgen Receptor Transactivation by Targeting at the Second Zinc Finger Module

“…In our study, we found that the fulllength of AR can be S-sulfhydrated in the presence of H 2 S. H 2 S suppressing AR downstream gene expression without changing AR interaction with HSPs as well as nuclear localization suggests to us that H 2 S may directly target at AR-DBD. AR-DBD contains two cysteine-rich zinc finger modules (6). The first zinc finger is responsible for the recognition of AREs, whereas the second zinc finger is involved in DNA-dependent dimerization and the stabilization and specificity of DNA binding (8).…”

“…The mechanisms underlying the development of CRPC are largely unclear, although AR signaling still plays important roles in CRPC (5). The AR consists of four structurally and functionally distinct domains, a poorly conserved N-terminal domain, a highly conserved DNA binding domain (DBD), a short hinge region, and a moderately conserved C-terminal ligand binding domain (6,7). The DBD of AR contains two cysteine 4-type zinc fingers, which enable the AR to specifically recognize androgen-responsive elements (AREs) (8).…”

A naturally occuring insertion of a single amino acid rewires transcriptional regulation by glucocorticoid receptor isoforms

“…Nonetheless, allosteric communication between receptor domains has been demonstrated in GR and other NRs (11)(12)(13)(14)(15). Notably, depending on the precise DNA sequences bound by retinoid X receptor (RXR)-vitamin D receptor (VDR) heterodimers, distinct domains outside of their DBDs adopt alternative conformations or changed dynamics, indicative of long-range allosteric communication (11).…”