Cyclin D1 binding to the androgen receptor (AR) NH2-terminal domain inhibits activation function 2 association and reveals dual roles for AR corepression

The androgen receptor (AR) is a member of the nuclear receptor superfamily, the activity of which is critical for the development and progression of prostate cancer. We and others have previously demonstrated that cyclin D1 is a potent corepressor of the AR. Although cyclin D1 is suspected to recruit histone deacetylases to the AR complex, previous studies have demonstrated that this activity alone is insufficient for cyclin D1 function. Here, we uncover a novel, secondary means of cyclin D1-mediated repression, through modulation of AR amino-carboxy terminal interactions. We show that cyclin D1 predominantly binds the N-terminal domain of the AR, dependent on the AR 23FxxLF27 motif. Through this motif, cyclin D1 abrogates the ability of the AR N-terminal domain to interact with the C terminus. Secondary amino-terminal domain sites capable of fostering interaction with the C terminus were refractory to cyclin D1 action, indicating that the ability of cyclin D1 to modulate AR amino-carboxy terminal interactions is specific to 23FxxLF27. Deletion of the N-terminal cyclin D1 binding site severely compromised AR activity (due to loss of FxxLF) but unmasked a repressor action through interaction with the AR C terminus. In summary, these data reveal novel, unexpected mechanisms of cyclin D1 activity and demonstrate that this function of cyclin D1 is critical for AR modulation.     

Androgen receptor ligand-binding domain interaction and nuclear receptor specificity of FXXLF and LXXLL motifs as determined by L/F swapping

The androgen receptor (AR) ligand-binding domain (LBD) binds FXXLF motifs, present in the AR N-terminal domain and AR-specific cofactors, and some LXXLL motifs of nuclear receptor coactivators. We demonstrated that in the context of the AR FXXLF motif many different amino acid residues at positions +2 and +3 are compatible with strong AR LBD interaction, although a preference for E at +2 and K or R at +3 was found. Pairwise systematic analysis of F/L swaps at +1 and +5 in FXXLF and LXXLL motifs showed: 1) F to L substitutions in natural FXXLF motifs abolished AR LBD interaction; 2) binding of interacting LXXLL motifs was unchanged or increased upon L to F substitutions; 3) certain noninteracting LXXLL motifs became strongly AR-interacting FXXLF motifs; whereas 4) other nonbinders remained unaffected by L to F substitutions. All FXXLF motifs, but not the corresponding LXXLL motifs, displayed a strong preference for AR LBD. Progesterone receptor LBD interacted with some FXXLF motifs, albeit always less efficiently than corresponding LXXLL motifs. AR LBD interaction of most FXXLF and LXXLL peptides depended on classical charge clamp residue K720, whereas E897 was less important. Other charged residues lining the AR coactivator-binding groove, K717 and R726, modulated optimal peptide binding. Interestingly, these four charged residues affected binding of individual peptides independent of an F or L at +1 and +5 in swap experiments. In conclusion, F residues determine strong and selective peptide interactions with AR. Sequences flanking the core motif determine the specific mode of FXXLF and LXXLL interactions.     

Novel FXXFF and FXXMF motifs in androgen receptor cofactors mediate high affinity and specific interactions with the ligand-binding domain

Upon hormone binding, a hydrophobic coactivator binding groove is induced in the androgen receptor (AR) ligand-binding domain (LBD). This groove serves as high affinity docking site for alpha-helical FXXLF motifs present in the AR N-terminal domain and in AR cofactors. Study of the amino acid requirements at position +4 of the AR FXXLF motif revealed that most amino acid substitutions strongly reduced or completely abrogated AR LBD interaction. Strong interactions were still observed following substitution of Leu+4 by Phe or Met residues. Leu+4 to Met or Phe substitutions in the FXXLF motifs of AR cofactors ARA54 and ARA70 were also compatible with strong AR LBD binding. Like the corresponding FXXLF motifs, interactions of FXXFF and FXXMF variants of AR and ARA54 motifs were AR specific, whereas variants of the less AR-selective ARA70 motif displayed increased AR specificity. A survey of currently known AR-binding proteins revealed the presence of an FXXFF motif in gelsolin and an FXXMF motif in PAK6. In vivo fluorescence resonance energy transfer and functional protein-protein interaction assays showed direct, efficient, and specific interactions of both motifs with AR LBD. Mutation of these motifs abrogated interaction of gelsolin and PAK6 proteins with AR. In conclusion, we have demonstrated strong interaction of FXXFF and FXXMF motifs to the AR coactivator binding groove, thereby mediating specific binding of a subgroup of cofactors to the AR LBD.     

Disrupted amino- and carboxyl-terminal interactions of the androgen receptor are linked to androgen insensitivity

We have compared the functional consequences of seven single-point mutations in the ligand-binding domain (LBD) of the androgen receptor (AR). The mutations span helices 3 to 11 and are present in patients suffering from androgen insensitivity syndromes (AIS) and other male-specific disorders. The mutants, except M742V, bound to androgen response elements in vivo and in vitro and showed a testosterone-dependent conformational change. With regard to functional activity, the mutant M742V had severely blunted ability to transactivate or exhibit the androgen-dependent amino/carboxyl-terminal (N/C) interaction; mutants F725L, G743V, and F754L showed reduced transactivation potential and attenuated N/C interaction; and mutants V715M, R726L, and M886V had minor functional impairments. The mutants belonging to the first two groups also displayed reduced response to coexpressed GRIP1. In addition, mutations of amino acids M894 and A896 in the putative core activation domain 2 (AF2) in helix 12 confirmed that this helix is important for N/C interactions. Thus, amino acids located between helices 3 and 4 (F725 and R726), in helix 5 (M742, G743, and F754), and in helix 12 (M894 and A896) play critical roles in mediating the N/C interaction of AR. The data also show that disrupted N/C interaction is a potential molecular abnormality in AIS cases in which LBD mutations have not resulted in markedly impaired ability to bind androgen.     

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.     

Elucidation of the essential amino acids involved in the binding of the cyanobacterial Orange Carotenoid Protein to the phycobilisome

The Orange Carotenoid Protein (OCP) is a soluble photoactive protein involved in cyanobacterial photoprotection. It is formed by the N-terminal domain (NTD) and C-terminal (CTD) domain, which establish interactions in the orange inactive form and share a ketocarotenoid molecule. Upon exposure to intense blue light, the carotenoid molecule migrates into the NTD and the domains undergo separation. The free NTD can then interact with the phycobilisome (PBS), the extramembrane cyanobacterial antenna, and induces thermal dissipation of excess absorbed excitation energy. The OCP and PBS amino acids involved in their interactions remain undetermined. To identify the OCP amino acids essential for this interaction, we constructed several OCP mutants (23) with modified amino acids located on different NTD surfaces. We demonstrated that only the NTD surface that establishes interactions with the CTD in orange OCP is involved in the binding of OCP to PBS. All amino acids surrounding the carotenoid β1 ring in the OCP^R-NTD (L51, P56, G57, N104, I151, R155, N156) are important for binding OCP to PBS. Additionally, modification of the amino acids influences OCP photoactivation and/or recovery rates, indicating that they are also involved in the translocation of the carotenoid.     

An investigation into CAG repeat length variation and N/C terminal interactions in the T877A mutant androgen receptor found in prostate cancer

Prostate cancer may progress by circumventing ablation therapy due to mutations in the androgen receptor (AR) gene. The most intensively studied is the T877A mutation in the ligand binding domain (LBD), which causes the AR to become promiscuous, i.e., respond to a number of different ligands. Our investigations have shown that the T877A mutation alters the inverse relationship between CAG repeat length and transactivation in a noticeable albeit minor manner, while increasing N/C terminal interactions. In the presence of beta-catenin, a coactivator over-expressed in prostate cancer, the inverse relationship between CAG repeat length and transactivation is reversed for the wild type (wt) AR as well. We have also used molecular modeling with the AR and FXXLF and LXXLL peptides to investigate N/C terminal and coactivator interactions. In T877A, this approach revealed an increase in the flexibility of amino acid residues in the activation function 2 (AF-2) domain in the LBD, and a larger solvent accessible surface in T877A compared to the wt AR AF-2 domain. Thus, the improved induced fit of the AR N-terminal domain FXXLF-containing peptide into the T877A LBD could be due to the increased flexibility and solvent accessibility of the AF-2 domain. These new observations suggest that the AR CAG effect can be overridden by prostate cancer mutations, and also further our understanding of hormone-refractory prostate cancer by helping to explain the promiscuity of the T877A mutation.     

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.     

The N-terminal region of the heme-regulated eIF2alpha kinase is an autonomous heme binding domain.

The N-terminal domain (NTD) of the heme-regulated eukaryotic initiation factor (eIF)2alpha kinase (HRI) was aligned to sequences in the NCBI data base using ENTREZ and a PAM250 matrix. Significant similarity was found between amino acids 11-118 in the NTD of rabbit HRI and amino acids 16-120 in mammalian alpha-globins. Several conserved amino acid residues present in globins are conserved in the NTD of HRI. His83 of HRI was predicted to be equivalent to the proximal heme ligand (HisF8) that is conserved in all globins. Molecular modeling of the NTD indicated that its amino acid sequence was compatible with the globin fold. Recombinant NTD (residues 1-159) was expressed in Escherichia coli. Spectral analysis of affinity purified recombinant NTD indicated that the NTD contained stably bound hemin. Mutational analysis indicated that His83 played a critical structural role in the stable binding of heme to the NTD, and was required to stabilize full length HRI synthesized de novo in the rabbit reticulocyte lysate. These results indicate that the NTD of HRI is an autonomous heme-binding domain, with His83 possibly serving as the proximal heme binding ligand.     

Identification of the WW domain-interaction sites in the unstructured N-terminal domain of EBV LMP 2A

Epstein-Barr virus latency is maintained by the latent membrane protein (LMP) 2A, which mimics the B-cell receptor (BCR) and perturbs BCR signaling. The cytoplasmic N-terminal domain of LMP2A is composed of 119 amino acids. The N-terminal domain of LMP2A (LMP2A NTD) contains two PY motifs (PPPPY) that interact with the WW domains of Nedd4 family ubiquitin-protein ligases. Based on our analysis of NMR data, we found that the LMP2A NTD adopts an overall random-coil structure in its native state. However, the region between residues 60 and 90 was relatively ordered, and seemed to form the hydrophobic core of the LMP2A NTD. This region resides between two PY motifs and is important for WW domain binding. Mapping of the residues involved in the interaction between the LMP2A NTD and WW domains was achieved by chemical shift perturbation, by the addition of WW2 and WW3 peptides. Interestingly, the binding of the WW domains mainly occurred in the hydrophobic core of the LMP2A NTD. In addition, we detected a difference in the binding modes of the two PY motifs against the two WW peptides. The binding of the WW3 peptide caused the resonances of five residues (Tyr(60), Glu(61), Asp(62), Trp(65), and Gly(66)) just behind the N-terminal PY motif of the LMP2A NTD to disappear. A similar result was obtained with WW2 binding. However, near the C-terminal PY motif, the chemical shift perturbation caused by WW2 binding was different from that due to WW3 binding, indicating that the residues near the PY motifs are involved in selective binding of WW domains. The present work represents the first structural study of the LMP2A NTD and provides fundamental structural information about its interaction with ubiquitin-protein ligase.     

RNA aptamers specifically interact with the prion protein PrP.

We have isolated RNA aptamers which are directed against the recombinant Syrian golden hamster prion protein rPrP23-231 (rPrPc) fused to glutathione S-transferase (GST). The aptamers did not recognize the fusion partner GST or the fusion protein GST::rPrP90-231 (rPrP27-30), which lacks 67 amino acids from the PrP N terminus. The aptamer-interacting region of PrPc was mapped to the N-terminal amino acids 23 to 52. Sequence analyses suggest that the RNA aptamers may fold into G-quartet-containing structural elements. Replacement of the G residues in the G quartet scaffold with uridine residues destroyed binding to PrP completely, strongly suggesting that the G quartet motif is essential for PrP recognition. Individual RNA aptamers interact specifically with prion protein in brain homogenates from wild-type mice (C57BL/6), hamsters (Syrian golden), and cattle as shown by supershifts obtained in the presence of anti-PrP antibodies. No interaction was observed with brain homogenates from PrP knockout mice (prn-p(0/0)). Specificity of the aptamer-PrP interaction was further confirmed by binding assays with antisense aptamer RNA or a mutant aptamer in which the guanosine residues in the G tetrad scaffold were replaced by uridine residues. The aptamers did not recognize PrP27-30 in brain homogenates from scrapie-infected mice. RNA aptamers may provide a first milestone in the development of a diagnostic assay for the detection of transmissible spongiform encephalopathies.