Novel FXXFF and FXXMF Motifs in Androgen Receptor Cofactors Mediate High Affinity and Specific Interactions with the Ligand-binding Domain

Wijngaart, Dennis J. van de; Royen, Martin E. van; Hersmus, Remko; Pike, Ashley C.W.; Houtsmuller, Adriaan B.; Jenster, Guido; Trapman, Jan; Dubbink, Hendrikus J.

doi:10.1074/jbc.m602567200

Cited by 62 publications

(65 citation statements)

References 52 publications

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“…It is comprised of a hydrophobic or amphipathic ␣-helical motif with a consensus sequence of LXXLL or FXXFF, although Val is tolerated in place of the Leu in position ϩ1 (16) and Met can functionally replace the Leu in position ϩ4 (where the core motif is numbered ϩ1 to ϩ5 from left to right) (36). The VXXLL motif in BAR is a predicted amphipathic ␣-helix, and its sequence and structural properties are consistent with the consensus NR box.…”

Section: Discussionmentioning

confidence: 99%

Interaction of Porphyromonas gingivalis with Oral Streptococci Requires a Motif That Resembles the Eukaryotic Nuclear Receptor Box Protein-Protein Interaction Domain

2008

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Porphyromonas gingivalis initially colonizes the oral cavity by interacting with organisms in supragingival plaque, such as the oralis group of oral streptococci. This interaction involves the association of the streptococcal antigen I/II with the minor fimbrial antigen (Mfa1) of P. gingivalis. Our previous studies showed that a peptide (BAR) derived from antigen I/II inhibits P. gingivalis adherence and subsequent biofilm formation on streptococcal substrates. In addition, screening a combinatorial peptide library identified select amino acid substitutions in the NITVK active region of BAR that increased the adherence of P. gingivalis to streptococci. Here we report that incorporating these residues in a synthetic peptide results in more-potent inhibition of P. gingivalis adherence and biofilm formation (I 50 [50% inhibition] at 0.52 M versus I 50 at 1.25 M for BAR). In addition, a second structural motif in BAR, comprised of the amino acids KKVQDLLKK, was shown to contribute to P. gingivalis adherence to streptococci. Consistent with this, the KKVQDLLKK and NITVK motifs are conserved only in antigen I/II proteins expressed by the oralis group of streptococci, which interact with P. gingivalis. Interestingly, the primary and secondary structures and the functional characteristics of the amphipathic VQDLL core ␣-helix resemble the consensus nuclear receptor (NR) box protein-protein interacting domain sequence (LXXLL) of eukaryotes. BAR peptides containing amino acid substitutions with the potential to disrupt the secondary structure of VQDLL were less-effective inhibitors of P. gingivalis adherence and biofilm formation, suggesting that the ␣-helical character of VQDLL is important. Furthermore, replacing the lysines that flank VQDLL with acidic amino acids also reduced inhibitory activity, suggesting that the association of VQDLL with Mfa1 may be stabilized by a charge clamp. These results indicate that the Mfa1-interacting interface of streptococcal antigen I/II encompasses both the KKVQDLLKK and NITVK motif and suggest that the adherence of P. gingivalis to streptococci is driven by a protein-protein interaction domain that resembles the eukaryotic NR box. Thus, both motifs must be taken into account in designing potential peptidomimetics that target P. gingivalis adherence and biofilm formation.Adult periodontitis is associated with elevated levels of several gram-negative organisms in the subgingival oral biofilm, including the asaccharolytic, obligate anaerobe Porphyromonas gingivalis (17,27). In this primary niche, P. gingivalis interacts with a variety of other gram-negative obligate and facultative anaerobes, such as Fusobacterium nucleatum (3, 22, 23), Treponema denticola (14,19,24), and Tannerella forsythus (18, 37), through specific receptor-ligand interactions. However, the initial colonization of the oral cavity by P. gingivalis likely occurs through adherence to organisms in the supragingival biofilm. The successful colonization of this niche by P. gingivalis is contingent upon a variety of factor...

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Section: Discussionmentioning

confidence: 99%

Interaction of Porphyromonas gingivalis with Oral Streptococci Requires a Motif That Resembles the Eukaryotic Nuclear Receptor Box Protein-Protein Interaction Domain

2008

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“…However, these same AF2 site mutations that disrupt AR FXXLF motif binding also inhibit AR AF2 recruitment of SRC/p160 coactivator LXXLL motifs, which bind with at least 5-to 10-fold-lower affinity than the FXXLF motifs present in AR (22,23), putative AR coregulators (27,32,58), and peptides identified in peptide display screens (8,14,31). Crystal structures of the AR ligand binding domain bound to natural or synthetic androgen agonists have demonstrated overlapping AF2 binding sites for the FXXLF and LXXLL motifs (1,22,23,32), supporting their physiological relevance in AR function.…”

Section: Discussionmentioning

confidence: 99%

Epidermal Growth Factor-Dependent Phosphorylation and Ubiquitinylation of MAGE-11 Regulates Its Interaction with the Androgen Receptor

Bai

Wilson

2008

Molecular and Cellular Biology

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The androgen receptor (AR) is a ligand-activated transcription factor that interacts with coregulatory proteins during androgen-dependent gene regulation. Melanoma antigen gene protein 11 (MAGE-11) is an AR coregulator that specifically binds the AR NH 2 -terminal FXXLF motif and modulates the AR NH 2 -and carboxyl-terminal N/C interaction to increase AR transcriptional activity. Here we demonstrate that epidermal growth factor (EGF) signaling increases androgen-dependent AR transcriptional activity through the posttranslational modification of MAGE-11. EGF in the presence of dihydrotestosterone stabilizes the AR-MAGE complex through the site-specific phosphorylation of MAGE-11 at Thr-360 and ubiquitinylation at Lys-240 and Lys-245. The time-dependent EGF-induced increase in AR transcriptional activity by MAGE-11 is mediated through AR activation functions 1 and 2 in association with the increased turnover of AR and MAGE-11. The results reveal a dynamic mechanism whereby growth factor signaling increases AR transcriptional activity through the covalent modification of an AR-specific coregulatory protein. Sequence conservation of the MAGE-11 phosphorylation and ubiquitinylation sites throughout the MAGE gene family suggests common regulatory mechanisms for this group of cancer-testis antigens.The androgen receptor (AR) is a ligand-dependent transcription factor that mediates the effects of the biologically active androgens testosterone and dihydrotestosterone (DHT) on gene regulation required for male sex development and function. Androgen-dependent gene regulation involves interactions between AR and androgen response element DNA and coregulatory proteins that link to the transcriptional machinery of chromatin. Like other steroid receptors, AR has a multidomain structure that includes the NH 2 -terminal activation domain 1 (AF1), which has evolved during mammalian evolution (9, 11) and is considered predominant in AR function. Activation function 2 (AF2) in the ligand binding domain functions as a binding site for SRC/p160 coactivator LXXLL motifs and for the FXXLF motif in the AR NH 2 terminus and in several putative AR coregulatory proteins (23,25,27). Cell-free fluorescence binding studies have shown that the AR20-30 FXXLF motif peptide binds to AF2 with 5-to 10-fold-higher affinity than an LXXLL motif containing a peptide from the most active ARSRC/p160 coactivator (23) and is the basis of the androgen-dependent AR NH 2 -terminal and carboxyl-terminal (N/C) interaction important for androgen-induced gene activation (7,25,26,30,40). The AR N/C interaction slows the dissociation rate of bound androgen (62), stabilizes AR binding to androgen response element DNA (60), and appears to be involved in domain swapping between an intramolecular AR monomer in the cytoplasm and an intermolecular antiparallel AR dimer in the nucleus (39, 51). The importance of the N/C interaction for AR function is supported by studies on the human androgen insensitivity syndrome, in which naturally occurring single amino acid mutations...

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“…The phenylalanine residues in the FxxLF motif are essential for strong N/C interaction and bind deep into the coactivator groove with van der Waals interactions, whereas the leucine residue in the peptide motif lies in a shallow ridge on the surface of the LBD, and the other two amino acid residues are exposed to the solvent Hur et al, 2004;van de Wijngaart et al, 2006). In other SRs, N/C interactions are absent or weak.…”

Section: Introductionmentioning

confidence: 99%

Stepwise androgen receptor dimerization

Royen

Cappellen

Vos

et al. 2012

Journal of Cell Science

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125

135

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SummaryAndrogen-regulated gene expression is a highly coordinated dynamic process mediated by androgen receptor (AR) ligand binding and DNA binding, and by specific AR protein-protein interactions. The latter include DNA-binding domain (D-box) interactions in AR homodimers, and the interaction of the FQNLF motif in the AR N-terminal domain and the coactivator groove in the ligand-binding domain (N/C interaction). We have studied these interactions in AR homodimerization using quantitative imaging techniques. We found that the initial cytoplasmic intramolecular AR N/C interaction after ligand binding is followed by a D-box-dimerization-dependent transition to intermolecular N/C interaction in a proportion of nuclear ARs. The consecutive steps leading to homodimerization are initiated prior to DNA binding. Our data indicate the presence of nuclear pools of both AR homodimers and monomers. On the basis of AR-regulated reporter assays we propose specificity in regulation of gene expression by AR homodimers and monomers mediated by AR domain interactions. Moreover, our findings elucidate important steps in the spatiotemporal organization of AR intra-and intermolecular interactions.

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Novel FXXFF and FXXMF Motifs in Androgen Receptor Cofactors Mediate High Affinity and Specific Interactions with the Ligand-binding Domain

Cited by 62 publications

References 52 publications

Interaction of Porphyromonas gingivalis with Oral Streptococci Requires a Motif That Resembles the Eukaryotic Nuclear Receptor Box Protein-Protein Interaction Domain

Interaction of Porphyromonas gingivalis with Oral Streptococci Requires a Motif That Resembles the Eukaryotic Nuclear Receptor Box Protein-Protein Interaction Domain

Epidermal Growth Factor-Dependent Phosphorylation and Ubiquitinylation of MAGE-11 Regulates Its Interaction with the Androgen Receptor

Stepwise androgen receptor dimerization

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