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FXXLF and WXXLF sequences mediate the NH2-terminal interaction with the ligand binding domain of the androgen receptor.

The nuclear receptor superfamily members of eukaryotic transcriptional regulators contain a highly conserved activation function 2 (AF2) in the hormone binding carboxyl-terminal domain and, for some, an additional activation function 1 in the NH2-terminal region which is not conserved. Recent biochemical and crystallographic studies revealed the molecular basis of AF2 is hormone-dependent recruitment of LXXLL motif-containing coactivators, including the p160 family, to a hydrophobic cleft in the ligand binding domain. Our previous studies demonstrated that AF2 in the androgen receptor (AR) binds only weakly to LXXLL motif-containing coactivators and instead mediates an androgen-dependent interaction with the AR NH2-terminal domain required for its physiological function. Here we demonstrate in a mammalian two-hybrid assay, glutathione S-transferase fusion protein binding studies, and functional assays that two predicted α-helical regions that are similar, but functionally distinct from the p160 coactivator interaction sequence, mediate the androgen-dependent, NH2- and carboxyl-terminal interaction. FXXLF in the AR NH2-terminal domain with the sequence23FQNLF27 mediates interaction with AF2 and is the predominant androgen-dependent interaction site. This FXXLF sequence and a second NH2-terminal WXXLF sequence 433WHTLF437 interact with different regions of the ligand binding domain to stabilize the hormone-receptor complex and may compete with AF2 recruitment of LXXLL motif-containing coactivators. The results suggest a unique mechanism for AR-mediated transcriptional activation.

Activation Function 2 in the Human Androgen Receptor Ligand Binding Domain Mediates Interdomain Communication with the NH2-terminal Domain

Activation function 2 in the ligand binding domain of nuclear receptors forms a hydrophobic cleft that binds the LXXLL motif of p160 transcriptional coactivators. Here we provide evidence that activation function 2 in the androgen receptor serves as the contact site for the androgen dependent NH2- and carboxyl-terminal interaction of the androgen receptor and only weakly interacts with p160 coactivators in an LXXLL-dependent manner. Mutagenesis studies indicate that it is the NH2-/carboxyl-terminal interaction that is required by activation function 2 to stabilize helix 12 and slow androgen dissociation critical for androgen receptor activityin vivo. The androgen receptor recruits p160 coactivators through its NH2-terminal and DNA binding domains in an LXXLL motif-independent manner. The results suggest a novel function for activation function 2 and a unique mechanism of nuclear receptor transactivation.

The FXXLF Motif Mediates Androgen Receptor-specific Interactions with Coregulators

The androgen receptor (AR) activation function 2 region of the ligand binding domain binds the LXXLL motifs of p160 coactivators weakly, engaging instead in an androgen-dependent, interdomain interaction with an FXXLF motif in the AR NH2 terminus. Here we show that FXXLF motifs are present in previously reported AR coactivators ARA70/RFG, ARA55/Hic-5, and ARA54, which account for their selection in yeast two-hybrid screens. Mammalian two-hybrid assays, ligand dissociation rate studies, and glutathioneS-transferase adsorption assays indicate androgen-dependent selective interactions of these FXXLF motifs with the AR ligand binding domain. Mutagenesis of residues within activation function 2 indicates distinct but overlapping binding sites where specificity depends on sequences within and flanking the FXXLF motif. Mutagenesis of the FXXLF motifs eliminated interaction with the ligand binding domain but only modestly reduced AR coactivation in transcription assays. The studies indicate that the FXXLF binding motif is specific for the AR and mediates interactions both within the AR and with coregulatory proteins.

Electrostatic modulation in steroid receptor recruitment of LXXLL and FXXLF motifs.

ABSTRACT Coactivator recruitment by activation function 2 (AF2) in the steroid receptor ligand binding domain takes place through binding of an LXXLL amphipathic α-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.

Probing the Functional Link between Androgen Receptor Coactivator and Ligand-binding Sites in Prostate Cancer and Androgen Insensitivity

The androgen receptor (AR) is a ligand-activated transcription factor required for male sex development and virilization and contributes to prostate cancer initiation and progression. High affinity androgen binding triggers conformational changes required for AR transactivation. Here we characterized naturally occurring AR gene mutations in the region of activation function 2 (AF2) that decrease or increase AR transcriptional activity by altering the region bounded by AF2 and the ligand binding pocket without affecting equilibrium androgen binding affinity. In the androgen insensitivity syndrome, germ line AR mutations increase the androgen dissociation rate and reduce AR FXXLF motif binding and the recruitment of steroid receptor coactivator (SRC)/p160 coactivator LXXLL motifs. In prostate cancer, somatic AR mutations in AF2 or near the bound ligand slow androgen dissociation and increase AR stabilization and coactivator recruitment. Crystal structures of the AR ligand binding domain bound to R1881 and FXXLF or LXXLL motif peptide indicate the mutations are proximal to the AF2 bound peptide, adjacent to the ligand pocket, or in a putative ligand gateway. The results suggest a bidirectional structural relay between bound ligand and coactivator that establishes AR functional potency in vivo.

Partial androgen insensitivity with phenotypic variation caused by androgen receptor mutations that disrupt activation function 2 and the NH2- and carboxyl-terminal interaction

Partial androgen insensitivity with sex phenotype variation in two unrelated families was associated with missense mutations in the androgen receptor (AR) gene that disrupted the AR NH(2)-terminal/carboxy terminal interaction. Each mutation caused a single amino acid change within the region of the ligand-binding domain that forms activation function 2 (AF2). In one family, the mutation I737T was in alpha helix 4 and in the other F725L was between helices 3 and 4. Neither mutation altered androgen binding as determined by assays of mutant AR in the patients cultured genital skin fibroblasts or of recombinant mutant receptors transfected into COS cells. In transient cotransfection assays in CV1 cells, transactivation with the AR mutants at low concentrations of DHT was reduced several fold compared with wild-type AR but increased at higher concentrations. Defects in NH(2)-terminal/carboxy terminal interactions were identified in mammalian two hybrid assays. In similar assays, there was reduced binding of the p160 coactivators TIF2/SRC2 and SRC1 to the mutant AR ligand binding domains (LBD). In the family with AR I737T, sex phenotype varied from severely defective masculinization in the proband to a maternal great uncle whose only manifestation of AIS was severe gynecomastia. He was fertile and passed the mutation to two daughters. The proband of the F725L family was also incompletely masculinized but was raised as a male while his half-sibling by a different father was affected more severely and reared as a female. These studies indicate that the function of an AR AF2 mutant in male development can vary greatly depending on the genetic background.

Methods for Detecting Domain Interactions in Nuclear Receptors

Publisher Summary This chapter focuses on the methods for detecting domain interactions in nuclear receptors. Amino- and carboxyl-terminal (N–C) domain interactions in the human androgen receptor (AR) are demonstrated in the studies based on the dissociation rate of bound ligand. An N–C interaction in the AR is also supported by the finding that NH2- and carboxyl-terminal fragments of the receptor, each containing the DNA-binding domain, exhibited androgen-dependent DNA binding and dimerization. Detection methods for the AR N–C interaction have changed over the years to optimize transfection efficiency and signal intensity and to facilitate tests for specific interacting sequences. Instead of the Chinese hamster ovary (CHO) cells used originally in the assay—humanepithelioid cervical carcinoma (HeLa) or human hepatocellular carcinoma (HepG2) cells—are employed. The Effectene TM reagent from Qiagen is used for transient transfection rather than DEAE dextran. The G5E1bLuc reporter vector is replaced by 5XGAL4Luc3, which yields a stronger signal. An additional method to confirm protein–protein interaction sites among nuclear receptor sequences involves determining the dissociation rate of bound radiolabeled ligand from chimeric receptor proteins. Another approach to demonstrate protein–protein interactions is GST affinity matrix or pull-down assays. GST affinity matrix assays demonstrate protein interactions, but it must be kept in mind that nonspecific binding can occur when high concentrations of protein are used. In studies with nuclear receptors, this potential complication is minimized by demonstrating hormone dependence for the interaction.