The atypical orphan nuclear receptor DAX-1 interacts with orphan nuclear receptor Nur77 and represses its transactivation

DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on the X chromosome, gene 1) (NROB1) is an atypical member of the nuclear receptor family, which lacks the classical zinc finger DNA binding domain and acts as a coregulator of a number of nuclear receptors. In this study, we have found that DAX-1 is a novel coregulator of the orphan nuclear receptor Nur77 (NR4A1). We demonstrate that DAX-1 represses the Nur77 transactivation by transient transfection assays. Specific interaction between Nur77 and DAX-1 was detected by coimmunoprecipitation, yeast two-hybrid, and glutathione-S-transferase pull-down assays. The ligand binding domain of DAX-1 and the activation function-2 domain of Nur77 were determined as the direct interaction domains between DAX-1 and Nur77. In vitro competition binding assay showed that DAX-1 repressed Nur77 transactivation through the competition with steroid receptor coactivator-1 for the binding of Nur77. Moreover, DAX-1 repressed Nur77- and LH-dependent increase of cytochrome P450 protein 17 promoter activity in transient transfection assays. Furthermore, Nur77-mediated transactivation was significantly increased by down-regulation of DAX-1 expression with DAX-1 small interfering RNA in testicular Leydig cell line, K28. LH treatment induced a transient increase in Nur77 mRNA, whereas LH repressed DAX-1 expression in a time- and dose-dependent manner in K28 cells. In addition, immunohistochemical analysis showed the expression of Nur77 in mouse testicular Leydig cells. These results suggest that DAX-1 acts as a novel coregulator of the orphan nuclear receptor Nur77, and that the DAX-1 may play a key role in the regulation of Nur77-mediated steroidogenesis in testicular Leydig cells.     

Androgen receptor corepressor-19 kDa (ARR19), a leucine-rich protein that represses the transcriptional activity of androgen receptor through recruitment of histone deacetylase

Androgen receptor (AR) that mediates androgen action is a crucial factor in male reproductive functions. Here, we report a novel AR corepressor ARR19 (androgen receptor corepressor-19 kDa), which has been isolated as a putative androgen-induced gene from murine testis. ARR19 encoding a leucine-rich protein is expressed only in male reproductive organs such as testis and prostate. ARR19 expression in the testis is developmentally regulated. Functional analysis conducted by the transient transfection of mammalian cells shows that ARR19 represses AR transactivation in a dose-dependent manner. Furthermore, yeast two-hybrid and glutathione S-transferase pull-down analyses reveal that ARR19 directly associates with AR through the N-terminal and leucine zipper-containing regions of ARR19 and the DNA binding-hinge domain of AR. Interestingly, ARR19 localized in the cytoplasmic compartment cotranslocates into the nucleus with AR upon androgen exposure. The ARR19 repression of AR transactivation is through the recruitment of histone deacetylase 4 (HDAC4) by ARR19. Overexpression of HDAC4 further inhibits the ARR19-repressed AR transactivation. In addition, ARR19 directly interacts with HDAC4 in vitro. Furthermore, DNA-protein complex immunoprecipitation assays reveal that HDAC4 is recruited to an androgen-regulated promoter through ARR19. Taken together, the results suggest that ARR19 may act as an AR corepressor in vivo and play an important role in male reproductive functions.     

The adenosine A2A receptor interacts with the actin-binding protein alpha-actinin

Recently, evidence has emerged that heptaspanning membrane or G protein-coupled receptors may be linked to intracellular proteins identified as regulators of receptor anchoring and signaling. Using a yeast two-hybrid screen, we identified alpha-actinin, a major F-actin-cross-linking protein, as a binding partner for the C-terminal domain of the adenosine A2A receptor (A2AR). Colocalization, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between A2AR and alpha-actinin in transfected HEK-293 cells and also in rat striatal tissue. A2AR activation by agonist induced the internalization of the receptor by a process that involved rapid beta-arrestin translocation from the cytoplasm to the cell surface. In the subsequent receptor traffic from the cell surface, the role of actin organization was shown to be crucial in transiently transfected HEK-293 cells, as actin depolymerization by cytochalasin D prevented its agonist-induced internalization. A2ADeltaCTR, a mutant version of A2AR that lacks the C-terminal domain and does not interact with alpha-actinin, was not able to internalize when activated by agonist. Interestingly, A2ADeltaCTR did not show aggregation or clustering after agonist stimulation, a process readily occurring with the wild-type receptor. These findings suggest an alpha-actinin-dependent association between the actin cytoskeleton and A2AR trafficking.     

Interaction of the corepressor Alien with DAX-1 is abrogated by mutations of DAX-1 involved in adrenal hypoplasia congenita

DAX-1 is an unusual member of the nuclear hormone receptor (NHR) superfamily. Lack of DAX-1-mediated silencing leads to adrenal hypoplasia congenita and hypogonadotropic hypogonadism. Gene silencing through NHRs such as the thyroid hormone receptor (TR) is mediated by corepressors. We have previously characterized a novel corepressor, termed Alien, which interacts with TR and the ecdysone receptor but not with the retinoic acid receptors RAR or RXR. Here, we show that DAX-1 interacts with the corepressor Alien but not with the corepressor SMRT. This interaction is mediated by the DAX-1-silencing domain. Naturally occurring mutants of the DAX-1 gene fail to interact with Alien and have lost silencing function. Because the silencing domain of DAX-1 is unusual for NHRs, we mapped the interaction of Alien with DAX-1 and with TR. We show that Alien exhibits different binding characteristics to DAX-1 and TR. Furthermore, Northern experiments demonstrate that Alien is expressed in the adrenal gland and testis in tissues where DAX-1 is specifically expressed. Interestingly, a novel adrenal gland-specific mRNA of Alien was discovered. Thus, the impairment of Alien binding seems to play an important role in the pathogenesis mediated by DAX-1 mutants.     

Structural basis for androgen receptor interdomain and coactivator interactions suggests a transition in nuclear receptor activation function dominance

The androgen receptor (AR) is required for male sex development and contributes to prostate cancer cell survival. In contrast to other nuclear receptors that bind the LXXLL motifs of coactivators, the AR ligand binding domain is preferentially engaged in an interdomain interaction with the AR FXXLF motif. Reported here are crystal structures of the ligand-activated AR ligand binding domain with and without bound FXXLF and LXXLL peptides. Key residues that establish motif binding specificity are identified through comparative structure-function and mutagenesis studies. A mechanism in prostate cancer is suggested by a functional AR mutation at a specificity-determining residue that recovers coactivator LXXLL motif binding. An activation function transition hypothesis is proposed in which an evolutionary decline in LXXLL motif binding parallels expansion and functional dominance of the NH(2)-terminal transactivation domain in the steroid receptor subfamily.