Structural and functional analysis of domains of the progesterone receptor

Steroid hormone receptors are multi-domain proteins composed of conserved well-structured regions, such as ligand (LBD) and DNA binding domains (DBD), plus other naturally unstructured regions including the amino-terminal domain (NTD) and the hinge region between the LBD and DBD. The hinge is more than just a flexible region between the DBD and LBD and is capable of binding co-regulatory proteins and the minor groove of DNA flanking hormone response elements. Because the hinge can directly participate in DNA binding it has also been termed the carboxyl terminal extension (CTE) of the DNA binding domain. The CTE and NTD are dynamic regions of the receptor that can adopt multiple conformations depending on the environment of interacting proteins and DNA. Both regions have important regulatory roles for multiple receptor functions that are related to the ability of the CTE and NTD to form multiple active conformations. This review focuses on studies of the CTE and NTD of progesterone receptor (PR), as well as related work with other steroid/nuclear receptors.     

Characterization and partial purification of androgen receptors from ram seminal vesicles

An androgen receptor has been demonstrated in the cytosol and in the nuclear fraction of ram seminal vesicles.The cytosol receptor was stabilized by sodium molybdate and 2 distinct [³H]methyltri-enolone-binding proteins, one sedimenting at 9S and one sedimenting at 3S, could be demonstrated by sucrose-gradient centrifugation in the presence of 50 mM molybdate. The slower sedimenting form could be partially purified by ADP-sepharose chromatography. The purified receptor still sedimented at 3S after centrifugation on sucrose gradients containing either 0.6 M KCl or 50 mM molybdate. The receptor was destroyed by heating at 50°C for 30 min and its complex with [³H]methyltrienolone dissociated slowly at low temperatures. The apparent equilibrium-dissociation constant (K_D) for the purified receptor was: 3.8 × 10^?10 M. The relative affinities for different steroids decreased in the following sequence: 5α-dihydrotestosterone ? methyltrienolone > testosterone > estradiol > R5020 > progesterone > diethylstilbestrol.The nuclear androgen receptor sedimented at 3S on sucrose gradients containing 0.6 M KCl. At pH 7.4 it behaved as an acidic protein with an electrophoretic mobility towards the anodic region of the agar gel.Because of the relatively large content of cytoplasmic and nuclear androgen receptors and the availability of large amounts of tissue the ram seminal vesicles could be a suitable source for large-scale purification of these receptors.     

Looking at nuclear receptors from a new angle

While the structures of the DNA- and ligand-binding domains of many nuclear receptors have been determined in great detail; the mechanisms by which these domains interact and possibly ‘communicate’ is still under debate. The first crystal structures of receptor dimers bound to ligand, DNA and coactivator peptides provided new insights in this matter. The observed binding modes revealed exciting new interaction surfaces between the different nuclear receptor domains. Such interfaces are proposed to be the route through which allosteric signals from the DNA are passed on to the ligand-binding domain and the activating functions of the receptor. The structural determinations of DNA-bound receptor dimers in solution, however, revealed an extended structure of the receptors. Here, we discuss these apparent contradictory structural data and their possible implications for the functioning of nuclear receptors.     

Interdomain interactions in the mineralocorticoid receptor

The potential for interaction between the N-terminal domain and the C-terminal region (hinge and ligand-binding domain) of the mineralocorticoid receptor (MR) was examined using the mammalian-2-hybrid assay. The MR C-terminal region was fused to the GAL4 DNA-binding domain (GAL4-MRC). To examine if the AF-2 is involved in the interaction, as has been reported for other steroid hormone receptors, it was inactivated by point mutation (E962A). The N-terminal domain was fused to the VP16 transactivation domain (VP16-MRNT). In the mammalian-2-hybrid assay both GAL4-MRC and GAL4-MRC(E962A) interact with VP16-MRNT in an aldosterone-dependent manner. The GAL4-MRC(E962A) construct was used in subsequent experiments to examine the AF-2-independent N/C-interaction. The MR antagonist spironolactone inhibits the aldosterone-mediated association of the two domains. GAL4-MRC(E962A) interacts weakly with the GR or AR N-terminal domains in the presence of aldosterone. No dimerization between GAL4-MRC(E962A) and VP16-MRC is observed. Interestingly, cortisol produces a much weaker N/C-interaction than aldosterone, and it is possible that the N/C-interaction may contribute to observed functional differences in the MR bound to the two ligands.     

慢性前列腺炎/盆腔疼痛综合征患者白细胞雄激素受体的改变及意义

目的探讨慢性前列腺炎/盆腔疼痛综合征(CP/CPPS)的发生与性激素及雄激素受体(AR)的关系。方法采用放射配体结合分析法检测78例CP/CPPS患者(CP/CPPS组)和32例正常对照者(对照组)外周血白细胞AR,同时采用放射免疫法检测患者血清睾酮(T)和雌二醇(E2)水平。结果CP/CPPS组与对照组血清E2和T水平无差异,外周血白细胞AR含量CP/CPPS组低于对照组(P<0.01),CP/CPPS组E2/T高于对照组,P<0.05。CP/CPPS患者CP症状指数评分(CPS I)与白细胞AR含量呈负相关(r=-0.512,P<0.01。结论CP/CPPS的发生、发展与E2、T比例失调及白细胞AR下调有密切的关系。     

A physiological role for androgen actions in the absence of androgen receptor DNA binding activity

We tested the hypothesis that androgens have physiological actions via non-DNA binding-dependent androgen receptor (AR) signaling pathways in males, using our genetically modified mice that express a mutant AR with deletion of the 2nd zinc finger of the DNA binding domain (AR^ΔZF2) that cannot bind DNA. In cultured genital skin fibroblasts, the mutant AR^ΔZF2 has normal ligand binding ability, phosphorylates ERK-1/2 in response to 1 min DHT treatment (blocked by the AR antagonist bicalutamide), but has reduced androgen-dependent nuclear localization compared to wildtype (WT). AR^ΔZF2 males have normal baseline ERK-1/2 phosphorylation, with a 1.5-fold increase in Akt phosphorylation in AR^ΔZF2 muscle vs WT. To identify physiological actions of non-DNA binding-dependent AR signaling, AR^ΔZF2 males were treated for 6 weeks with dihydrotestosterone (DHT). Cortical bone growth was suppressed by DHT in AR^ΔZF2 mice (6% decrease in periosteal and 7% decrease in medullary circumference vs untreated AR^ΔZF2 males). In conclusion, these data suggest that non-DNA binding dependent AR actions suppress cortical bone growth, which may provide a mechanism to fine-tune the response to androgens in bone.     

Functional and structural analysis of R607Q and R608K androgen receptor substitutions associated with male breast cancer

We previously described an androgen receptor (AR) point mutation located in the DNA-binding domain (DBD), adjacent to another AR substitution. Both were observed in two unrelated families with male breast cancer (MBC) and partial androgen insensitivity syndrome. This work was designed to determine the potential role of these two residues by in vitro study of the consequences of these two substitutions on biological functions and their structural impact at the atomic level. Mutant ARs revealed normal androgen-binding affinities and weaker DNA binding to an isolated androgen-responsive element. In cotransfection assays the mutant ARs displayed a reduced transactivation efficiency at 0.3·10⁻¹⁰ M. Neither binding to an estrogen-responsive element nor transactivation efficiency of an ERE reporter gene was observed. Molecular modeling revealed that Arg607 and Arg608 were partially surface-exposed and located in adjacent areas in the AR-DBD complex with DNA. This is in favor of a protein–protein interaction. It is conceivable that such an interaction could be affected by mutation of one of these two arginines.     

A surface on the androgen receptor that allosterically regulates coactivator binding

Current approaches to inhibit nuclear receptor (NR) activity target the hormone binding pocket but face limitations. We have proposed that inhibitors, which bind to nuclear receptor surfaces that mediate assembly of the receptor's binding partners, might overcome some of these limitations. The androgen receptor (AR) plays a central role in prostate cancer, but conventional inhibitors lose effectiveness as cancer treatments because anti-androgen resistance usually develops. We conducted functional and x-ray screens to identify compounds that bind the AR surface and block binding of coactivators for AR activation function 2 (AF-2). Four compounds that block coactivator binding in solution with IC(50) approximately 50 microM and inhibit AF-2 activity in cells were detected: three nonsteroidal antiinflammatory drugs and the thyroid hormone 3,3',5-triiodothyroacetic acid. Although visualization of compounds at the AR surface reveals weak binding at AF-2, the most potent inhibitors bind preferentially to a previously unknown regulatory surface cleft termed binding function (BF)-3, which is a known target for mutations in prostate cancer and androgen insensitivity syndrome. X-ray structural analysis reveals that 3,3',5-triiodothyroacetic acid binding to BF-3 remodels the adjacent interaction site AF-2 to weaken coactivator binding. Mutation of residues that form BF-3 inhibits AR function and AR AF-2 activity. We propose that BF-3 is a previously unrecognized allosteric regulatory site needed for AR activity in vivo and a possible pharmaceutical target.     

Amplification of exons 4 and 5 of androgen receptor gene by testosterone in aged female mouse brain cortex

We have investigated the effect of testosterone on theamplification of androgen receptor (AR) gene in the brain cortex ofaging female mice. For this purpose, high molecular weight (HMW) DNApurified from the brain cortex of intact, gonadectomized, testosterone-and estradiol-treated adult and old female mice was digested withdifferent restriction enzymes and used for Southern hybridization with³²P-labeled AR cDNA fragments representing different domainsof AR. The results reveal that only exons 4 and 5 corresponding toamino-terminal part of the hormone binding domain of AR are amplified intestosterone-treated old female but not in adult mice. Densitometricanalysis further shows that testosterone increases the copy number ofexons 4 and 5 of mouse AR gene by four-fold. Reprobing of slot blotswith estrogen receptor and cathepsin D cDNA as probes supports theobservation that amplification occurs only in AR gene. The tissuespecificity is also confirmed when the slot blot hybridization of mouseliver HMW DNA with AR cDNA fails to show similar amplification. As therestriction map analysis of Southern blots does not show restrictionfragment length polymorphism, the possibility of structuralrearrangement leading to amplification of AR gene is ruled out. Thus ourresults suggest that the in vivo induction of mouse AR geneamplification by testosterone is tissue- and age-specific, and mightcontribute to the progress of genetic instability in the brain of agedfemale mice.