Molecular Mechanisms of Androgen Receptor Interactions

The androgen receptor (AR) mediates the effects of the male sex-steroid hormones (androgens), testosterone and 5?-dihydrotestosterone. Androgens are critical in the development and maintenance of male sexual characteristics. AR is a member of the steroid receptor ligand-inducible transcription factor family. The steroid receptor family is a subgroup of the nuclear receptor superfamily that also includes receptors for the active forms of vitamin A, vitamin D3, and thyroid hormones. Like all nuclear receptors, AR has a conserved modular structure consisting of a non-conserved amino-terminal domain (NTD), containing the intrinsic activation function 1, a highly conserved DNA-binding domain, and a conserved ligand-binding domain (LBD) that harbors the activation function 2. Each of these domains plays an important role in receptor function and signaling, either via intra- and inter-receptor interactions, interactions with specific DNA sequences, termed hormone response elements, or via functional interactions with domain-specific proteins, termed coregulators (coactivators and corepressors). Upon binding androgens, AR acquires a new conformational state, translocates to the nucleus, binds to androgen response elements, homodimerizes and recruits sequence-specific coregulatory factors and the basal transcription machinery. This set of events is required to activate gene transcription (expression). Gene transcription is a strictly modulated process that governs cell growth, cell homeostasis, cell function and cell death. Disruptions of AR transcriptional activity caused by receptor mutations and/or altered coregulator interactions are linked to a wide spectrum of androgen insensitivity syndromes, and to the pathogenesis of prostate cancer (CaP). The treatment of CaP usually involves androgen depletion therapy (ADT). ADT achieves significant clinical responses during the early stages of the disease. However, under the selective pressure of androgen withdrawal, androgen-dependent CaP can progress to an androgen-independent CaP. Androgen-independent CaP is invariably a more aggressive and untreatable form of the disease. Advancing our understanding of the molecular mechanisms behind the switch in androgen-dependency would improve our success of treating CaP and other AR related illnesses. This study evaluates how clinically identified AR mutations affect the receptor s transcriptional activity. We reveal that a potential molecular abnormality in androgen insensitivity syndrome and CaP patients is caused by disruptions of the important intra-receptor NTD/LBD interaction. We demonstrate that the same AR LBD mutations can also disrupt the recruitment of the p160 coactivator protein GRIP1. Our investigations reveal that 30% of patients with advanced, untreated local CaP have somatic mutations that may lead to increases in AR activity. We report that somatic mutations that activate AR may lead to early relapse in ADT. Our results demonstrate that the types of ADT a CaP patient receives may cause a clustering of mutations to a particular region of the receptor. Furthermore, the mutations that arise before and during ADT do not always result in a receptor that is more active, indicating that coregulator interactions play a pivotal role in the progression of androgen-independent CaP. To improve CaP therapy, it is necessary to identify critical coregulators of AR. We screened a HeLa cell cDNA library and identified small carboxyl-terminal domain phosphatase 2 (SCP2). SCP2 is a protein phosphatase that directly interacts with the AR NTD and represses AR activity. We demonstrated that reducing the endogenous cellular levels of SCP2 causes more AR to load on to the prostate specific antigen (PSA) gene promoter and enhancer regions. Additionally, under the same conditions, more RNA polymerase II was recruited to the PSA promoter region and overall there was an increase in androgen-dependent transcription of the PSA gene, revealing that SCP2 could play a role in the pathogenesis of CaP.

Orphan nuclear receptor subfamily NR4A their interplay with other nuclear receptors and functions in osteoblasts

Nurr1, NGFI-B and Nor1 (NR4A2, NR4A1 and NR4A3, respectively) belong to the NR4A subfamily of nuclear receptors. The NR4A receptors are orphan nuclear receptors which means that activating or repressing ligands for these receptors have not been found. NR4A expression is rapidly induced in response to various stimuli including growth factors and the parathyroid hormone (PTH). The studies concerning the NR4A receptors in the central nervous system have demonstrated that they have a major role in the development and function of the dopaminergic neurons of the midbrain and in regulating hypothalamus-pituitary-adrenal-axis. However, the peripheral functions of the NR4A family are largely unknown. Cultured mouse primary osteoblasts, a preosteoblastic cell line and several osteoblastic cell lines were used to investigate the role of NR4A receptors in osteoblasts. NR4A receptors were shown to directly bind to and activate the promoter of the osteopontin gene (OPN) in osteoblastic cells, thus regulating its expression. OPN is a major bone matrix protein expressed throughout the differentiation of preosteoblastic cells into osteoblasts. The activation of the OPN promoter was shown to be dependent on the activation function-1 located in the N-terminal part of Nurr1 and to occur in both monomeric and RXR heterodimeric forms of NR4A receptors. Furthermore, PTH was shown to upregulate OPN expression through the NR4A family. It was also demonstrated that the fibroblast growth factor-8b (FGF-8b) induces the expression of NR4A receptors in osteoblasts as immediate early genes. This induction involved phosphatidylinositol-3 kinase, protein kinase C, and mitogen activated protein kinase, which are all major pathways of FGF signalling. Nurr1 and NGFI-B were shown to induce the proliferation of preosteoblastic cells and to reduce their apoptosis. FGF-8b was shown to stimulate the proliferation of osteoblastic cells through the NR4A receptors. These results suggest that NR4A receptors have a role both in the differentiation of osteoblasts and in the proliferation and apoptosis of preosteoblast. The NR4A receptors were found to bind to the same response element on OPN as the members of the NR3B family of orphan receptors do. Mutual repression was observed between the NR4A receptors and the NR3B receptors. This repression was shown to be dependent on the DNA-binding domains of both receptor families, but to result neither from the competition of DNA binding nor from the competition for coactivators. As the repression was dependent on the relative expression levels of the NR4As and NR3Bs, it seems likely that the ratio of the receptors mediates their activity on their response elements. Rapid induction of the NR4As in response to various stimuli and differential expression of the NR3Bs can effectively control the gene activation by the NR4A receptors. NR4A receptors can bind DNA as monomers, and Nurr1 and NGFI-B can form permissive heterodimers with the retinoid X receptor (RXR). Permissive heterodimers can be activated with RXR agonists, unlike non-permissive heterodimers, which are formed by RXR and retinoic acid receptor or thyroid hormone receptor (RAR and TR, respectively). Non-permissive heterodimers can only be activated by the agonists of the heterodimerizing partner. The mechanisms behind differential response to RXR agonists have remained unresolved. As there are no activating or repressing ligands for the NR4A receptors, it would be important to find out, how they are regulated. Permissiviness of Nurr1/RXR heterodimers was linked to the N-terminal part of Nurr1 ligand-binding domain. This region has previously been shown to mediate the interaction between NRs and corepressors. Non-permissive RAR and TR, permissive Nurr1 and NGFI-B, and RXR were overexpressed with corepressors silencing mediator for retinoic acid and thyroid hormone receptors (SMRT), and with nuclear receptor corepressor in several cell lines. Nurr1 and NGFI-B were found to be repressed by SMRT. The interaction of RXR heterodimers with corepressors was weak in permissive heterodimers and much stronger in non-permissive heterodimers. Non-permissive heterodimers also released corepressors only in response to the agonist of the heterodimeric partner of RXR. In the permissive Nurr1/RXR heterodimer, however, SMRT was released following the treatment with RXR agonists. Corepressor release in response to ligands was found to differentiate permissive heterodimers from non-permissive ones. Corepressors were thus connected to the regulation of NR4A functions. In summary, the studies presented here linked the NR4A family of orphan nuclear receptors to the regulation of osteoblasts. Nurr1 and NGFI-B were found to control the proliferation and apoptosis of preosteoblasts. The studies also demonstrated that cross-talk with the NR3B receptors controls the activity of these orphan receptors. The results clarified the mechanism of permissiviness of RXR-heterodimers. New information was obtained on the regulation and functions of NR4A receptors, for which the ligands are unknown.

A genome-wide association study of caffeine-related sleep disturbance: Confirmation of a role for a common variant in the adenosine receptor

OBJECTIVES To identify common genetic variants that predispose to caffeine-induced insomnia and to test whether genes whose expression changes in the presence of caffeine are enriched for association with caffeine-induced insomnia. DESIGN A hypothesis-free, genome-wide association study. SETTING Community-based sample of Australian twins from the Australian Twin Registry. PARTICIPANTS After removal of individuals who said that they do not drink coffee, a total of 2,402 individuals from 1,470 families in the Australian Twin Registry provided both phenotype and genotype information. MEASUREMENTS AND RESULTS A dichotomized scale based on whether participants reported ever or never experiencing caffeine-induced insomnia. A factor score based on responses to a number of questions regarding normal sleep habits was included as a covariate in the analysis. More than 2 million common single nucleotide polymorphisms (SNPs) were tested for association with caffeine-induced insomnia. No SNPs reached the genome-wide significance threshold. In the analysis that did not include the insomnia factor score as a covariate, the most significant SNP identified was an intronic SNP in the PRIMA1 gene (P = 1.4 x 10(-)(6), odds ratio = 0.68 [0.53 - 0.89]). An intergenic SNP near the GBP4 gene on chromosome 1 was the most significant upon inclusion of the insomnia factor score into the model (P = 1.9 x 10(-)(6), odds ratio = 0.70 [0.62 - 0.78]). A previously identified association with a polymorphism in the ADORA2A gene was replicated. CONCLUSIONS Several genes have been identified in the study as potentially influencing caffeine-induced insomnia. They will require replication in another sample. The results may have implications for understanding the biologic mechanisms underlying insomnia.

Identification of the semaphorin receptor PLXNA2 as a candidate for susceptibility to schizophrenia

The discovery of genetic factors that contribute to schizophrenia susceptibility is a key challenge in understanding the etiology of this disease. Here, we report the identification of a novel schizophrenia candidate gene on chromosome 1q32, plexin A2 (PLXNA2), in a genome-wide association study using 320 patients with schizophrenia of European descent and 325 matched controls. Over 25,000 single-nucleotide polymorphisms (SNPs) located within approximately 14,000 genes were tested. Out of 62 markers found to be associated with disease status, the most consistent finding was observed for a candidate locus on chromosome 1q32. The marker SNP rs752016 showed suggestive association with schizophrenia (odds ratio (OR) = 1.49, P = 0.006). This result was confirmed in an independent case-control sample of European Americans (combined OR = 1.38, P = 0.035) and similar genetic effects were observed in smaller subsets of Latin Americans (OR = 1.26) and Asian Americans (OR = 1.37). Supporting evidence was also obtained from two family-based collections, one of which reached statistical significance (OR = 2.2, P = 0.02). High-density SNP mapping showed that the region of association spans approximately 60 kb of the PLXNA2 gene. Eight out of 14 SNPs genotyped showed statistically significant differences between cases and controls. These results are in accordance with previous genetic findings that identified chromosome 1q32 as a candidate region for schizophrenia. PLXNA2 is a member of the transmembrane semaphorin receptor family that is involved in axonal guidance during development and may modulate neuronal plasticity and regeneration. The PLXNA2 ligand semaphorin 3A has been shown to be upregulated in the cerebellum of individuals with schizophrenia. These observations, together with the genetic results, make PLXNA2 a likely candidate for the 1q32 schizophrenia susceptibility locus.

Cross-sectional study of a microsatellite marker in the low density lipoprotein receptor gene in obese normotensives

1. The low density lipoprotein receptor is an important regulator of serum cholesterol which may have implications for the development of both hypertension and obesity. In this study, genotypes for a low density lipoprotein receptor gene (LDLR) dinucleotide polymorphism were determined in both lean and obese normotensive populations. 2. In previous cross-sectional association studies an ApaLI and a HincII polymorphism for LDLR were shown to be associated with obesity in essential hypertensives. However, these polymorphisms did not show an association with obesity in normotensives. 3. In contrast, this study reports that preliminary results for an LDLR microsatellite marker, located more towards the 3' end of the gene, show a significant association with obesity in the normotensive population studied. These results indicate that LDLR could play an important role in the development of obesity, which might be independent of hypertension.

Cross Talk between Receptor Guanylyl Cyclase C and c-src Tyrosine Kinase Regulates Colon Cancer Cell Cytostasis

Increased activation of c-src seen in colorectal cancer is an indicator of a poor clinical prognosis, suggesting that identification of downstream effectors of c-src may lead to new avenues of therapy. Guanylyl cyclase C (GC-C) is a receptor for the gastrointestinal hormones guanylin and uroguanylin and the bacterial heat-stable enterotoxin. Though activation of GC-C by its ligands elevates intracellular cyclic GMP (cGMP) levels and inhibits cell proliferation, its persistent expression in colorectal carcinomas and occult metastases makes it a marker for malignancy. We show here that GC-C is a substrate for inhibitory phosphorylation by c-src, resulting in reduced ligand-mediated cGMP production. Consequently, active c-src in colonic cells can overcome GC-C-mediated control of the cell cycle. Furthermore, docking of the c-src SH2 domain to phosphorylated GC-C results in colocalization and further activation of c-src. We therefore propose a novel feed-forward mechanism of activation of c-src that is induced by cross talk between a receptor GC and a tyrosine kinase. Our findings have important implications in understanding the molecular mechanisms involved in the progression and treatment of colorectal cancer.

Inhibitory action of norepinephrine on the induction of tryptophan-2,3-dioxygenase by cortisol: Mediation by the I-receptor

Induction of hepatic tryptophan-2,3-dioxygenase in rats by cortisol or corticosterone was inhibited on treatment with norepinephrine. The I-adrenergic blockers showed a small potentiating effect of the norepinephrine-mediated inhibition. The I-adrenergic blockers significantly reversed this inhibition, suggesting that norepinephrine acts Image the I-receptor in inhibition of the cortisol-mediated induction of this enzyme.

The Linker Region in Receptor Guanylyl Cyclases Is a Key Regulatory Module MUTATIONAL ANALYSIS OF GUANYLYL CYCLASE C

Receptor guanylyl cyclases are multidomain proteins, and ligand binding to the extracellular domain increases the levels of intracellular cGMP. The intracellular domain of these receptors is composed of a kinase homology domain (KHD), a linker of similar to 70 amino acids, followed by the C-terminal guanylyl cyclase domain. Mechanisms by which these receptors are allosterically regulated by ligand binding to the extracellular domain and ATP binding to the KHD are not completely understood. Here we examine the role of the linker region in receptor guanylyl cyclases by a series of point mutations in receptor guanylyl cyclase C. The linker region is predicted to adopt a coiled coil structure and aid in dimerization, but we find that the effects of mutations neither follow a pattern predicted for a coiled coil peptide nor abrogate dimerization. Importantly, this region is critical for repressing the guanylyl cyclase activity of the receptor in the absence of ligand and permitting ligand-mediated activation of the cyclase domain. Mutant receptors with high basal guanylyl cyclase activity show no further activation in the presence of non-ionic detergents, suggesting that hydrophobic interactions in the basal and inactive conformation of the guanylyl cyclase domain are disrupted by mutation. Equivalent mutations in the linker region of guanylyl cyclase A also elevated the basal activity and abolished ligand-and detergent-mediated activation. We, therefore, have defined a key regulatory role for the linker region of receptor guanylyl cyclases which serves as a transducer of information from the extracellular domain via the KHD to the catalytic domain.

A comparative structural bioinformatics analysis of the insulin receptor family ectodomain based on phylogenetic information

The insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R) and the insulin receptor-related receptor (IRR) are covalently-linked homodimers made up of several structural domains. The molecular mechanism of ligand binding to the ectodomain of these receptors and the resulting activation of their tyrosine kinase domain is still not well understood. We have carried out an amino acid residue conservation analysis in order to reconstruct the phylogeny of the IR Family. We have confirmed the location of ligand binding site 1 of the IGF1R and IR. Importantly, we have also predicted the likely location of the insulin binding site 2 on the surface of the fibronectin type III domains of the IR. An evolutionary conserved surface on the second leucine-rich domain that may interact with the ligand could not be detected. We suggest a possible mechanical trigger of the activation of the IR that involves a slight ‘twist’ rotation of the last two fibronectin type III domains in order to face the likely location of insulin. Finally, a strong selective pressure was found amongst the IRR orthologous sequences, suggesting that this orphan receptor has a yet unknown physiological role which may be conserved from amphibians to mammals.

Diaryl dihydropyrazole-3-carboxamides with significant in vivo antiobesity activity related to CB1 receptor antagonism: Synthesis, biological evaluation, and molecular modeling in the homology model

A number of analogues of diaryl dihydropyrazole-3-carboxamides have been synthesized. Their activities were evaluated for appetite suppression and body weight reduction in animal models. Depending on the chemical modification of the selected dihydropyrazole scaffold, the lead compoundsthe bisulfate salt of (±)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid morpholin-4-ylamide 26 and the bisulfate salt of (−)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid morpholin-4-ylamide 30showed significant body weight reduction in vivo, which is attributed to their CB1 antagonistic activity and exhibited a favorable pharmacokinetic profile. The molecular modeling studies also showed interactions of two isomers of (±)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid morpholin-4-ylamide 9 with CB1 receptor in the homology model similar to those of N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (rimonabant) 1 and 4S-(−)-3-(4-chlorophenyl)-N-methyl-N‘-[(4-chlorophenyl)-sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamidine (SLV-319) 2.

Mapping of assembled epitopes with microgram quantities of antigen: Identification of an epitope at the receptor binding region of human follicle stimulating hormone

Identification of epitopes by modification studies has been reported by us recently. The method requires milligram quantities of antigen and since several proteins are not available in large quantities they are not amenable for such an investigation. One such protein is human follicle stimulating hormone (hFSH) whose mapping of epitopes is of importance in reproductive biology. Here we report a method that uses microgram quantities of hFSH to map a beta-specific epitope located at the receptor binding region. This identification has also been validated by the chemical modification method using heterologous antigen ovine follicle stimulating hormone (oFSH).

Blockade of PD-1/PD-L1 promotes adoptive T-Cell immunotherapy in a tolerogenic environment

Adoptive cellular immunotherapy using in vitro expanded CD8+ T cells shows promise for tumour immunotherapy but is limited by eventual loss of function of the transferred T cells through factors that likely include inactivation by tolerogenic dendritic cells (DC). The coinhibitory receptor programmed death-1 (PD-1), in addition to controlling T-cell responsiveness at effector sites in malignancies and chronic viral diseases is an important modulator of dendritic cell-induced tolerance in naive T cell populations. The most potent therapeutic capacity amongst CD8+ T cells appears to lie within Tcm or Tcm-like cells but memory T cells express elevated levels of PD-1. Based on established trafficking patterns for Tcm it is likely Tcm-like cells interact with lymphoid-tissue DC that present tumour-derived antigens and may be inherently tolerogenic to develop therapeutic effector function. As little is understood of the effect of PD-1/PD-L1 blockade on Tcm-like CD8+ T cells, particularly in relation to inactivation by DC, we explored the effects of PD-1/PD-L1 blockade in a mouse model where resting DC tolerise effector and memory CD8+ T cells. Blockade of PD-1/PDL1 promoted effector differentiation of adoptively-transferred Tcm-phenotype cells interacting with tolerising DC. In tumour-bearing mice with tolerising DC, effector activity was increased in both lymphoid tissues and the tumour-site and anti-tumour activity was promoted. Our findings suggest PD-1/PD-L1 blockade may be a useful adjunct for adoptive immunotherapy by promoting effector differentiation in the host of transferred Tcmlike cells. © 2015 Blake et al.

Expression of glucose transporter isoforms and the insulin receptor during hamster preimplantation embryo development

During preimplantation development, embryos of many species are known to express up to five isoforms of the facilitative glucose transporter proteins (GLUT). Development of hamster blastocysts is inhibited by glucose. We therefore investigated GLUT isoform and insulin receptor (IR) expression in hamster preimplantation embryos cultured in glucose-free medium from the 8-cell stage onwards. We show that GLUT1, 3 and 8 mRNA are constitutively expressed from the 8-cell to the blastocyst stage. The IR is expressed from the morula stage onwards. Messenger RNA of the insulin-responsive GLUT4 was not detected at any stage. GLUT1 and 3 were localised by immunocytochemistry. GLUT1 was expressed in both embryoblast and trophoblast, in the latter, mainly in basal and lateral membranes directed towards the blastocoel. and embryoblast. GLUT3 was exclusively localised in the apical. membrane of trophoblast cells. We show that hamster preimplantation embryos express several GLUT isoforms thus closely resembling embryos of other mammalian species. Despite endogenous IR expression, the insulin-sensitive isoform GLUT4 was not expressed, indicating that the insulin-mediated glucose uptake known from classical insulin target cells may not be relevant for hamster blastocysts.

Structure and function of GDNF receptor alpha splice variants

The growth factors of the glial cell line-derived neurotrophic factor (GDNF) family consisting of GDNF, neurturin (NRTN), artemin (ARTN) and persephin (PSPN), are involved in the development, differentiation and maintenance of many types of neurons. They also have important functions outside the nervous system in the development of kidney, testis and thyroid gland. Each of these GFLs preferentially binds to one of the glycosylphosphatidylinositol (GPI)-anchored GDNF family receptors α (GFRα). GDNF binds to GFRα1, NRTN to GFRα2, ARTN to GFRα3 and PSPN to GFRα4. The GFLs in the complex with their cognate GFRα receptors all bind to and signal through the receptor tyrosine kinase RET. Alternative splicing of the mouse GFRα4 gene yields three splice isoforms. These had been described as putative GPI-anchored, transmembrane and soluble forms. My goal was to characterise the function of the different forms of mouse GFRα4. I firstly found that the putative GPI-anchored GFRα4 (GFRα4-GPI) is glycosylated, membrane-bound, GPI-anchored and interacts with PSPN and RET. We also showed that mouse GFRα4-GPI mediates PSPN-induced phosphorylation of RET, promotes PSPN-dependent neuronal differentiation of the rat pheochromocytoma cell line PC6-3 and PSPN-dependent survival of cerebellar granule neurons (CGN). However, although this receptor can mediate PSPN-signalling and activate RET, GFRα4-GPI does not recruit RET into lipid rafts. The recruitment of RET into lipid rafts has previously been thought to be a crucial event for GDNF- and GFL-mediated signalling via RET. I secondly demonstrated that the putative transmembrane GFRα4 (GFRα4-TM) is indeed a real transmembrane GFRα4 protein. Although it has a weak binding capacity for PSPN, it can not mediate PSPN-dependent phosphorylation of RET, neuronal differentiation or survival. These data show that GFRα4-TM is inactive as a receptor for PSPN. Surprisingly, GFRα4-TM can negatively regulate PSPN-mediated signalling via GFRα4-GPI. GFRα4-TM interacts with GFRα4-GPI and blocks PSPN-induced phosphorylation of RET, neuronal differentiation as well as survival. Taken together, our data show that GFRα4-TM may act as a dominant negative inhibitor of PSPN-mediated signaling. The most exciting part of my work was the finding that the putative soluble GFRα4 (GFRα4-sol) can form homodimers and function as an agonist of the RET receptor. In the absence of PSPN, GFRα4-sol can promote the phosphorylation of RET, trigger the activation of the PI-3K/AKT pathway, induce neuronal differentiation and support the survival of CGN. Our findings are in line with a recent publication showing the GFRα4-sol might contribute to the inherited cancer syndrome multiple endocrine neoplasia type 2. Our data provide an explanation to how GFRα4-sol may cause or modify the disease. Mammalian GFRα4 receptors all lack the first Cys-rich domain which is present in other GFRα receptors. In the final part of my work I have studied the function of this particular domain. I created a truncated GFRα1 construct lacking the first Cys-rich domain. Using binding assays in both cellular and cell-free systems, phosphorylation assays with RET, as well as neurite outgrowth assays, we found that the first Cys-rich domain contributes to an optimal function of GFRα1, by stabilizing the interaction between GDNF and GFRα1.