8 research outputs found
Structural features discriminate androgen receptor N/C terminal and coactivator interactions
Human androgen receptor (AR) transcriptional activity involves interdomain and coactivator interactions with the agonist-bound AR ligand binding domain (LBD). Structural determinants of the AR NH2- and carboxyl-terminal interaction between the AR NH2-terminal FXXLF motif and activation function 2 (AF2) in the LBD were shown previously by crystallography. In this report, we provide evidence for a region in AR LBD helix 12 outside the AF2 binding cleft that facilitates interactions with the FXXLF and LXXLL motifs. Mutagenesis of glutamine 902 to alanine in AR LBD helix 12 (Q902A) disrupted AR FXXLF motif binding to AF2, but enhanced coactivator LXXLL motif binding. Functional compensation for defective FXXLF motif binding by AR-Q902A was suggested by the slower dissociation rate of bound androgen. Functional importance of glutamine 902 was indicated by the charged residue germline mutation Q902R that caused partial androgen insensitivity, and a similar somatic mutation Q902K reported in prostate cancer, both of which increased the androgen dissociation rate and decreased AR transcriptional activity. High affinity equilibrium androgen binding was retained by alanine substitution mutations at Tyr-739 in AR LBD helix 5 or Lys-905 in helix 12 structurally adjacent to AF2, whereas transcriptional activity decreased and the androgen dissociation increased. Deleterious effects of these loss of function mutations were rescued by the helix stabilizing AR prostate cancer somatic mutation H874Y. Sequence NH2-terminal to the AR FXXLF motif contributed to the AR NH2- and carboxyl-terminal interaction based on greater AR-2-30 FXXLF motif peptide binding to the agonist-bound AR LBD than a shorter AR-20-30 FXXLF motif peptide. We conclude that helix 12 residues outside the AF2 binding cleft modulate AR transcriptional activity by providing flexibility to accommodate FXXLF or LXXLL motif binding
An Androgen Receptor NH 2 -terminal Conserved Motif Interacts with the COOH Terminus of the Hsp70-interacting Protein (CHIP)
The NH2-terminal sequence of steroid receptors is highly variable between different receptors and in the same receptor from different species. In this study, a primary sequence homology comparison identified a 14-amino acid NH2-terminal motif of the human androgen receptor (AR) that is common to AR from all species reported, including the lower vertebrates. The evolutionarily conserved motif is unique to AR, with the exception of a partial sequence in the glucocorticoid receptor of higher species. The presence of the conserved motif in AR and the glucocorticoid receptor and its absence in other steroid receptors suggests convergent evolution. The function of the AR NH2-terminal conserved motif was suggested from a yeast two-hybrid screen that identified the COOH terminus of the Hsp70-interacting protein (CHIP) as a binding partner. We found that CHIP functions as a negative regulator of AR transcriptional activity by promoting AR degradation. In support of this, two mutations in the AR NH2-terminal conserved motif previously identified in the transgenic adenocarcinoma of mouse prostate model reduced the interaction between CHIP and AR. Our results suggest that the AR NH2-terminal domain contains an evolutionarily conserved motif that functions to limit AR transcriptional activity. Moreover, we demonstrate that the combination of comparative sequence alignment and yeast two-hybrid screening using short conserved peptides as bait provides an effective strategy to probe the structure-function relationships of steroid receptor NH2-terminal domains and other intrinsically unstructured transcriptional regulatory proteins
Androgen Receptor Exon 1 Mutation Causes Androgen Insensitivity by Creating Phosphorylation Site and Inhibiting Melanoma Antigen-A11 Activation of NH 2 - and Carboxyl-terminal Interaction-dependent Transactivation
Naturally occurring germ line mutations in the X-linked human androgen receptor (AR) gene cause incomplete masculinization of the external genitalia by disrupting AR function in males with androgen insensitivity syndrome. Almost all AR missense mutations that cause androgen insensitivity syndrome are located in the highly structured DNA and ligand binding domains. In this report we investigate the functional defect associated with an AR exon 1 missense mutation, R405S, that caused partial androgen insensitivity. The 46,XX heterozygous maternal carrier had a wild-type Arg-405 CGC allele but transmitted an AGC mutant allele coding for Ser-405. At birth, the 46,XY proband had a bifid scrotum, hypospadias, and micropenis consistent with clinical stage 3 partial androgen insensitivity. Androgen-dependent transcriptional activity of AR-R405S expressed in CV1 cells was less than wild-type AR and refractory in androgen-dependent AR NH2- and carboxyl interaction transcription assays that depend on the coregulator effects of melanoma antigen-A11. This mutation created a Ser-405 phosphorylation site evident by the gel migration of an AR-R405S NH2-terminal fragment as a double band that converted to the wild-type single band after treatment with λ-phosphatase. Detrimental effects of the R405S mutation were related to the proximity of the AR WXXLF motif 433WHTLF437 required for melanoma antigen-A11 and p300 to stimulate transcriptional activity associated with the AR NH2- and carboxyl-terminal interaction. We conclude that the coregulator effects of melanoma antigen-A11 on the AR NH2- and carboxyl-terminal interaction amplify the androgen-dependent transcriptional response to p300 required for normal human male sex development in utero
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
Assessment of Key Health and Wellness Indicators Among North Carolina Emergency Medical Service Providers
Structural features discriminate androgen receptor N/C terminal and coactivator interactions
Melanoma Antigen Gene Protein-A11 (MAGE-11) F-box Links the Androgen Receptor NH2-terminal Transactivation Domain to p160 Coactivators*
Androgen-dependent transcriptional activity by the androgen receptor (AR) and its coregulators is required for male reproductive development and function. In humans and other primates, melanoma antigen gene protein-A11 (MAGE-11) is an AR selective coregulator that increases AR transcriptional activity. Here we show that the interaction between AR and MAGE-11 is mediated by AR NH2-terminal FXXLF motif binding to a highly conserved MAGE-11 F-box in the MAGE homology domain, and is modulated by serum stimulation of mitogen-activated protein kinase phosphorylation of MAGE-11 Ser-174. The MAGE-11-dependent increase in AR transcriptional activity is mediated by a direct interaction between MAGE-11 and transcriptional intermediary factor 2 (TIF2) through the NH2-terminal region of TIF2, and by a MAGE-11 FXXIF motif interaction with an F-box-like region in activation domain 1 of TIF2. The results suggest that MAGE-11 functions as a bridging factor to recruit AR coactivators through a novel FXX(L/I)F motif-F-box interaction paradigm
Overview of physics results from MAST
Substantial advances have been made on the Mega Ampère Spherical Tokamak (MAST). The parameter range of the MAST confinement database has been extended and it now also includes pellet-fuelled discharges. Good pellet retention has been observed in H-mode discharges without triggering an ELM or an H/L transition during peripheral ablation of low speed pellets. Co-ordinated studies on MAST and DIII-D demonstrate a strong link between the aspect ratio and the beta scaling of H-mode energy confinement, consistent with that obtained when MAST data were merged with a subset of the ITPA database. Electron and ion ITBs are readily formed and their evolution has been investigated. Electron and ion thermal diffusivities have been reduced to values close to the ion neoclassical level. Error field correction coils have been used to determine the locked mode threshold scaling which is comparable to that in conventional aspect ratio tokamaks. The impact of plasma rotation on sawteeth has been investigated and the results have been well-modelled using the MISHKA-F code. Alfvén cascades have been observed in discharges with reversed magnetic shear. Measurements during off-axis NBCD and heating are consistent with classical fast ion modelling and indicate efficient heating and significant driven current. Central electron Bernstein wave heating has been observed via the O-X-B mode conversion process in special magnetically compressed plasmas. Plasmas with low pedestal collisionality have been established and further insight has been gained into the characteristics of filamentary structures at the plasma edge. Complex behaviour of the divertor power loading during plasma disruptions has been revealed by high resolution infra-red measurements