DNA dependent recruitment of DDX17 and other interacting proteins by the human androgen receptor.

An oligonucleotide-based assay (OBA) was used to identify novel co-factors that can be recruited by the deoxyribonucleic acid (DNA)-bound androgen receptor (AR). Nuclear extracts obtained from LNCaP cells, after incubation with R1881, were incubated with biotinylated oligonucleotides bound to streptavidin coated beads. The oligonucleotides contain 3 copies in tandem of the androgen responsive element ARE1 from the prostate specific antigen (PSA) gene promoter. As control incubation, a scrambled version of the tandem ARE1 was used. Immunoblots of the eluents revealed that the AR was bound to the ARE1 oligonucleotide and to a much lesser extent to the scrambled oligonucleotide. Proteins eluted from the oligonucleotides, were separated on a 5-15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gradient gel, followed by identification using mass spectrometry. Identified proteins were scored for having one or more of the following known properties: nuclear localization, involved in transcription regulation, involvement in steroid hormone receptor (SHR) function, or specifical involvement in AR function. A total number of 85 nuclear proteins were found in two separate OBAs. Based on peptide counting, we found enrichment of 7 proteins eluted from the ARE1 oligonucleotide, compared to the scrambled oligonucleotide. Taken together with the obtained scores, these proteins are considered putative AR co-factors. One of these proteins, DDX17, is known to be a co-factor for estrogen receptor alpha (ERalpha), but has never been associated with AR function. The results indicate that the ARE oligonucleotide-based assay may allow enrichment of new candidate DNA-bound AR interacting proteins.

A novel mutation F826L in the human androgen receptor in partial androgen insensitivity syndrome; increased NH2-/COOH-terminal domain interaction and TIF2 co-activation.

A novel mutation F826L located within the ligand binding domain (LBD) of the human androgen receptor (AR) was investigated. This mutation was found in a boy with severe penoscrotal hypospadias (classified as 46,XY DSD). The AR mutant F826L appeared to be indistinguishable from the wild-type AR, with respect to ligand binding affinity, transcriptional activation of MMTV-luciferase and ARE2-TATA-luciferase reporter genes, protein level in genital skin fibroblasts (GSFs), and sub-cellular distribution in transfected cells. However, an at least two-fold higher NH2-/COOH-terminal domain interaction was found in luciferase and GST pull-down assays. A two-fold increase was also observed for TIF2 (transcription intermediary factor 2) co-activation of the AR F826L COOH-terminal domain. This increase could not be explained by a higher stability of the mutant protein, which was within wild-type range. Repression of transactivation by the nuclear receptor co-repressor (N-CoR) was not affected by the AR F826L mutation. The observed properties of AR F826L would be in agreement with an increased activity rather than with a partial defective AR transcriptional activation. It is concluded that the penoscrotal hypospadias in the present case is caused by an as yet unknown mechanism, which still may involve the mutant AR.

Phosphorylation of androgen receptor isoforms.

Phosphorylation of the human AR (androgen receptor) is directly correlated with the appearance of at least three AR isoforms on an SDS/polyacrylamide gel. However, it is still not clear to what extent phosphorylation is involved in the occurrence of isoforms, which sites are phosphorylated and what are the functions of these phosphosites. The human AR was expressed in COS-1 cells and AR phosphorylation was studied further by mutational analyses and by using reversed-phase HPLC and MS. The reversed-phase HPLC elution pattern of the three isoforms revealed that Ser-650 was phosphorylated constitutively. After de novo synthesis, only Ser-650 was phosphorylated in the smallest isoform of 110 kDa and both Ser-650 and Ser-94 were phosphorylated in the second isoform of 112 kDa. The hormone-induced 114 kDa isoform shows an overall increase in phosphorylation of all the isolated peptides. The activities of the Ser-Ala substitution mutant S650A (Ser-650-->Ala) was found to be identical with wild-type AR activation in four different cell lines and three different functional analyses, e.g. transactivation, N- and C-terminal-domain interaction and co-activation by transcriptional intermediary factor 2. This was also found for mutants S94A and S515A with respect to transactivation. However, the S515A mutation, which should eliminate phosphorylation of the potential mitogen-activated protein kinase site, Ser-515, resulted in an unphosphorylated form of the peptide containing Ser-650. This suggests that Ser-515 can modulate phosphorylation at another site. The present study shows that the AR isoform pattern from AR de novo synthesis is directly linked to differential phosphorylation of a distinct set of sites. After mutagenesis of these sites, no major change in functional activity of the AR was observed.