Systematic and functional characterization of novel androgen receptor variants arising from alternative splicing in the ligand-binding domain.
The presence of intact ligand-binding domain (LBD) ensures the strict androgen-dependent regulation of androgen receptor (AR): binding of androgen induces structural reorganization of LBD resulting in release of AR from HSP90, suppression of nuclear export which otherwise dominates over import and nuclear translocation of AR as a transcription factor. Thus, loss or defects of the LBD abolish constraint from un-liganded LBD as exemplified by constitutively active AR variants (AR-Vs), which are associated with emerging resistance mechanism to anti-AR therapy in castration-resistant prostate cancer (mCRPC). Recent analysis of the AR splicing landscapes revealed mCRPC harboring multiple AR-Vs with diverse patterns of inclusion/exclusion of exons (exons 4-8) corresponding to LBD to produce namely exon-skipping variants. In silico construction for these AR-Vs revealed four novel AR-Vs having unique features: Exclusion of specified exons introduces a frameshift in variants v5es, v6es and v7es. ARv56es maintains the reading frame resulting in the inclusion of the C-terminal half of the LBD. We systematically characterized these AR-Vs regarding their subcellular localization, affinity for HSP90 and transactivation capability. Notably, ARv5es was free from HSP90, exclusively nuclear, and constitutively active similarly as previously reported for v567es. In contrast, v6es and v7es were similar in that they are cytoplasmic, transcriptionally inactive and bind HSP90, ARv56es was present in both nucleus and cytoplasm, does not bind HSP90 and is transcriptionally inactive. Converting these transcriptionally inactive AR-Vs into active forms, we identified the two separate elements that allosterically suppress otherwise constitutively active AR-Vs; one in exon 5 for v6es and v7es and the other in exon 8 for v56es. Our findings identify a novel constitutively active AR-V, ARv5es and establish a method to predict potential activities of AR-Vs carrying impaired LBD.
Department of Medicine, University of Washington, Seattle, WA, USA.
T
T
Uo
Computational Biology Program, Public Health Sciences Division and Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
H
H
Dvinge
Department of Medicine, University of Washington, Seattle, WA, USA.
C C
CC
Sprenger
Computational Biology Program, Public Health Sciences Division and Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
R K
RK
Bradley
Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
P S
PS
Nelson
Department of Medicine, University of Washington, Seattle, WA, USA.
S R
SR
Plymate
D017398:Alternative Splicing; D002460:Cell Line; D005091:Exons; D000072669:Gene Editing; D015870:Gene Expression; D017930:Genes, Reporter; D056426:Genetic Loci; D006801:Humans; D042541:Intracellular Space; D007438:Introns; D008024:Ligands; D059365:Nonsense Mediated mRNA Decay; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics*; D021381:Protein Transport; D011944:Receptors, Androgen / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D008024:Ligands; D011944:Receptors, Androgen
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