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Transcriptional activation and transient expression of the human androgen receptor
T Gao1, M Marcelli, M J McPhaul
1Department of Internal Medicine, The University of Texas Southwestern Medical Center at Dallas, 75235-8857, USA.
Abstract:
A series of cDNAs containing deletions within the open-reading frame of the human androgen receptor (AR) were constructed and transiently expressed in CV1 cells to investigate the effects of these alterations on the level of expression of the protein and on its capacity to activate a model reporter gene (MMTV-luciferase). The levels of AR expression were assayed using immunoblots made using an antibody directed at an epitope (amino acids 1-21) preserved in all of the deletions. Treatment of the transfected cells with androgen increased the level of normal or mutant AR approximately five-fold in all constructs in which the hormone-binding domain was intact. This finding indicates that an intact hormone-binding domain is necessary and sufficient for the androgen-dependent increase in AR levels. Contraction of expansion or the glutamine repeat or deletion of the glycine repeat in the amino terminus diminished the capacity of the mutant ARs to activate the MMTV luciferase gene. The presence of a large-scale deletion within the amino terminus (amino acid residues 96-483), abolished receptor function, and two smaller deletions (bounded by residues 80-93 and 245-485) within the amino terminus substantially impaired receptor function. As previously described, deletion of the hormone-binding domain (amino acids 708-917) resulted in a constitutively active receptor. Unexpectedly, the large-scale deletion within the amino terminus (amino acids 96-483), in combination with deletion of the carboxy terminus also produced a constitutively active receptor that was almost as active as ligand-activated normal AR. None of the alterations in AR function could be explained by changes in the level of AR expression and the function of some mutant receptors was even more defective when the relative levels of mutant ARs expressed was considered. These findings imply that interaction of the sequences within the amino- and carboxy-terminal portions of the AR, or proteins that interact with these segments, is critical for regulation of transcription by the AR.
Insights
Altering the human androgen receptor (AR) affects its function. Specific deletions in the amino or carboxy terminus impact AR
Area of Science:
- Molecular Biology
- Endocrinology
- Genetics
Background:
- The human androgen receptor (AR) plays a crucial role in gene regulation.
- Understanding AR structure-function relationships is vital for hormone-related research.
Purpose of the Study:
- To investigate the impact of deletions in the human androgen receptor (AR) on its expression and transcriptional activity.
- To identify specific domains critical for AR function and androgen-dependent regulation.
Main Methods:
- Construction and transient expression of AR cDNAs with defined deletions in CV1 cells.
- Assay of AR expression levels using immunoblots.
- Measurement of reporter gene activation (MMTV-luciferase) to assess AR transcriptional capacity.
Main Results:
- An intact hormone-binding domain is necessary and sufficient for androgen-dependent AR level increases.
- Deletions in the amino terminus, particularly large ones, significantly impair or abolish AR transcriptional activity.
- Deletion of the hormone-binding domain results in a constitutively active AR.
- A combined deletion in the amino and carboxy termini unexpectedly yields a highly active, constitutively active AR.
Conclusions:
- AR amino- and carboxy-terminal interactions are critical for transcriptional regulation.
- Mutant AR function defects are not explained by altered expression levels.
- Specific domains within the AR are essential for its proper function and response to androgens.