Generation of a Mammalian Cell Line Stably Expressing a Tetracycline-Regulated Epitope-Tagged Human Androgen Receptor: Implications for Steroid Hormone Receptor Research

Wang, Qianben; Fondell, Joseph D.

doi:10.1006/abio.2000.4960

Cited by 10 publications

(11 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The pGEX-2TK-AR-AF2 vector was constructed by PCR, generating BamHI and SmaI restriction sites at amino acids 622 and 919, respectively, and subsequently subcloning the fragment into the corresponding sites of pGEX-2TK (Amersham Biosciences). The pGEX-2TK-AR-AF1 construct was generated by PCR by generating BamHI/AflII sites at AR amino acids 1 and 170 and subcloning the corresponding fragment, together with a AflII/HindIII-blunted fragment from pSV-AR 0 (amino acids 171-564) (39), into BamHI/SmaIdigested pGEX-2TK. The androgen-responsive luciferase reporter genes pMMTV-Luc, pARE 2 -DS-Luc, and pPB(Ϫ285/ϩ32)-Luc were described previously (39).…”

Section: Methodsmentioning

confidence: 99%

“…The pGEX-2TK-AR-AF1 construct was generated by PCR by generating BamHI/AflII sites at AR amino acids 1 and 170 and subcloning the corresponding fragment, together with a AflII/HindIII-blunted fragment from pSV-AR 0 (amino acids 171-564) (39), into BamHI/SmaIdigested pGEX-2TK. The androgen-responsive luciferase reporter genes pMMTV-Luc, pARE 2 -DS-Luc, and pPB(Ϫ285/ϩ32)-Luc were described previously (39). The pGL3-promoter vector was obtained from Promega Corp.…”

Section: Methodsmentioning

confidence: 99%

“…We recently generated a HeLa-derived cell line (termed E19) that stably expresses a tetracycline-regulated FLAG-tagged human AR (f:AR), thereby rendering the cells responsive to androgens (39). As evidenced by our ability to purify f:AR in association with specific p160 proteins 2 and the coactivator CBP (39), the E19 line can serve as a useful tool for isolating AR-coregulatory factor complexes that assemble in vivo.…”

Section: Ar Interacts With Components Of the Trap-mediator Complex Inmentioning

confidence: 99%

See 2 more Smart Citations

A Coregulatory Role for the TRAP-Mediator Complex in Androgen Receptor-mediated Gene Expression

Wang

Sharma

Ren

et al. 2002

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Ar Interacts With Components Of the Trap-mediator Complex Inmentioning

confidence: 99%

See 1 more Smart Citation

A Coregulatory Role for the TRAP-Mediator Complex in Androgen Receptor-mediated Gene Expression

Wang

Sharma

Ren

et al. 2002

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

“…Human GRg is ubiquitously expressed, but only accounts for 5% of all GR transcripts (Rivers et al 1999, Stevens et al 2004a The addition of an arginine as in the human GRg or the marmoset monkey (see below) would create a new hydrogen bond and, therefore, allow a new 3-turn to be formed, which could affect affinity of receptor-DNA binding (Wickert & Selbig 2002). Indeed, the human GRg (Arg 453 ) is half maximally activated compared to wild-type with a similar EC 50 in an in vitro co-transfection assay with GR arg 453 and pMMTV-luciferase reporter plasmid in COS7 (African green monkey kidney) cells (Ray et al 1996, Wang & Fondell 2001. Alternative splicing comparable to that in human GRg is also seen in the cotton-top marmoset (Saguinus oedipus) and thus is of wider occurrence (Brandon et al 1991).…”

Section: Non-fish Vertebratesmentioning

confidence: 99%

Evolution of glucocorticoid receptors with different glucocorticoid sensitivity

Stolte¹,

Kemenade²,

Savelkoul³

et al. 2006

Journal of Endocrinology

148

View full text Add to dashboard Cite

Glucocorticoids (GCs) are commonly used to treat a variety of immune diseases. However, the efficacy of treatment is greatly influenced by an individual variation in sensitivity to GCs, which is caused by differences in the glucocorticoid receptor (GR). The variable receptor profile results from variations in the GR gene, or alternative splicing of the gene coded. We investigated the evolution of the GR gene by comparing genomic GR sequences of vertebrates. Exon length and amino acid sequence are conserved among all classes of vertebrates studied, which indicates strong evolutionary pressure on conservation of this gene. Interestingly, teleostean fishes have two different GR proteins. One of the duplicate fish GR genes has a nine-amino-acid insert in the DNA binding region that results from alternative splicing. The duplicate GR genes and products of alternative splicing in teleostean fishes are differentially expressed in vivo and show different transactivation capacity in vitro. The presence of two GR genes appears to be a result of divergence of receptors rather than of ligands. Teleostean fishes express different, evolutionarily related, functional GR proteins within a single organism. Hereby, teleostean fishes present a model that facilitates investigation of the molecular basis of cortisol resistance and different regulatory functions of cortisol.

show abstract

“…The Probasin (À285/ þ 32) luciferase reporter and the pSG5-hAR expression construct were gifts of Dr O Janne. The MMTVluciferase plasmid was obtained from Dr Joseph Fondell (Wang and Fondell, 2001). The ebp1 expression construct has been previous described (Xia et al, 2001a).…”

Section: Plasmidsmentioning

confidence: 99%

Specificity and heregulin regulation of Ebp1 (ErbB3 binding protein 1) mediated repression of androgen receptor signalling

Zhang

Hamburger

2004

Br J Cancer

View full text Add to dashboard Cite

Although ErbB receptors have been implicated in the progression of prostate cancer, little is known about proteins that may mediate their interactions with the androgen receptor (AR). Ebp1, a protein cloned via its association with the ErbB3 receptor, binds the AR and inhibits androgen-regulated transactivation of wild-type AR in COS cells. As the complement of coregulators in different cells are important for AR activity, we determined the effect of Ebp1 on AR function in prostate cancer cell lines. In addition, we examined the regulation of Ebp1 function by the ErbB3/4 ligand heregulin (HRG). In this study, we demonstrate, using several natural AR-regulated promoters, that Ebp1 repressed transcriptional activation of wild-type AR in prostate cancer cell lines. Downregulation of Ebp1 expression in LNCaP cells using siRNA resulted in activation of AR in the absence of androgen. Ebp1 associated with ErbB3 in LNCaP cells in the absence of HRG, but HRG induced the dissociation of Ebp1 from ErbB3. In contrast, HRG treatment enhanced both the association of Ebp1 with AR and also the ability of Ebp1 to repress AR transactivation. These studies suggest that Ebp1 is an AR corepressor whose biological activity can be regulated by the ErbB3 ligand, HRG.

show abstract

Generation of a Mammalian Cell Line Stably Expressing a Tetracycline-Regulated Epitope-Tagged Human Androgen Receptor: Implications for Steroid Hormone Receptor Research

Cited by 10 publications

References 48 publications

A Coregulatory Role for the TRAP-Mediator Complex in Androgen Receptor-mediated Gene Expression

A Coregulatory Role for the TRAP-Mediator Complex in Androgen Receptor-mediated Gene Expression

Evolution of glucocorticoid receptors with different glucocorticoid sensitivity

Specificity and heregulin regulation of Ebp1 (ErbB3 binding protein 1) mediated repression of androgen receptor signalling

Contact Info

Product

Resources

About