Abstract
Promoter interference assay was employed to examine in intact cells the roles of the functional domains of androgen receptor (AR) and the ligand for specific DNA interactions using a cytomegalovirus-(androgen response element)-chloramphenicol acetyltransferase reporter (pCMV-ARE2-CAT). Native rat and human ARs interfered with pCMV-ARE2-CAT expression in a hormone-dependent fashion. Low steroid-independent interference seemed to occur because of the ligand binding domain (LBD), which was transcriptionally inhibitory also in a heterologous context. AR devoid of LBD (rARDelta641-902) decreased pCMV-ARE2-CAT activity by 50%. The rARDelta46-408 mutant devoid of the NH2-terminal transcription activation region exhibited ligand-dependent promoter interference of a similar magnitude. Ligand and DNA binding-deficient mutants (hARM807R and rARC562G, respectively) did not influence pCMV-ARE2-CAT expression, although hARM807R binds to ARE in vitro. Non-steroidal anti-androgens casodex and hydroxyflutamide antagonized agonist-dependent promoter interference, whereas cyproterone acetate, RU 56187, RU 57073, and RU 59063 were partial agonists/antagonists. Collectively, interaction of ARs with ARE in intact cells does not require the presence of the COOH-terminal or NH2-terminal domain and/or their interaction. In the context of native AR, however, the androgen-induced conformational change in LBD is mandatory for generation of a transcriptionally competent receptor that binds to DNA in intact cells.
Highlights
Promoter interference assay was employed to examine in intact cells the roles of the functional domains of androgen receptor (AR) and the ligand for specific DNA interactions using a cytomegalovirus-(androgen response element)-chloramphenicol acetyltransferase reporter
We have examined which functional domains of the wild-type receptor, in addition to the DNA binding domain (DBD), are mandatory for specific DNA binding in vivo
Even though the weak ligand-independent activity of AR was not specific for the hormone response element inserted in the promoter interference construct, it was specific for the promoter interference assay itself, as a similar androgen-independent activity was not observed in transactivation experiments
Summary
(Received for publication, November 15, 1996, and in revised form, April 17, 1997). From the ‡Institute of Biomedicine, Department of Physiology, and the §Department of Clinical Chemistry, University of Helsinki, FIN-00014 Helsinki, Finland. Promoter interference assay was employed to examine in intact cells the roles of the functional domains of androgen receptor (AR) and the ligand for specific DNA interactions using a cytomegalovirus-(androgen response element)-chloramphenicol acetyltransferase reporter (pCMV-ARE2-CAT). The COOH-terminal ligand binding domain (LBD) provides the means to regulate the function of native AR in a steroid-dependent fashion. Removal of this region generates a constitutively active receptor form. In the present work we have used promoter interference assays to investigate whether binding of AR to specific DNA sequences is ligand-dependent in intact CV-1 cells. Immunocytochemical analysis of cultured cells was employed to rule out the possibility that some of the results were compromised by altered subcellular distribution of receptor proteins
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