Filamin-A fragment localizes to the nucleus to regulate androgen receptor and coactivator functions -- Data Supplement - Supporting Information -- Proceedings of the National Academy of Sciences

Supporting information for Loy et al. (2003) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0736237100

Supporting Materials and Methods

GAL–androgen receptor (AR) hinge (622–670) was generated by inserting a PCR fragment encoding residues 622–670 of the AR in-frame into the EcoRI/BamHI site of pM vector (CLONTECH). To make pG5tkLuc, the Herpes simplex virus thymidine kinase (hsvtk) promoter was PCR amplified and inserted into HindIII site upstream of the luciferase gene in pG5Luc. ERE₄-Luc contains four copies of the vitellogenin estrogen response element driving luciferase gene expression.

Repressor Function of AR Hinge Subdomain.
To examine the inhibitory function in the AR subdomain defined by residues 628–646, chimeric AR ligand-binding domain (LBD) fragments (with or without residues 628–646) were linked to the GAL4-DBD and coexpressed with a constitutively expressed GAL4tkLUC reporter (Fig. 6). The reporter alone exhibited significant intrinsic activity, which was suppressed by the intact hinge LBD (628–919) chimeric construct in the presence of 5α-dihydrotestosterone (DHT). In contrast, deletion of the hinge subdomain (residues 628–646) unmasked hormone-dependent activation function of the ARLBD, which was not evident when the LBD was attached to the entire hinge domain. Filamin A (FLNa) Specifically Represses AR Transactivation Activity.
To ascertain whether the inhibitory effect of FLNa was specific for the AR, FLNa was tested for its ability to inhibit transcription activation by other nuclear receptors, namely the estrogen, progesterone, and glucocorticoid receptors (ER, PR, and GR, respectively). Fig. 7 demonstrates that ligand-dependent PR, GR, Erα, and Erβ-mediated transcription activities were not affected by FLNa.