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Figure 1.
FIGURE 1. AF2 activity in the AR LBD. A, schematic diagram
of AR LBD deletion mutants. Full-length human AR (amino acid
residues 1-919) contains activation function 1 (AF1, amino acid
residues 142-337), DNA binding domain (DBD residues 559-623),
hinge region residues 624-670, and LBD residues 671-919 that
includes AF2. AR hinge region residues 624-639 contain the
carboxyl-terminal portion of the bipartite AR nuclear targeting
signal residues Arg-629, Lys-630, Lys-632, and Lys-633
(underlined) (38). AR-(624-919), -(640-919), and -(658-919) with
WT and mutant sequence were expressed as GAL4 DNA binding domain
fusion proteins. AR residues 663-919 and H874Y mutant were
expressed for crystallography as His[6]-tagged fusion proteins
with intervening thrombin cleavage site. B, similar expression
of GAL-AR-LBD fusion proteins. COS cells were transfected with
10 µg of GAL-0 (lane 1), GAL-AR-(658-919) (lanes 2-5), and
GAL-AR-(624-919) (lanes 6-9) with WT or mutant sequence. Protein
extracts (60 µg of protein/lane) were separated on a 10%
acrylamide gel containing SDS and the blot probed using an
anti-GAL antibody. C, androgen-dependent activity of
GAL-AR-(624-919), GAL-AR-(658-919) WT and H874Y mutants in CV1
cells requires coexpression of TIF2. CV1 cells plated in 6-cm
dishes were transfected by calcium phosphate DNA precipitation
with 5 µg of 5XGAL4Luc3 reporter vector and 0.1 µg
of GAL-AR-(624-919) or GAL-AR-(658-919) with WT or H874Y
sequence in the absence and presence of 2 µg of pSG5-TIF2.
Cells were treated with and without increasing concentrations of
DHT (D) and T as indicated and luciferase activity was
determined. Data are representative of three independent
experiments. D, inhibition of the AR N/C and coactivator
interactions by AR hinge residues 624-639. HeLa cells were
transfected using FuGENE 6 by adding per well 0.1 µg of
5XGAL4Luc, 50 ng of VP16, VP-AR-(1-660), or VP-TIF2-(624-1287)
with 0.1 µg of GAL-AR-(624-919), -(640-919), or
-(658-919). Cells were incubated with and without 0.1-10 nM DHT
for 24 h as indicated and assayed for luciferase activity. Data
are representative of three independent experiments. E,
androgen-dependent transcriptional activity of GAL-AR-(624-919),
GAL-AR-(658-919), and H874Y mutants in CWR-R1 cells. CWR-R1
cells (2 x 10^5/well) were transfected using Effectene by adding
per well 0.1 µg of GAL-AR-(624-919), GAL-AR-(658-919), or
H874Y mutants and 0.25 µg of 5XGAL4Luc3. Cells were
treated with and without increasing concentrations of T and DHT
for 24 h as indicated, and luciferase activity was determined.
Data are representative of three independent experiments. F,
androgen-dependent AF2 activity of GAL-AR-(658-919) in CWR-R1
cells. CWR-R1 cells (2 x 10^5/well) were transfected using
Effectene by adding per well 0.1 µg of GAL-AR-(658-919) or
H874Y, K720A, or E897K mutants and 0.25 µg of 5XGAL4Luc3.
Lys-720 and Glu-897 are charge clamp residues in AF2. Cells were
incubated with and without 0.1, 1, and 10 nM T for 24 h, and
luciferase activity was determined. Data are representative of
at least three independent experiments.
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