TABLE 1.

Binding activity and transcriptional activity of helix 12 mutant androgen receptor constructs^a

Mutation	R1881 binding		Binding to GAL4/AR LBD (% of wild-type binding)			MMTV LTR transcriptional activity (% of wild-type activity)
	Kd (nM)	Bmax (dpm/0.09 ml of cells)	VP16/β-catenin	TIF2	VP16/AR (1-660)	VP16/β-catenin	TIF2	AR (1-503)
None	0.0832	2,630
V889M	0.222	1,779	−0.14 ± 0.18	15.76 ± 0.43	2.94 ± 0.36	2.09 ± 0.96	43.03 ± 4.96	9.24 ± 1.56
F891A	0.739	1,201	0.2 ± 0.10	0.05 ± 0.23	0.06 ± 0.07	−0.01 ± 0.10	−0.04 ± 0.04	0.24 ± 0.09
P892L	0.333	762	−0.03 ± 0.02	0.05 ± 0.10	0 ± 0.05	0.17 ± 0.06	0.26 ± 0.05	0.45 ± 0.16
E893A	0.0512	2,120	10.26 ± 3.8	58.1 ± 7.36	9.68 ± 1.98	5.19 ± 1.35	27.31 ± 4.07	27.51 ± 7.47
M894A	0.133	1,893	31.15 ± 6.8	0.15 ± 0.13	34.6 ± 8.47	23.82 ± 3.27	2.16 ± 1.44	52.04 ± 7.89
M894D	0.166	2,326	17.38 ± 4.64	−0.02 ± 0.26	4.13 ± 0.08	42.41 ± 0.24	0.16 ± 0.20	7.22 ± 4.49
M895T	0.222	1,454	9.63 ± 1.52	3.4 ± 1.02	16.09 ± 3.45	30.98 ± 4.98	31.2 ± 5.32	47.37 ± 4.58
A896V	0.166	1,944	114 ± 23.08	29.77 ± 9.23	43.43 ± 10.3	47.81 ± 7.05	40.79 ± 10.62	69.44 ± 8.94
E897V	0.111	2,170	207.6 ± 60.15	2.47 ± 1.97	0.13 ± 0.05	84.5 ± 13.7	0.38 ± 0.35	0.23 ± 0.04
I898A	0.166	2,149	18.09 ± 1.31	43.91 ± 6.61	0.68 ± 0.31	43.78 ± 13.57	73.45 ± 17.39	26.1 ± 5.31
I899A	0.222	2,163	15.7 ± 6.02	45.33 ± 5.02	15.98 ± 7.01	59.6 ± 30.4	85.8 ± 16.3	69.04 ± 1.2
Q902R	0.0739	2,691	74.31 ± 21.43	0.06 ± 0.30	1.03 ± 0.11	27.45 ± 1.53	0.44 ± 0.25	6.75 ± 1.79
V903M	0.0832	2,146	85.76 ± 29.0	26.04 ± 11.92	34.71 ± 5.37	66.99 ± 13.58	27.67 ± 4.56	64.64 ± 11.04
P904S	0.665	3,014	0.87 ± 0.37	1.64 ± 1.14	8.06 ± 0.73	6.4 ± 0.75	14.54 ± 0.50	46.44 ± 2.69
K905A	0.111	1,838	65.67 ± 25.25	46.27 ± 7.87	45.61 ± 9.68	66.8 ± 4.77	1.02 ± 0.14	13.61 ± 2.13
L907F	0.222	2,420	13.61 ± 7.21	40.32 ± 11.63	19.89 ± 4.4	22.54 ± 5.80	47.07 ± 6.49	52.67 ± 10.18
G909R	0.333	2,266	59.29 ± 9.41	17.24 ± 0.56	17.38 ± 1.04	31.02 ± 4.41	17.7 ± 2.12	48.08 ± 8.73

^aTwo-hybrid interaction was determined with 10 ng of wild-type GAL4/AR LBD or the indicated point mutants and 50 ng of either VP16/β-catenin, TIF2, or VP16/AR(1-660), together with 100 ng of pFR-LUC and 10 ng of Renilla null luciferase reporters. Transactivation assay was determined by transfecting 10 ng of wild-type pCMVhAR507-919 or the indicated mutants, 100 ng of MMTV-LUC, and 50 ng of VP16/β-catenin, TIF2, or pCMVhAR1-503, together with 100 ng of pFR-LUC and 10 ng of Renilla null luciferase reporters. Cells were treated with 100 nM R1881 in phenol red-free medium for 24 h and assayed for firefly and Renilla luciferase activities as described in Materials and Methods. The induction by R1881 was determined for each mutant in the presence of individual factors, and the induction of wild-type GAL4/AR LBD and pCMVhAR507-919 were set at 100%, The percent binding activity to GAL/AR LBD mutants or percent transcriptional activity mediated by pCMVhAR507-919 mutants was then calculated as (induction of mutant − 1)/(induction of wild type − 1) × 100. Negative values indicate that the induction of the mutant by R1881 was less than 1 (the ethanol control value taken as 1).