A BACTERIALLY EXPRESSED MINERALOCORTICOID RECEPTOR IS ASSOCIATED IN-VITRO WITH THE 90-KILODALTON HEAT-SHOCK PROTEIN AND SHOWS TYPICAL HORMONE-BINDING AND DNA-BINDING CHARACTERISTICS

A recombinant system was developed for generation of steroid-receptor complexes in vitro. The DNA- and steroid-binding domains of the rat mi neralocorticoid receptor were expressed in Escherichia coli as a fusio n protein with glutathione S-transferase. The identity of the expresse d recombinant protein was confirmed by Western blot analysis. Protein preparations purified by affinity chromatography, avoiding the use of detergents or high ionic strength buffers, exhibited negligible steroi d binding. However, after incubation of these preparations with rabbit reticulocyte lysate, known to promote the association of isolated ste roid receptors with heat shock proteins, the [H-3]aldosterone-binding activity gradually increased. This temperature-dependent effect reache d a maximum after 1 h at 30-degrees-C and was favored by ATP supplemen tation (B(max) = 22 +/- 3 pmol/mg of protein). The apparent K(d) value for aldosterone (0.6 +/- 0.2 nM) and the steroid-binding specificity of the recombinant protein were in accordance with those reported for the native mineralocorticoid receptor. The sedimentation and DNA-cellu lose-binding characteristics of the radioactive complexes were also in agreement with those reported for the native heteromeric receptor. Co mplexes sedimented at 8.9 +/- 0.2 or 4.2 +/- 0.2 S in sucrose gradient s containing 20 mM sodium molybdate or 0.4 M KCl, respectively. Monocl onal antibody 8D3 against the 90-kDa heat shock protein (hsp90) was ab le to bind to the 8.9S complexes, increasing its sedimentation coeffic ient. Treatment of the complexes with 100 mM sodium thiocyanate, known to activate the native receptor to a DNA-binding state, caused a 79% increase in DNA-cellulose binding over the control values. This last e ffect was prevented when 20 mM molybdate was also present. This is the first report of the use of a recombinant steroid receptor for the gen eration in vitro of complexes showing typical characteristics of nativ e receptors associated to hsp90. This novel strategy is apt to facilit ate future structural studies of steroid receptors.