A bacterially expressed mineralocorticoid receptor is associated in vitro with the 90-kilodalton heat shock protein and shows typical hormone- 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 mineralocorticoid receptor were expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The identity of the expressed 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 steroid binding. However, after incubation of these preparations with rabbit reticulocyte lysate, known to promote the association of isolated steroid receptors with heat shock proteins, the [3H]aldosterone-binding activity gradually increased. This temperature-dependent effect reached a maximum after 1 h at 30 degrees C and was favored by ATP supplementation (Bmax = 22 +/- 3 pmol/mg of protein). The apparent Kd 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-cellulose-binding characteristics of the radioactive complexes were also in agreement with those reported for the native heteromeric receptor. Complexes sedimented at 8.9 +/- 0.2 or 4.2 +/- 0.2 S in sucrose gradients containing 20 mM sodium molybdate or 0.4 M KCl, respectively. Monoclonal antibody 8D3 against the 90-kDa heat shock protein (hsp90) was able to bind to the 8.9S complexes, increasing its sedimentation coefficient. 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Caamaño
C A
CA
Mental Health Research Institute, University of Michigan, Ann Arbor 48109-0720.
Morano
M I
MI
Patel
P D
PD
Watson
S J
SJ
Akil
H
H
D000450:Aldosterone / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015153:Blotting, Western; D002482:Cellulose / Q000031:analogs & derivatives; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004247:DNA; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D014176:Protein Biosynthesis; D011817:Rabbits; D018161:Receptors, Mineralocorticoid; D011987:Receptors, Steroid / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012156:Reticulocytes / Q000378:metabolism
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; C033190:DNA-cellulose; D006360:Heat-Shock Proteins; D018161:Receptors, Mineralocorticoid; D011987:Receptors, Steroid; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000450:Aldosterone; D002482:Cellulose; D004247:DNA; D005982:Glutathione Transferase