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)

Molecular cloning of a mineralocorticoid (type I) receptor complementary DNA from rat hippocampus.

Rat brain expresses two types of corticosteroid-binding proteins. The type I receptor binds corticosterone with high affinity and is structurally related to the kidney mineralocorticoid receptor (MR), while the type II or classical glucocorticoid receptor binds corticosterone with lower affinity and displays an in vivo preference for dexamethasone. Here we describe the isolation and characterization of a cDNA coding for the MR, from a rat hippocampus cDNA library, by low stringency hybridization to radiolabeled human glucocorticoid receptor cDNA. The nucleotide and deduced amino acid sequence for rat hippocampal MR displays extensive homology to a MR cDNA isolated from human kidney, suggesting that they are orthologous genes. Southern analysis suggests that there is only one gene for the MR, and in vitro expression of the receptor generates a high affinity corticosterone-binding protein. These data provide evidence to support the contention that a single gene gives rise to the MR in renal tissues and type I receptors in the brain.