Overexpression and characterization of the human mineralocorticoid receptor.
The full-length human renal mineralocorticoid receptor (hMR) has been overproduced in Spodoptera frugiperda (Sf9) insect cells using baculovirus-mediated expression. The overproduced hMR binds aldosterone with high affinity (Kd = 1.36 nM) and has high affinity for cortisol, cortexolone, and progesterone. Immunoprecipitation and immunoblot analysis of the recombinant hMR with MR-specific antibodies reveal three major protein bands with molecular masses of 115, 119, and 125 kDa. hMR isoforms show maximal accumulation at 48 h post-infection with the recombinant baculovirus. Maximal aldosterone binding was detected at 24 h rather than at 48 h post-infection, suggesting that the assembly of hMR monomers into the nonactivated steroid-binding receptor complexes and/or their stability deteriorates after 24 h post-infection. It is estimated by specific aldosterone binding that 1.2 x 10(6) hMR molecules are expressed per Sf9 cell (equivalent to 7 pmol/mg of cytosolic protein) at 24 h post-infection. 5-Fold more receptor molecules/cell are expressed but not detected by steroid binding at 48 h post-infection as determined by immunoblot analysis. Using the MR-specific H10E anti-idiotypic monoclonal antibody, immunoprecipitation of cytosol from recombinant baculovirus-infected Sf9 cells pulse-labeled with 32Pi demonstrated for the first time that the recombinant hMR is highly phosphorylated. The hMR is expressed as 9-10 S oligomeric complexes (Stokes radii approximately 67-85 A) that are slightly heavier than the unactivated glucocorticoid receptor and can be converted to smaller 4 S receptor monomers (Stokes radii approximately 25-55 A) by elevated temperature, pH, and ionic strength. Unlike the glucocorticoid receptor, the oligomeric hMR complex can bind DNA-cellulose without prior activation. Finally, indirect immunofluorescence demonstrated that the hMR is expressed primarily as a cytoplasmic protein that can be induced to translocate to the nucleus upon treatment with hormone.
Alnemri
E S
ES
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Maksymowych
A B
AB
Robertson
N M
NM
Litwack
G
G
D000450:Aldosterone / Q000494:pharmacology; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002848:Chromatography, DEAE-Cellulose; D004268:DNA-Binding Proteins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005814:Genes, Viral; D006801:Humans; D007668:Kidney / Q000737:chemistry; D008901:Mineralocorticoids / Q000378:metabolism*; D009036:Moths; D010766:Phosphorylation; D011233:Precipitin Tests; D018161:Receptors, Mineralocorticoid; D011987:Receptors, Steroid / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D008901:Mineralocorticoids; D018161:Receptors, Mineralocorticoid; D011987:Receptors, Steroid; D011994:Recombinant Proteins; D000450:Aldosterone