Characterization of human mineralocorticosteroid receptor expressed in the baculovirus system.
To investigate the structure and function of the mineralocorticosteroid receptor (MR), one has to circumvent the major difficulty related to its very low abundance. For this purpose, the full-length human MR (hMR) has been produced using the efficient baculovirus system. The recombinant protein is overexpressed in Sf9 insect cells at a concentration of approximately 2 pmol/mg of protein, which is 50-100 times more than the concentration in aldosterone target tissues. It binds aldosterone with high affinity (Kd approximately 1 nM) and clearly displays a mineralocorticosteroid specificity as evidenced by competition studies with steroid ligands and by the monoclonal anti-idiotypic antibody H10E interacting with the steroid-binding domain of MR. After [35S]methionine labeling, a single polypeptide band at approximately 120 kDa is detected and further identified as hMR by immunoblotting with A4, an anti-peptide antibody. Sedimentation analyses show that the native form of MR is recognized by A4 and B174, an antibody to the 90-kDa heat shock protein, since they both induce a shift of the receptor from 9S to 11S sedimentation coefficient. These results clearly demonstrate that MR is a heterooligomer containing the insect equivalent of the 90-kDa heat shock protein. This 9S receptor complex is converted to a 4S form during high-salt gradient ultracentrifugation, suggesting that MR can undergo a complete in vitro transformation. Immunofluorescence studies indicate that hMR, which is almost exclusively a cytoplasmic protein in Sf9 cells, is translocated to the nucleus after aldosterone exposure. Therefore, the recombinant hMR seems to behave as the native receptor. Given the possibility of large-scale protein production, the baculovirus system should prove useful in studies of the molecular basis of MR function as a transcription factor.
Binart
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N
Institut National de la Santé et de la Recherche Médicale Unité 33, Bicêtre, France.
Lombes
M
M
Rafestin-Oblin
M E
ME
Baulieu
E E
EE
D000450:Aldosterone / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001667:Binding, Competitive; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D007313:Insecta; D007700:Kinetics; D008970:Molecular Weight; D018161:Receptors, Mineralocorticoid; D011987:Receptors, Steroid / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D018161:Receptors, Mineralocorticoid; D011987:Receptors, Steroid; D011994:Recombinant Proteins; D000450:Aldosterone