title: Characterization of human mineralocorticosteroid receptor expressed in the baculovirus system.
abstract: 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.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale Unité 33, Bicêtre, France.
authors.forename: N
authors.initials: N
authors.lastname: Binart
authors.forename: M
authors.initials: M
authors.lastname: Lombes
authors.forename: M E
authors.initials: ME
authors.lastname: Rafestin-Oblin
authors.forename: E E
authors.initials: EE
authors.lastname: Baulieu
mesh_terms: 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
chemical_list: D018161:Receptors, Mineralocorticoid; D011987:Receptors, Steroid; D011994:Recombinant Proteins; D000450:Aldosterone