title: Modification of an essential amino group in the mineralocorticoid receptor evidences a differential conformational change of the receptor protein upon binding of antagonists, natural agonists and the synthetic agonist 11,19-oxidoprogesterone.
abstract: The alkylation of amino groups of the mineralocorticoid receptor (MR) with pyridoxal 5'-phosphate or 2,4,6-trinitrobenzenesulphonate (TNBS) under controlled conditions modifies only one lysyl residue, which accounts for a 70% inhibition of steroid binding capacity. The Kd of aldosterone for MR is not affected by the treatment, but the total number of binding sites is greatly decreased. The modified receptor is capable of dynamically conserving its association with the hsp90-based heterocomplex. Importantly, the binding of natural agonists protects the hormone binding capacity of the MR from the inactivating action of alkylating agents. In contrast, antagonistic steroids are totally incapable of providing such protection. Like the antagonistic ligands, and despite its potent mineralocorticoid biological effect, the sole MR specific synthetic agonist known to date, 11,19-oxidoprogesterone (11-OP), shows no protective effect upon treatment of the MR with pyridoxal 5'-phosphate or TNBS. Limited digestion of the MR with alpha-chymotrypsin generates a 34 kDa fragment, which becomes totally resistant to digestion upon binding of natural agonists, but not upon binding of antagonists. Interestingly, the synthetic 21-deoxypregnanesteroid 11-OP exhibits an intermediate pattern of proteolytic degradation, suggesting that the conformational change generated in the MR is not equivalent to that induced by antagonists or natural agonists. We conclude that in the first steps of activation, the MR changes its conformation upon binding of the ligand. However, the nature of this conformational change depends on the nature of the ligand. The experimental evidence shown in this work suggests that a single lysyl group can determine the hormone specificity of the MR.
authors.affiliation: Department of Physiology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors.forename: Graciela
authors.initials: G
authors.lastname: Piwien-Pilipuk
authors.forename: Kimon C
authors.initials: KC
authors.lastname: Kanelakis
authors.forename: Alberto A
authors.initials: AA
authors.lastname: Ghini
authors.forename: Carlos P
authors.initials: CP
authors.lastname: Lantos
authors.forename: Gerald
authors.initials: G
authors.lastname: Litwack
authors.forename: Gerardo
authors.initials: G
authors.lastname: Burton
authors.forename: Mario D
authors.initials: MD
authors.lastname: Galigniana
mesh_terms: D000450:Aldosterone / Q000494:pharmacology; D000477:Alkylating Agents / Q000494:pharmacology; D000588:Amines / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002918:Chymotrypsin; D003600:Cytosol / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000378:metabolism*; D008297:Male; D000451:Mineralocorticoid Receptor Antagonists; D011374:Progesterone / Q000031:analogs & derivatives* / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D018161:Receptors, Mineralocorticoid / Q000819:agonists* / Q000737:chemistry; D013997:Time Factors; D014302:Trinitrobenzenesulfonic Acid / Q000494:pharmacology; D014316:Tritium
chemical_list: D000477:Alkylating Agents; D000588:Amines; D018841:HSP90 Heat-Shock Proteins; D000451:Mineralocorticoid Receptor Antagonists; D018161:Receptors, Mineralocorticoid; D014316:Tritium; C092753:11,19-oxidoprogesterone; D000450:Aldosterone; D011374:Progesterone; D011732:Pyridoxal Phosphate; D014302:Trinitrobenzenesulfonic Acid; D002918:Chymotrypsin; C117608:alpha-chymotrypsin