Mineralocorticosteroid receptor of the chick intestine. Oligomeric structure and transformation.
The binding of [3H]aldosterone in the chick intestine cytosol was analyzed in terms of affinity and specificity. In this tissue, aldosterone binds to the mineralocorticosteroid receptor, with a high affinity (Kd approximately 0.3 nM) and low capacity (approximately 50 fmol/mg protein), and to the glucocorticosteroid receptor. The selective labeling of the mineralocorticosteroid receptor was achieved by incubating the cytosol with [3H]aldosterone in the presence of RU 486. This synthetic steroid completely inhibited the binding of [3H]aldosterone to the glucocorticosteroid receptor and did not bind to the mineralocorticosteroid receptor. The oligomeric structure of the mineralocorticosteroid receptor was studied by using BF4, a monoclonal antibody which reacts with the 90-kDa heat shock protein (hsp 90), a nonhormone-binding component of nontransformed steroid receptors. The mineralocorticosteroid receptor sedimented at 8.5 +/- 0.4 S (n = 8) in a 15-40% glycerol gradient. This peak was shifted to 11.2 +/- 0.6 S (n = 5) after incubation with BF4, indicating that, in the cytosol, hsp 90 was associated with the mineralocorticosteroid receptor. Dissociation of the complex was observed on gradients containing 0.4 M KCl, as judged by the absence of displacement by BF4 of the 4.3 +/- 0.4 S (n = 10) peak. The effect of molybdate and tungstate ions, and of dimethyl pimelimidate, an irreversible cross-linking agent, on the stability of the hsp 90-receptor complex was investigated. Complexes recovered in the presence of 20 mM molybdate ions dissociated on gradients containing 0.4 M KCl (5.2 +/- 0.6 S (n = 4), whereas complexes prepared in the presence of 20 mM tungstate ions sedimented at 8.5 +/- 0.4 S (n = 7). Similarly, complexes prepared in the presence of molybdate ions dissociated during high pressure liquid chromatography (HPLC) gel filtration analysis performed in 0.4 M KCl (RS (Stokes radius) = 3.9 +/- 0.5 nm (n = 3) versus 7.3 +/- 0.2 nm (n = 3) in the presence of 20 mM molybdate ions), whereas complexes prepared in the presence of tungstate ions did not dissociate (RS = 6.9 +/- 0.2 nm (n = 3]. As observed for the tungstate-stabilized receptor, the cross-linked receptor dissociated neither on gradient containing 0.4 M KCl (9.5 +/- 0.1 S (n = 3] nor during HPLC performed in 0.4 M KCl (RS = 6.5 +/- 0.3 (n = 4]. Furthermore, the cross-linked receptor was more resistant to the inactivating effect of urea on aldosterone binding than the noncross-linked receptor prepared in the presence of either molybdate or tungstate ions.
Rafestin-Oblin
M E
ME
Institut National de la Santé et de la Recherche Médicale U33, Bicêtre, France.
Couette
B
B
Radanyi
C
C
Lombes
M
M
Baulieu
E E
EE
D000450:Aldosterone / Q000378:metabolism; D000818:Animals; D002499:Centrifugation, Density Gradient; D002645:Chickens; D002850:Chromatography, Gel; D006360:Heat-Shock Proteins / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism; D007700:Kinetics; D008901:Mineralocorticoids / Q000378:metabolism*; D011189:Potassium Chloride; D011487:Protein Conformation; D011965:Receptors, Glucocorticoid / Q000032:analysis; D018161:Receptors, Mineralocorticoid; D011987:Receptors, Steroid / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D013861:Thiocyanates; D014170:Transformation, Genetic; D014508:Urea
D006360:Heat-Shock Proteins; D008901:Mineralocorticoids; D011965:Receptors, Glucocorticoid; D018161:Receptors, Mineralocorticoid; D011987:Receptors, Steroid; D013861:Thiocyanates; D000450:Aldosterone; C024553:sodium thiocyanate; D011189:Potassium Chloride; D014508:Urea