Photoaffinity labelling of the human mineralocorticoid receptor with steroids having a reactive group at position 3, 18 or 21.
The ability of a glucocorticoid (triamcinolone acetonide: TA) and three progesterone derivatives with photoreactive groups at different positions (promegestone: R5020; 18-oxo-18-vinylprogesterone: 18OVP; 21-diazoprogesterone: 21DP) to bind covalently to the human mineralocorticoid receptor (hMR) expressed in Sf9 insect cells was assessed. Sedimentation gradient analysis and exchange assays with aldosterone showed that [3H]TA, a partial mineralocorticoid agonist, and [3H]R5020, a pure antimineralocorticoid, were covalently bound to hMR after UV irradiation, with a labelling efficiency of approx. 3-5%. UV irradiation did not alter the heterooligomeric structure of the hMR, since the irradiated [3H]TA- and [3H]R5020-hMR complexes sedimented at approx. 9-10 S, as did the non-irradiated complexes. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed a band labelled by [3H]TA or [3H]R5020, having a molecular mass of 120 kDa. This band was not detected in the presence of an excess of the corresponding unlabelled steroid or when the cytosol was recovered from non-infected Sf9 cells. Electrophoresis of a truncated hMR (hMRDelta(1-351)) photolabelled with [3H]TA revealed a 80 kDa band, compatible with the molecular mass of the truncated hMR. Limited chymotrypsin proteolysis of the [3H]TA photolabelled hMR generated a 30 kDa fragment covalently associated with [3H]TA. As the 30 kDa fragment generated by chymotrypsin has been shown to encompass the entire ligand-binding domain of the hMR (B. Couette, J. Fagart, S. Jalaguier, M. Lombès, A. Souque, M.E. Rafestin-Oblin, Biochem. J. 315 (1996) 421-427), the present experiments provide evidence that [3H]TA is covalently bound to the ligand binding domain of the hMR. Exchange assays with [3H]A also revealed that unlabelled 18OVP and 21DP, two mineralocorticoid agonists bearing photoreactive groups at skeleton positions crucial for the ligand-MR interaction, are covalently bound to hMR with an approx. 30-35% labelling efficiency.
Fagart
J
J
INSERM U478, Faculté de Médecine Xavier Bichat, Institut Fédératif de Recherche 02, PO Box 416, 16, rue Henri Huchard, 75780 Paris Cedex 18, France.
Couette
B
B
Souque
A
A
Davioud
E
E
Marquet
A
A
Rafestin-Oblin
M E
ME
D000818:Animals; D002522:Chlorocebus aethiops; D002918:Chymotrypsin / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D051379:Mice; D019920:Photoaffinity Labels / Q000378:metabolism* / Q000494:pharmacology / Q000528:radiation effects; D011282:Pregnenediones / Q000378:metabolism / Q000494:pharmacology; D011374:Progesterone / Q000031:analogs & derivatives / Q000378:metabolism; D011397:Promegestone / Q000378:metabolism / Q000494:pharmacology / Q000528:radiation effects; D018161:Receptors, Mineralocorticoid / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000528:radiation effects; D013256:Steroids / Q000378:metabolism* / Q000494:pharmacology; D014162:Transfection; D014222:Triamcinolone Acetonide / Q000378:metabolism / Q000494:pharmacology; D014466:Ultraviolet Rays
C115465:18-oxo-18-vinylprogesterone; D003432:Cross-Linking Reagents; D019920:Photoaffinity Labels; D011282:Pregnenediones; D018161:Receptors, Mineralocorticoid; D011994:Recombinant Proteins; D013256:Steroids; D011374:Progesterone; C100802:21-diazopregn-4-ene-3,20-dione; D011397:Promegestone; D002918:Chymotrypsin; D014222:Triamcinolone Acetonide