Photoaffinity labelling of the human mineralocorticoid receptor with steroids having a reactive group at position 3, 18 or 21.
Biochim Biophys Acta
; 1388(1): 35-44, 1998 Oct 14.
Article
em En
| MEDLINE
| ID: mdl-9774704
ABSTRACT
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Esteroides
/
Receptores de Mineralocorticoides
/
Marcadores de Fotoafinidade
/
Reagentes de Ligações Cruzadas
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Biochim Biophys Acta
Ano de publicação:
1998
Tipo de documento:
Article
País de afiliação:
França