Interaction of unsaturated fatty acids with rat liver glucocorticoid receptors: studies to localize the site of interaction.
Polyunsaturated fatty acids have been shown to decrease the binding of [3H]dexamethasone to rat liver glucocorticoid receptors by mixed non-competitive inhibition, suggesting that these fatty acids interact at a site on the receptor different from the hormone binding site. The present study was undertaken to localize the site of interaction of polyunsaturated fatty acids on the receptor by comparing the differential effects of docosahexaenoic acid (a 22-carbon polyunsaturated fatty acid of the series n-3) on antagonist (RU486) and agonist binding, by covalent cross-linking of the hsp 90 and other proteins to the receptor to attempt to mask the site of interaction, by limited trypsinization to cleave the site and by using antibodies against specific epitopes to prevent fatty acid access by steric hindrance. Binding [3H]RU486 was not inhibited by docosahexaenoic acid at a concentration (60 mumol/l) that increases the dissociation constant of [3H]dexamethasone eightfold. Covalent stabilization of the hetero-oligomeric glucocorticoid receptor structure did not keep the fatty acid from inhibiting [3H]dexamethasone binding. The binding to the receptor of monoclonal and polyclonal antibodies against different domains of the receptor did not sterically hinder the fatty acid interaction with the receptor. After limited trypsinization of the receptor, the fatty acid still increased the dissociation rate constant of [3H]dexamethasone binding, indicating that the site of interaction of polyunsaturated fatty acids is on a fragment of the receptor containing the hormone-binding domain and some sequences C-terminal of the DNA-binding domain.
Sumida
C
C
INSERM U224, Faculté de Médecine Xavier Bichat, Paris, France.
Vallette
G
G
Nunez
E A
EA
D000818:Animals; D000911:Antibodies, Monoclonal; D003600:Cytosol / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism; D004281:Docosahexaenoic Acids / Q000494:pharmacology; D005231:Fatty Acids, Unsaturated / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008297:Male; D015735:Mifepristone / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D014018:Tissue Distribution
D000911:Antibodies, Monoclonal; D005231:Fatty Acids, Unsaturated; D011965:Receptors, Glucocorticoid; D004281:Docosahexaenoic Acids; D015735:Mifepristone; D003907:Dexamethasone