Antibodies to steroid receptor deoxyribonucleic acid binding domains and their reactivity with the human glucocorticoid receptor.
Functional properties of the DNA-binding domain of the human glucocorticoid receptor were investigated using high titer polyclonal antibodies produced against single synthetic peptides or a mixture of peptides whose sequences were derived from the DNA-binding domain of steroid receptor proteins. Three of seven antisera recognized both native and denatured forms of the glucocorticoid receptor, although considerably lower antisera dilutions were required for antibody binding to native receptor. Activation of the glucocorticoid receptor to its DNA-binding form was required for antibody recognition of the native receptor. Antisera to the second finger region of the DNA-binding domain caused a portion of the activated 4S glucocorticoid receptor to sediment as 7 or 9S in sucrose gradients containing 0.4 M KCl, but did not alter the sedimentation of the nontransformed 8S receptor. Specificity of the glucocorticoid receptor-antibody interaction was demonstrated by loss of reactivity after preabsorption with peptide antigens. Antisera that interacted specifically with the glucocorticoid receptor inhibited DNA binding of the activated receptor by as much as 80%. Thus, antibody probes directed against DNA-binding domain sequences provide immunological evidence that glucocorticoid receptor activation exposes the DNA-binding region of the receptor.
Wilson
E M
EM
Department of Pediatrics, University of North Carolina, Chapel Hill 27599.
Lubahn
D B
DB
French
F S
FS
Jewell
C M
CM
Cidlowski
J A
JA
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D001667:Binding, Competitive; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011965:Receptors, Glucocorticoid / Q000276:immunology / Q000378:metabolism*; D014614:Vaccines, Synthetic / Q000276:immunology
D000906:Antibodies; D011965:Receptors, Glucocorticoid; D014614:Vaccines, Synthetic; D004247:DNA