Region-specific antiglucocorticoid receptor antibodies selectively recognize the activated form of the ligand-occupied receptor and inhibit the binding of activated complexes to deoxyribonucleic acid.
A synthetic 18-amino acid peptide (Cys500-Lys517) was used to raise polyclonal antibodies in rabbits to the glucocorticoid receptor (GR). The sequence of this peptide is identical to that of residues 500-517 of the rat and 481-498 of the human GR. This sequence overlaps the carboxy-terminal end of the core DNA-binding domain and the amino-terminus of the hinge region of the receptor. Antiserum (AP64) was obtained which recognized both human and rat GR, as determined by immunoblots of receptors immunopurified with authentic anti-GR antibodies, immunoadsorption of both specific [3H]dexamethasone-bound GR and 98K receptors that were specifically covalently labeled by [3H]dexamethasone mesylate, and AP64-induced shifts in the elution position of monomeric [3H]dexamethasone-bound GR from Sephacryl S-300. The specificity of AP64 was demonstrated by the ability of the immunizing peptide, but not a peptide of similar length, to inhibit both the antibody-induced change in elution position from Sephacryl S-300 and the antibody-mediated immobilization of [3H]dexamethasone-bound complexes by protein-A. Further studies indicated that AP64 did not react with native steroid-free GR or with steroidbound (or affinity-labeled) unactivated GR, but did selectively associated with monomeric activated, steroid-bound (or affinity labeled) complexes. AP64 also inhibited the DNA binding of activated complexes in a manner that was specifically blocked by the immunizing peptide. Collectively, these data allow the direct localization of a structural region of the GR that is occluded in the unactivated complex but exposed as a result of activation.
Urda
L A
LA
Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Yen
P M
PM
Simons
S S
SS
Harmon
J M
JM
D000818:Animals; D000906:Antibodies / Q000502:physiology*; D004247:DNA / Q000378:metabolism*; D006801:Humans; D008024:Ligands / Q000378:metabolism; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000276:immunology*
D000906:Antibodies; D008024:Ligands; D011965:Receptors, Glucocorticoid; D004247:DNA