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.
Journal Article (Journal Article)
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.
Full Text
Duke Authors
Cited Authors
- Urda, LA; Yen, PM; Simons, SS; Harmon, JM
Published Date
- February 1989
Published In
Volume / Issue
- 3 / 2
Start / End Page
- 251 - 260
PubMed ID
- 2710132
International Standard Serial Number (ISSN)
- 0888-8809
Digital Object Identifier (DOI)
- 10.1210/mend-3-2-251
Language
- eng
Conference Location
- United States