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.

Published

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

Pubmed Central 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