Characterization of a monoclonal antibody that probes the functional domains of the glucocorticoid receptor.
Monoclonal antibodies to the rat hepatic glucocorticoid receptor (GR) were produced by using 4000-fold-purified unactivated rat hepatic GR as the immunogen in an immunization in vitro. Hybridomas were screened for anti-GR antibody production by using an enzyme-linked immunosorbent assay. The antibody, 3A6, described here, is an IgM (lambda). The interaction of 3A6 with the purified GR was explored by sedimentation analysis, where a shift of the 9 S GR to a form with a higher s20,w value was demonstrated. Binding specificity and sensitivity were demonstrated by protein immunoblotting. 3A6 cross-reacted with all rat tissue glucocorticoid receptors (GRs) examined, except those of the brain. Species cross-reactivity was observed with other mammalian GRs (from human CEM-C7 cells and from pig and mouse liver). Immunocytochemical localization of the GR was assessed by indirect immunofluorescence in intact fixed cells, which demonstrated intense cytoplasmic staining in the absence of pretreatment with glucocorticoids and nuclear localization when cells were pretreated with glucocorticoids. This monoclonal antibody significantly inhibited steroid binding to unoccupied receptor and DNA binding of activated steroid-receptor complexes. Furthermore, preincubation of the purified activated GR complex with 3A6 prevented phosphorylation of the GR in vitro. Thus 3A6 differs from previous monoclonal antibodies to the GR in its capacity to cross-react with the human GR and by its specificity for an epitope on or near a functional domain of the GR.
Robertson
N M
NM
Fels Research Institute, Temple University School of Medicine, Philadelphia, PA 19140.
Kusmik
W F
WF
Grove
B F
BF
Miller-Diener
A
A
Webb
M L
ML
Litwack
G
G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D000936:Antigen-Antibody Complex / Q000302:isolation & purification; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D003429:Cross Reactions; D004247:DNA / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007122:Immunoelectrophoresis; D008024:Ligands; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D011965:Receptors, Glucocorticoid / Q000276:immunology* / Q000378:metabolism*; D013256:Steroids / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; C033190:DNA-cellulose; D008024:Ligands; D011965:Receptors, Glucocorticoid; D013256:Steroids; D002482:Cellulose; D004247:DNA