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Characterization of a monoclonal antibody that probes the functional domains of the glucocorticoid receptor.

ABSTRACT: 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.

SUBMITTER: Robertson NM

PROVIDER: S-EPMC1148239 | biostudies-other | 1987 Aug

REPOSITORIES: biostudies-other

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Project description:We describe the use of four monoclonal antibodies (MAbs) to the rabbit liver growth hormone (GH) receptor and one raised against purified rat liver GH receptor to characterize liver receptor subtypes which differ in their hormone-binding regions. The anti-(rat liver GH receptor) MAb both inhibited and precipitated rat and rabbit GH receptors, but only one-half of 125I-oGH (ovine GH) binding to liver microsomes could be inhibited by excess antibody. Conversely, only one-half of 125I-anti-(rat GH receptor) MAb binding was inhibited by excess oGH and Scatchard plots for this MAb exhibited two components. Although only 50% of 125I-oGH binding to membranes was inhibited by this MAb, all solubilized receptor could be immunoprecipitated. We postulate two epitopes for the anti-(rat GH receptor) MAb, one located at the hormone-binding site (inhibitory site) and one elsewhere (immunoprecipitating site). A second, rabbit-specific antibody (MAb 7) inhibited 85% of hormone binding but only 30% of 125I-anti-(rat GH receptor) MAb binding to rabbit liver microsomes. A combination of this MAb with the anti-(rat GH receptor) MAb totally inhibited 125I-oGH binding. MAb 7 alone totally inhibited 125I-rat GH binding to rabbit liver microsomes, as it did with 125I-oGH binding to purified receptor. On the basis of these results and others we postulate three types of GH receptor in rabbit liver membranes and ascribe approximate extents of 125I-oGH binding to each. A cytosolic 'GH receptor' which is not poly(ethylene glycol)-precipitable is shown to share five epitopes with 'type 2' microsomal receptors. Purified plasma membrane and endoplasmic reticulum fractions derived from a rabbit liver microsomal preparation have identical antigenic characteristics with respect to the GH-binding region, indicating that the heterogeneity we describe is not related to receptor processing. Of the three types of GH receptor in the plasma membrane of the rabbit (and possibly rat) we postulate that one (type 1) corresponds to the GH receptor involved in stimulating growth and possesses all of the epitopes studied here. A second (type 2) appears to be identical with the cytosolic 'GH receptor' and lacks the epitope for the anti-(rat GH receptor) MAb in the hormone binding site region. A third (type 3) does not possess the epitope for the inhibitory anti-(rabbit GH receptor) MAb, appears not to bind rat GH and is lost during purification. The availability of type-specific MAbs will facilitate assignment of specific functions to liver receptor subtypes which mediate the multiple functions of GH.

Project description:Purpose of reviewTo summarize the in-vivo efficacy of neutralizing human monoclonal antibodies against HIV-1, to discuss the recent finding that an engineered human antibody VH domain, domain antibody (dAb), exhibits exceptionally potent and broadly cross-reactive neutralizing activity against HIV-1 primary isolates by targeting a hidden conserved epitope that is not accessible by larger antibodies and to suggest the possibility of developing a novel class of potent HIV-1 inhibitors based on human dAbs.Recent findingsHIV-1 has evolved a number of strategies to evade humoral immunity, including protecting highly conserved and important structures from the access of antibodies generated by the immune system. We have recently demonstrated that a human dAb (size approximately 15 kDa), m36, targets a highly protected structure on the HIV-1 envelope glycoprotein (Env), gp120, and exhibits exceptionally potent neutralizing activity against HIV-1 primary isolates, with potency on average higher than those of the broadly cross-reactive neutralizing human monoclonal antibody, scFv m9, and the inhibitory peptide, C34.SummaryThe efficacy of the anti-HIV-1 therapy is significantly compromised by resistance to the currently used US Food and Drug Administration-approved antiretroviral drugs, which suggests an urgent need to develop novel classes of potent inhibitors. Several broadly cross-reactive neutralizing human monoclonal antibodies are highly effective against HIV-1 infection in vitro, but their administration to HIV-1-infected humans has only resulted in modest antiviral effects. Engineered human antibody fragments, dAbs, could be more potent because of their small size (about 10-fold smaller than that of an IgG), which allows targeting of highly conserved structures on the HIV-1 envelope glycoprotein that are not accessible by full-size antibodies and relatively efficient penetration into the densely packed lymphoid environment in which HIV-1 mostly replicates and spreads.

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Characterization of a monoclonal antibody that probes the functional domains of the glucocorticoid receptor.

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