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Abstract
Full length human glucocorticoid receptor and truncated receptor derivatives lacking the major amino-terminal trans-activating domain were expressed in stably transfected Chinese hamster ovary (CHO) cells. The receptors were co-expressed together with human metallothionein IIa, and the expression levels were amplified in the presence of increasing concentrations of metal. In amplified cells, both synthesized receptor forms showed the expected molecular weights, as assayed by affinity labeling and immunoblotting. They were expressed at concentrations of about 350,000-520,000 molecules/cell which corresponds to a 10-fold increase in receptor levels as compared to rat liver cells. The hormone (agonist or antagonist) binding properties of the expressed proteins were very similar to those characteristic of authentic glucocorticoid receptors in tissues or cultured cells. Moreover, the expressed proteins specifically recognized a glucocorticoid-response element sequence motif in in vitro protein-DNA binding experiments. The activation of a glucocorticoid-responsive reporter gene by the expressed full length receptor was dramatic (about 75-fold) and strictly ligand-dependent. In contrast, the expressed amino-terminal deletion mutant exhibited considerably weaker functional activity but showed normal hormone-binding properties. Upon exposure to dexamethasone in vivo, the expressed receptor mRNAs and proteins were down-regulated about 2- to 6-fold, indicating that regulatory signals important for autoregulation may be contained within structures corresponding to the ligand and DNA-binding domains. Transcription from the expression vector was not negatively regulated from the hormone, strongly arguing that receptor down-regulation was due to a post-transcriptional mechanism. In conclusion, this expression system should be a useful tool for further structural and functional studies of the receptor, including the biochemistry of its activation from a cryptic to a functional species, and its ligand-dependent autoregulation.
Highlights
From Karo BioAB, Box 4032, S-141 04 Huddinge, Sweden and the $Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital F-60, Novum, s-14186 Huddinge, Sweden
Full length human glucocorticoid receptor andtrun- erted via a specific intracellular receptor protein which, like cated receptor derivatives lacking the major amino- many other steroid receptors, functions as a signal-activated terminal trans-activating domain were expressed in nuclear transcriptional regulator (Beato, 1989; Freedman et stably transfectedChinese hamster ovary (CHO) cells. al., 1989, and references therein)
The amplified in the presence of increasing concentrations response is known to be mediated by a positive control eleof metal. Both synthesized receptor forms showed the expected molecular weights, as assayed by affinity labeling and immunoblotting. They were expressed at concentrations of about 350,000520,000 molecules/cellwhich corresponds toa 10-fold increase in receptor levels as compared to rat liver ment’ which is present in single or multiple copies upstream of or within target genes(reviewed in Yamamoto, 1985)
Summary
From Karo BioAB, Box 4032, S-141 04 Huddinge, Sweden and the $Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital F-60, Novum, s-14186 Huddinge, Sweden. The effect of dexamethasoonne cellular levels of the expressed full length receptor protein was analyzed in immunoblot assays employing the monoclonal antibody 5 (Okret et al, 1984) which is directed aangainst weipthitionpe the aminoterminus of thereceptor(Rusconiand Yamamoto, s full length receptor, AGR, or the ligand-binding domain, respectively, were treated with 1 V M dexamethasone in uiuo prior to analysis of RNpArotaenidn the at indicated points of time.
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