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J. Biol. Chem., Vol. 266, Issue 16, 10078-10085, Jun, 1991
M Alksnis, T Barkhem, PE Stromstedt, H Ahola, E Kutoh, JA Gustafsson, L Poellinger and S Nilsson
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.
High level expression of functional full length and truncated glucocorticoid receptor in Chinese hamster ovary cells. Demonstration of ligand-induced down-regulation of expressed receptor mRNA and protein
Karo Bio AB, Huddinge, Sweden.
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