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.
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.
Alksnis
M
M
Karo Bio AB, Huddinge, Sweden.
Barkhem
T
T
Strömstedt
P E
PE
Ahola
H
H
Kutoh
E
E
Gustafsson
J A
JA
Poellinger
L
L
Nilsson
S
S
D000345:Affinity Labels; D000818:Animals; D001345:Autoradiography; D001483:Base Sequence; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics*; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D013256:Steroids / Q000378:metabolism; D014162:Transfection
D000345:Affinity Labels; D008024:Ligands; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; D013256:Steroids