Tightly regulated and inducible expression of a yoked hormone-receptor complex in HEK 293 cells.
We have previously reported the construction of a constitutively active luteinizing hormone receptor by covalently linking a fused heterodimeric hormone to the extracellular domain of the G protein-coupled receptor. This yoked hormone-receptor complex (YHR) was found to produce high levels of cAMP in the absence of exogenous hormone. Stable lines expressing YHR were generated in HEK 293 cells to obtain lines with different expression levels; however, in a relatively short time of continued passage, it was found that YHR expression was greatly reduced. Herein, we describe the development of clonal lines of HEK 293 cells in which the expression of YHR is under the control of a tetracycline-regulated system. Characterization of clonal lines revealed tight control of YHR expression both by dose and time of incubation with doxycycline. These experiments demonstrated a good correlation between expression levels of the receptor and basal cAMP production. Moreover, the reduction in receptor expression following doxycycline removal revealed that YHR mRNA and protein decayed at similar rates, again suggesting a strong linkage between mRNA and protein levels. The controlled expression of YHR in this cell system will allow for a more detailed analysis of the signaling properties associated with constitutive receptor activation and may prove to be advantageous in developmental studies with transgenic animals.
Meehan
T P
TP
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
Puett
D
D
Narayan
P
P
D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D004318:Doxycycline / Q000494:pharmacology; D005640:Follicle Stimulating Hormone / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics; D011962:Receptors, FSH / Q000378:metabolism*; D011974:Receptors, LH / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013752:Tetracycline / Q000494:pharmacology
D006063:Chorionic Gonadotropin; D012333:RNA, Messenger; D011962:Receptors, FSH; D011974:Receptors, LH; D011993:Recombinant Fusion Proteins; C102587:yoked hormone receptor, recombinant; D005640:Follicle Stimulating Hormone; D000242:Cyclic AMP; D013752:Tetracycline; D004318:Doxycycline