Mu-opioid receptor expression in High Five insect cells is regulated by 5' untranslated region (5'UTR).
There is increasing evidence that the 5'UTR of mRNAs affects regulation of gene expression in eukaryotic cells. We examined the overexpression of the mu-opioid receptor in High Five insect cells, employing rat mu-receptor cDNA linked to variable lenghts of their native 5'UTR. The sequences employed consist of either 209 nucleotides (termed ,,long") upstream the translation initiation site of the mu-receptor mRNA, or a truncated 5'UTR comprising only 11 nucleotides (,,short"). These constructs served to generate recombinant baculovirus for the expression of mu-receptor protein in High Five insect cells. 48 hours after baculovirus infection cells were harvested for mu-receptor characterization or RNA analysis. Scatchard analysis of radioligand binding consistently revealed three to four fold higher concentrations of the mu-opioid receptors expressed with the ,,long" over the ,,short" UTR containing baculovirus. The distinct expression rates of mu-receptors paralleled the amounts of mRNAs determined by RNase protection assay. Regardless of the distinct 5'UTR regions, the expressed opioid receptors displayed identical high affinity binding characteristics for the opioid antagonist diprenorphine and similar EC50 values to inhibit forskolin (10(-5) M) stimulated cAMP synthesis. Our results demonstrate that the native 5'UTR of the mu-opioid receptor has an enhancing effect on expression in the baculovirus/insect cell system.
Belke-Louis
G F
GF
Institute of Pharmacology, Toxicology and Pharmacy, Ludwig Maximilians University, Muenchen, Germany. Belke_Louis@pharmtox.vetmed.uni-muenchen.de
Wehmeyer
A
A
Schulz
R
R
D020121:5' Untranslated Regions / Q000502:physiology*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D000242:Cyclic AMP / Q000096:biosynthesis; D007313:Insecta; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017450:Receptors, Opioid, mu / Q000096:biosynthesis / Q000235:genetics*
D020121:5' Untranslated Regions; D012333:RNA, Messenger; D017450:Receptors, Opioid, mu; D000242:Cyclic AMP