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.

Detection, cDNA cloning and sequencing of canine interleukin 12.

In canine peripheral blood mononuclear cells (PBMC) the mRNAs coding for both subunits of canine interleukin 12 (IL-12) were identified using reverse transcription polymerase chain reaction (RT-PCR). Stimulation of canine PBMC with Staphylococcus aureus strain Cowan plus Concanavalin A for 5 h resulted in significant mRNA synthesis. Likewise, inactivated vaccinia virus induced IL-12 mRNA synthesis, however with different kinetics. The complete nucleotide sequence for both IL-12 subunits was determined using rapid amplification of cDNA ends (RACE)-PCR and cloning of amplified specific cDNAs. Computer-aided amino acid (aa) sequence comparison of both canine IL-12 subunits revealed more than 80% identity with the amino acid sequences of six other mammalian species. Closest relationship was found to human, porcine, bovine and cervine IL-12. However, no reactivity was found with antibodies directed against human IL-12, when supernatants of stimulated canine PBMC were tested. Supernatants of canine PBMC stimulated for IL-12 release also induced interferon gamma (IFN-gamma) mRNA as detectable by RT-PCR; however, it was not clear whether IFN-gamma mRNA synthesis was due to an IL-12 specific effect or other stimuli. As to the stimulating effect of IL-12 on canine IFN-gamma mRNA synthesis, recombinant human IL-12 was found to be a good inducer. Since IL-12 is regarded a major regulatory molecule of T-cell-mediated immune response and cell growth our work on the cloning and sequencing of this cytokine from dogs lays the basis for future investigations on the biological and possible therapeutic role of canine IL-12.

Rat beta-adrenergic receptor kinases 1 and 2 in mouse neuroblastoma X rat glioma NG 108-15 hybrid cells.

Beta-adrenergic receptor kinase (betaARK, EC 2.7.1.-) has been implicated in the phosphorylation of G protein-coupled receptors, including opioid receptors. Since delta-opioid receptors of mouse neuroblastoma x rat glioma hybrid cells (NG 108-15) desensitize upon activation, this investigation was designed to find out whether NG 108-15 cells contain betaARK activity. Using the reverse transcription polymerase chain reaction technique, we identified two mRNAs, one coding for rat betaARK1 and the other for rat betaARK2. No hint was found for the presence of mouse betaARK. Examining the cytosolic betaARK activity in these hybrid cells using rhodopsin as substrate, we found a strict functional dependence on the presence of exogenous G protein subunit Gbetagamma. This relationship reflects a characteristic for betaARK1 and 2 out of the known G protein-coupled receptor kinases. Finally, highly purified recombinant betaARK1 proved active to phosphorylate enriched delta-opioid receptor preparations in an opioid agonist-dependent manner. The results reported here provide the basis to study more closely the molecular function of G protein-coupled receptor kinases in a cell line (NG 108-15) most frequently used to investigate acute and chronic opioid actions.