Overexpression of delta-opioid receptors in recombinant baculovirus-infected Trichoplusia ni "High 5" insect cells.
"High 5" cells derived from Trichoplusia ni ovaries were infected with baculovirus bearing the cDNA of the mouse delta-opioid receptor. The maximal binding capacity for the narcotic antagonist [3H] naltrindole was 1.4 pmol/mg of membrane protein, and that for the agonist [3H][D-penicillamine2,D-penicillamine5]enkephalin (DPDPE) was 0.3 pmol/mg. DPDPE proved highly potent in competing with its tritiated analogue at delta-receptors of NG108-15 hybrid cells and of High 5 and Sf9 insect cells. However, in insect cells the opioid was more than 100-fold less effective in competing with [3H]naltrindole as compared with the mammalian cells. This decline in potency was counteracted in a dose-dependent manner by exposure of High 5 membranes to the exogenous G protein G(o), which increased the binding capacity for DPDPE. Functional studies revealed a dose-dependent inhibition (up to 30%) by opioids on forskolin-stimulated cyclic AMP synthesis, and this effect was potentiated by G(o). Quantification of G alpha o and G alpha i disclosed striking differences between Sf9 and High 5 insect cells, both of which overexpressed the cloned delta-opioid receptor. Although no inhibitory G proteins were detected in membranes of Sf9 cells, High 5 cells contained 0.5 pmol of G alpha o/mg of membrane protein, and a 20-fold higher concentration for G alpha i. The distinct G-protein expression in insect cells may be considered an advantage for studying functions of G protein-coupled receptors.
Wehmeyer
A
A
Institute of Pharmacology, Toxicology, and Pharmacy, University of Munich, Germany.
Schulz
R
R
D000262:Adenylyl Cyclases / Q000378:metabolism; D000700:Analgesics / Q000494:pharmacology; D000818:Animals; D016367:Baculoviridae; D001667:Binding, Competitive / Q000502:physiology; D015153:Blotting, Western; D020881:Enkephalin, D-Penicillamine (2,5)-; D004745:Enkephalins / Q000494:pharmacology; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D015151:Immunoblotting; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism; D051379:Mice; D009271:Naltrexone / Q000031:analogs & derivatives / Q000494:pharmacology; D009292:Narcotic Antagonists / Q000494:pharmacology; D010053:Ovary / Q000737:chemistry / Q000166:cytology / Q000201:enzymology; D017465:Receptors, Opioid, delta / Q000819:agonists / Q000032:analysis / Q000235:genetics*; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D018411:Spodoptera / Q000166:cytology / Q000821:virology*; D014316:Tritium; D014764:Viral Proteins / Q000235:genetics
D000700:Analgesics; D004745:Enkephalins; D008565:Membrane Proteins; D009292:Narcotic Antagonists; D017465:Receptors, Opioid, delta; D011994:Recombinant Proteins; D014764:Viral Proteins; D014316:Tritium; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009271:Naltrexone; D020881:Enkephalin, D-Penicillamine (2,5)-; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; C055382:naltrindole