Overexpression of delta-opioid receptors in recombinant baculovirus-infected Trichoplusia ni "High 5" insect cells.

Abstract

"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.