Overexpression of δ‐Opioid Receptors in Recombinant Baculovirus‐Infected <i>Trichoplusia ni</i>“High 5” Insect Cells

Abstract

Abstract: “High 5” cells derived from Trichoplusia ni ovaries were infected with baculovirus bearing the cDNA of the mouse δ‐opioid receptor. The maximal binding capacity for the narcotic antagonist [ 3 H]naltrindole was 1.4 pmol/mg of membrane protein, and that for the agonist [ 3 H][ d ‐penicillamine 2 , d ‐penicillamine 5 ]enkephalin (DPDPE) was 0.3 pmol/mg. DPDPE proved highly potent in competing with its tritiated analogue at δ‐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 [ 3 H]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 Gα o and Gα i disclosed striking differences between Sf9 and High 5 insect cells, both of which overexpressed the cloned δ‐opioid receptor. Although no inhibitory G proteins were detected in membranes of Sf9 cells, High 5 cells contained 0.5 pmol of Gα o /mg of membrane protein, and a 20‐fold higher concentration for Gα i . The distinct G‐protein expression in insect cells may be considered an advantage for studying functions of G protein‐coupled receptors.