EXPRESSION OF MU-OPIOID, DELTA-OPIOID AND KAPPA-OPIOID RECEPTORS IN BACULOVIRUS-INFECTED INSECT CELLS

The mu-, delta- and kappa-opioid receptors have been expressed in Sf9 and 'High Five' insect cells using the baculovirus expression system. In both cell lines highest receptor levels (pmol/mg membrane protein) were observed 48 h after infection. Concomitant exposure to the narcot ic antagonist naloxone (1 mu M) enhanced the production of each recept or type. However, 'High Five' cells differed from Sf9 cells in a 2-3-f old higher receptor density in the cell membrane and were therefore em ployed for receptor characterization. In membranes of 'High Five' cell s opioid receptor levels ranged from 1.0 +/- 0.2 pmol/mg protein for t he kappa-opioid receptor, 1.7 +/- 0.2 pmol/mg for the delta-opioid rec eptor to 2.1 +/- 0.5 pmol/mg for the mu-opioid receptor. The mu-, delt a- and kappa-opioid receptor agonists [D-Ala(2),N-methyl-Phe(4)-Gly-ol (5)]enkephalin ([H-3]DAMGO), [D-Pen(2),D-Pen(5)]enkephalin ([H-3]DPDPE ) and (5 alpha,7 alpha,8 thyl-N-(7-(1-pyrrolidinyl-1-oxaspiro(4,5)dec- 8-yl) benzeneacetamide ([H-3]U69,563) bound to the opioid receptors wi th K-d values of 3.4 +/- 0.3 nM, 4.5 +/- 0.1 nM and 1.2 +/- 0.3 nM, re spectively, resembling those reported for opioid receptors expressed i n mammalian cells. Testing the functionality of the receptors in 'High Five' cells, we found that high affinity agonist binding was strongly reduced in the presence of GTP gamma S/sodium, indicating their coupl ing to G proteins. Furthermore, activation of the three receptor types inhibited forskolin-stimulated cAMP formation. The results presented here suggest that the 'High Five' cell/baculovirus system provides a c onvenient method for high level expression of functionally intact opio id receptors as judged by receptor binding studies, their G-protein co upling and inhibition of adenylyl cyclase.