Expression of mu-, delta- 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 narcotic antagonist naloxone (1 microM) enhanced the production of each receptor type. However, "High Five' cells differed from Sf9 cells in a 2-3-fold higher receptor density in the cell membrane and were therefore employed for receptor characterization. In membranes of 'High Five' cells opioid receptor levels ranged from 1.0 +/- 0.2 pmol/mg protein for the kappa-opioid receptor, 1.7 +/- 0.2 pmol/mg for the delta-opioid receptor to 2.1 +/- 0.5 pmol/mg for the mu-opioid receptor. The mu-, delta- and kappa-opioid receptor agonists [D-Ala2,N-methyl-Phe4-Gly-ol5]enkephalin ([3H]DAMGO), [D-Pen2,D-Pen5]enkephalin ([3H]DPDPE) and (5 alpha, 7 alpha, 8 beta)-(+)-N-methyl-N-(7-(1-pyrrolidinyl-1-oxaspiro(4,5)dec-8-yl) benzeneacetamide ([3H]U69,563) bound to the opioid receptors with Kd values of 3.4 +/- 0.3 nM, 4.5 +/- 0.1 nM and 1.2 +/- 0.3 nM, respectively, resembling those reported for opioid receptors expressed in 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 coupling 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 convenient method for high level expression of functionally intact opioid receptors as judged by receptor binding studies, their G-protein coupling and inhibition of adenylyl cyclase.
Obermeier
H
H
Institute of Pharmacology, Toxicology and Pharmacy, Ludwig-Maximilians University Munich, Germany.
Wehmeyer
A
A
Schulz
R
R
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D044482:Benzeneacetamides; D002460:Cell Line; D000242:Cyclic AMP / Q000096:biosynthesis; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D020881:Enkephalin, D-Penicillamine (2,5)-; D004745:Enkephalins / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D007313:Insecta; D007700:Kinetics; D011485:Protein Binding; D011759:Pyrrolidines / Q000494:pharmacology; D011957:Receptors, Opioid / Q000819:agonists / Q000096:biosynthesis* / Q000378:metabolism; D017465:Receptors, Opioid, delta / Q000096:biosynthesis; D017473:Receptors, Opioid, kappa / Q000096:biosynthesis; D017450:Receptors, Opioid, mu / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000819:agonists / Q000096:biosynthesis / Q000378:metabolism
D044482:Benzeneacetamides; D004745:Enkephalins; D011759:Pyrrolidines; D011957:Receptors, Opioid; D017465:Receptors, Opioid, delta; D017473:Receptors, Opioid, kappa; D017450:Receptors, Opioid, mu; D011994:Recombinant Proteins; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D020881:Enkephalin, D-Penicillamine (2,5)-; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; C045444:U 69593