EXPRESSION OF THE HUMAN OXYTOCIN RECEPTOR IN BACULOVIRUS-INFECTED INSECT CELLS - HIGH-AFFINITY BINDING IS INDUCED BY A CHOLESTEROL CYCLODEXTRIN COMPLEX

We have expressed a c-myc epitope-tagged human oxytocin receptor in th e baculovirus/Sf9 cell system. The receptor was identified by SDS-PAGE and subsequent immunoblot as a similar to 50 kDa protein which decrea sed to about 44 kDa upon treatment with tunicamycin, Binding studies s howed that the human oxytocin receptor was expressed in a low-affinity state (K-d = 215 nM, B-max = 1.66 pmol/mg). After addition of cholest erol in the form of a soluble cholesterol-methyl-beta-cyclodextrin com plex to the membranes, we obtained part of the human oxytocin receptor in its high-affinity state for oxytocin (K-d = 0.96 nM and B-max = 31 8 is fmol/mg of protein). In subsequent studies, we added the choleste rol-methyl-beta-cyclodextrin complex to the Sf9 cell culture medium at various times post infection. Binding analysis showed that this resul ts in a more than 3-fold further increase in functional receptor bindi ng sites of high-affinity state (B-max = 1.08 pmol/mg), The cholestero l effect was dose-dependent, with an EC(50) of about 50 mu M cholester ol. Due to these findings, we determined the cholesterol and phospholi pid content in purified Sf9 plasma membranes. The untreated naturally cholesterol auxotroph insect cells grown in medium with 2% fetal calf serum had a molar cholesterol/phospholipid ratio of about 0.04, which is approximately 20-fold lower than normally found in plasma membranes of higher eukaryotic cells. The high-affinity binding of the oxytocin receptor increased in parallel with the cholesterol levels present in the corresponding plasma membranes, Here we show for the first time t hat cholesterol can be a critical factor for the function of membrane proteins expressed in the baculovirus/Sf9 cell system.