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 the baculovirus/Sf9 cell system. The receptor was identified by SDS-PAGE and subsequent immunoblot as a approximately 50 kDa protein which decreased to about 44 kDa upon treatment with tunicamycin. Binding studies showed that the human oxytocin receptor was expressed in a low-affinity state (Kd = 215 nM; Bmax = 1.66 pmol/mg). After addition of cholesterol in the form of a soluble cholesterol-methyl-beta-cyclodextrin complex to the membranes, we obtained part of the human oxytocin receptor in its high-affinity state for oxytocin (Kd = 0.96 nM and Bmax = 318 fmol/mg of protein). In subsequent studies, we added the cholesterol-methyl-beta-cyclodextrin complex to the Sf9 cell culture medium at various times post infection. Binding analysis showed that this results in a more than 3-fold further increase in functional receptor binding sites of high-affinity state (Bmax = 1.08 pmol/mg). The cholesterol effect was dose-dependent, with an EC50 of about 50 microM cholesterol. Due to these findings, we determined the cholesterol and phospholipid 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Gimpl
G
G
Max-Planck-Institut für Biophysik, Frankfurt, Germany.
Klein
U
U
Reiländer
H
H
Fahrenholz
F
F
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology*; D003505:Cyclodextrins / Q000378:metabolism / Q000494:pharmacology*; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D008563:Membrane Lipids / Q000032:analysis; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010121:Oxytocin / Q000378:metabolism; D010743:Phospholipids / Q000032:analysis; D011485:Protein Binding; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics; D018045:Receptors, Oxytocin / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection / Q000235:genetics; D047392:beta-Cyclodextrins
D003505:Cyclodextrins; D008563:Membrane Lipids; D008565:Membrane Proteins; D010743:Phospholipids; D021382:Protein Sorting Signals; D018045:Receptors, Oxytocin; D011994:Recombinant Proteins; D047392:beta-Cyclodextrins; D010121:Oxytocin; D002784:Cholesterol; C031215:betadex