EXPRESSION OF THE HUMAN OXYTOCIN RECEPTOR IN BACULOVIRUS-INFECTED INSECT CELLS - HIGH-AFFINITY BINDING IS INDUCED BY A CHOLESTEROL CYCLODEXTRIN COMPLEX
EXPRESSION OF THE HUMAN OXYTOCIN RECEPTOR IN BACULOVIRUS-INFECTED INSECT CELLS - HIGH-AFFINITY BINDING IS INDUCED BY A CHOLESTEROL CYCLODEXTRIN COMPLEX
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DOI：
10.1021/bi00042a010
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发表时间：
1995-10-24
期刊：
影响因子：
2.9
通讯作者：
FAHRENHOLZ, F
中科院分区：
文献类型：
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作者：
GIMPL, G;KLEIN, U;FAHRENHOLZ, F
我们在杆状病毒/Sf 9细胞系统中表达了c-myc表位标记的人催产素受体。通过SDS-PAGE和随后的免疫印迹鉴定该受体为类似于50 kDa的蛋白质，其在用衣霉素处理后降低至约44 kDa。结合研究显示人催产素受体以低亲和力状态表达（Kd = 215 nM，B-max = 1.66 pmol/mg）。在以可溶性胆固醇-甲基-β-环糊精复合物的形式将胆固醇添加到膜后，我们获得了对催产素处于高亲和力状态的部分人催产素受体（Kd = 0.96 nM，B-max = 318是fmol/mg蛋白质）。在随后的研究中，我们在感染后的不同时间将胆固醇-甲基-β-环糊精复合物添加到Sf 9细胞培养基中。结合分析表明，这导致高亲和力状态的功能性受体结合位点进一步增加3倍以上（B-max = 1.08 pmol/mg）。胆固醇效应是剂量依赖性的，EC（50）约为50 μ M胆固醇。由于这些发现，我们测定了纯化的Sf 9质膜中的胆固醇和磷脂含量。在含有2%胎牛血清的培养基中生长的未处理的天然胆固醇营养缺陷型昆虫细胞具有约0.04的胆固醇/磷脂摩尔比，其比在高等真核细胞的质膜中正常发现的低约20倍。催产素受体的高亲和力结合与相应质膜中存在的胆固醇水平平行增加，在这里，我们首次表明胆固醇可以是杆状病毒/Sf 9细胞系统中表达的膜蛋白功能的关键因素。
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 similar to 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 (K-d = 215 nM, B-max = 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 (K-d = 0.96 nM and B-max = 318 is 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 (B-max = 1.08 pmol/mg), The cholesterol effect was dose-dependent, with an EC(50) of about 50 mu M 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, Here we show for the first time that cholesterol can be a critical factor for the function of membrane proteins expressed in the baculovirus/Sf9 cell system.