Purification and mass spectrometric analysis of the kappa opioid receptor.
Purification and mass spectrometric analysis of the kappa opioid receptor.
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kappa 阿片受体的纯化和质谱分析。
DOI：
10.1016/j.brainres.2008.06.121
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发表时间：
2008
期刊：
影响因子：
2.9
通讯作者：
Howells,RichardD
中科院分区：
文献类型：
--
作者：
Wannemacher,KennethM;Terskiy,Alexandra;Bian,Shengjie;Yadav,PremN;Li,Hong;Howells,RichardD
建立了稳定表达大鼠κ-阿片受体（rKOR）的克隆人胚肾（HEK）293细胞系，所述大鼠κ-阿片受体（rKOR）在氨基末端具有FLAG表位。[3 H]二丙诺啡的Kd为1.1±0.2 nM，Bmax为2.6±0.4 pmol/mg。强啡肽A（1-13），U69，593和纳洛酮竞争性抑制[3 H]二丙诺啡结合，Ki值分别为2.0，18和18 nM，与先前报道的未修饰受体的亲和力一致。U69，593以浓度依赖性方式刺激[35 S]GTPγS结合，并引起丝裂原活化蛋白（MAP）激酶磷酸化，表明活化的表位标记受体触发了适当的信号通路。免疫印迹分析表明，两个免疫反应性受体物种的表观分子量为42和52 kDa的表达。先前的研究表明，42 kDa蛋白定位于细胞内，是52 kDa受体的前体，该受体存在于细胞表面。用n-十二烷基-β-d-麦芽吡喃糖苷从转染的HEK 293细胞膜中提取rKOR。依次使用麦胚凝集素层析，Sephacryl S300凝胶过滤层析，抗FLAG免疫亲和层析和SDS/PAGE允许纯化的52 kDa受体。MALDI-TOF质谱法用于鉴定用胰蛋白酶和溴化氰连续凝胶内消化后来自rKOR的肽。检测到18个rKOR肽，对应于受体的27.1%覆盖率。前体选择性MS/MS证实了大多数这些肽的身份。此外，我们已经确定热休克蛋白70（HSP 70）作为rKOR相互作用的蛋白质。
A clonal human embryonic kidney (HEK) 293 cell line was established that stably expressed the rat κ-opioid receptor (rKOR) with a FLAG epitope at the amino terminus. The Kdfor [3H]diprenorphine was 1.1±0.2 nM, and the Bmaxwas 2.6±0.4 pmol/mg. Dynorphin A (1–13), U69,593 and naloxone competitively inhibited [3H]diprenorphine binding with Kivalues of 2.0, 18 and 18 nM, respectively, in good agreement with previously reported affinities for the unmodified receptor. U69,593 stimulated [35S]GTPγS binding in a concentration-dependent manner and caused phosphorylation of mitogen-activated protein (MAP) kinase, indicating that the activated epitope-tagged receptor triggered appropriate signaling pathways. Immunoblot analysis demonstrated that two immunoreactive receptor species with apparent molecular masses of 42 and 52 kDa were expressed. Previous studies indicated that the 42 kDa protein was localized intracellularly and was a precursor of the 52 kDa receptor, which was present at the cell surface. rKOR was extracted from transfected HEK 293 cell membranes with n-dodecyl-β-d-maltopyranoside. Sequential use of wheat germ agglutinin chromatography, Sephacryl S300 gel filtration chromatography, anti-FLAG immunoaffinity chromatography and SDS/PAGE permitted purification of the 52 kDa receptor. MALDI-TOF mass spectrometry was used to identify peptides derived from rKOR following sequential in-gel digestion with trypsin and cyanogen bromide. Eighteen rKOR peptides were detected, corresponding to 27.1% coverage of the receptor. Precursor-selective MS/MS confirmed the identity of most of these peptides. In addition, we have identified heat shock protein 70 (HSP70) as a rKOR-interacting protein.