HUMAN SEROTONIN(1B) RECEPTOR EXPRESSION IN SF9 CELLS - PHOSPHORYLATION, PALMITOYLATION, AND ADENYLYL-CYCLASE INHIBITION
HUMAN SEROTONIN(1B) RECEPTOR EXPRESSION IN SF9 CELLS - PHOSPHORYLATION, PALMITOYLATION, AND ADENYLYL-CYCLASE INHIBITION
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DOI：
10.1021/bi00094a032
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发表时间：
1993-11-02
期刊：
影响因子：
2.9
通讯作者：
ODOWD, BF
中科院分区：
文献类型：
--
作者：
NG, GYK;GEORGE, SR;ODOWD, BF
对人降钙素1B（5-HT 1B）受体的一级蛋白结构的分析揭示了磷酸化的共有位点和棕榈酰化的推定位点。为了研究这些翻译后修饰，我们在Sf 9细胞中表达了c-myc表位标记的5-HT 1B（m5-HT 1B）受体。该策略使得受体能够通过免疫印迹分析来检测，并通过使用对c-myc表位具有特异性的单克隆抗体9 E10的免疫沉淀来纯化。激动剂放射性配体[H-3]5-HT结合研究表明，表达的5-HT 1B和m5-HT 1B受体显示出神经元5-HT 1B受体的特征药理学特征。表达的受体显示高和低亲和力状态的[H-3]5-HT，这表明受体耦合到内源性G-蛋白。事实上，激动剂与高亲和力受体状态的结合在GTP γ S、Gpp（NH）p和百日咳毒素的存在下受到调节。[P-32] ADP-核糖基化实验鉴定了Sf 9膜中存在的主要约41-kDa ADP-核糖基化蛋白，其与部分纯化的牛脑G（i α）/G（o α）亚基共迁移。腺苷酸环化酶活性的测量表明，腺苷酸能激动剂介导的腺苷酸环化酶活性的抑制与其亲和力常数的效力的等级顺序。从表达m5-HT 1B受体的细胞制备的膜的免疫印迹分析和免疫沉淀材料的光亲和标记揭示了在约95和约42 kDa处的光标记物质。在约95和约42 kDa处迁移的免疫沉淀物质显示在用[P-32 i]磷酸盐或[H-3]棕榈酸进行全细胞代谢标记后被后修饰，这提供了5 HT 1B血清素受体被磷酸化和棕榈酰化的第一个证据。
Analysis of the primary protein structure ot the human serotonin1B (5-HT1B) receptor reveals consensus sites for phosphorylation and a putative site for palmitoylation. To investigate these posttranslational modifications, we have expressed a c-myc epitope-tagged 5-HT1B (m5-HT1B) receptor in Sf9 cells. This strategy enabeled receptors to be detected by immunoblot analysis and purified by immunoprecipitation using a monoclonal antibody, 9E10, specific for the c-myc epitope. Agonist radio ligand [H-3]5-HT binding studies showed that the expressed 5-HT1B and m5-HT1B receptors displayed the characteristic pharmacological profile of the neuronal 5-HT1B receptor. The expressed receptors displayed both high- and low-affinity states for [H-3]5-HT, suggesting that the receptors were coupled to endogenous G-proteins. Indeed, agonist binding to the high-affinity receptor state was regulated in the presence of GTPgammaS, Gpp(NH)p, and pertussis toxin. [P-32]ADP-ribosylation experiments identified a major approximately 41-kDa ADP-ribosylated protein present in Sf9 membranes that comigrated with partially purified bovine brain G(ialpha)/G(oalpha) subunits. Measurements of adenylyl cyclase activity in membranes from cells expressing m5-HT1B receptors showed that serotonergic agonists mediated the inhibition of adenylyl cyclase activity with a rank order of potency comparable to their affinity constants. Immunoblot analysis of membranes prepared from cells expressing m5-HT1B receptors and photoaffinity labeling of the immunoprecipitated material revealed photolabeled species at approximately 95 and at approximately 42 kDa. Immunoprecipitated material migrating at approximately 95 and approximately 42 kDa was shown to be posttranslationally modified following whole cell metabolic labeling with [P-32i]phosphate or [H-3]palmitic acid, and this provides the first evidence that the 5HT1B serotonin receptor is phosphorylated and palmitoylated.