Serotonin binding protein: synthesis, secretion, and recycling.
Serotonin binding protein: synthesis, secretion, and recycling.
复制标题
血清素结合蛋白：合成、分泌和回收。
DOI：
10.1046/j.1471-4159.1994.63010097.x
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发表时间：
1994
影响因子：
4.7
通讯作者：
Gershon,MD
中科院分区：
文献类型：
--
作者：
Tamir,H;Liu,KP;Hsiung,S;Adlersberg,M;Gershon,MD
5-羟色胺结合蛋白（SBP）存在于所有储存5-羟色胺（5-HT）的神经外胚层衍生细胞中。已检测到三种形式的SBP（68、56和45 kDa），干扰5-HT结合的SBP抗体与这些蛋白质中的每一种反应。目前的实验检验了两个假设：（a）56-和45-kDa形式的SBP是由68-kDa前体分子的翻译后裂解产生的;（B）45-kDa SBP是肾上腺素能分泌囊泡的组成部分。使用甲状腺髓样癌细胞作为模型进行脉冲追踪实验。这些神经直肠皮来源的细胞产生5-HT和所有三种形式的SBP。用l-[35S]甲硫氨酸脉冲标记20 min后，在37 ° C下，在存在过量未标记l-甲硫氨酸的情况下孵育细胞0、30、60或90 min。或者，在延迟或停止新合成的蛋白质从粗面内质网通过高尔基体的转运的条件（20 ° C，抑制ATP产生）下进行追踪。孵育后，洗涤并溶解细胞，并免疫沉淀SBP。对免疫沉淀物中的放射性蛋白进行电泳分离和定量。在脉冲后，发现三种形式的SBP中的每一种都被标记为35S。35S标记的68-、56-和45-kDa SBP的相对比例在每个追踪间期保持相同。当在20 ° C或在阻断ATP生物合成的条件下进行追踪时，这些比例没有改变。这些观察结果表明，每种形式的SBP都是翻译的初级产物，较小形式的SBP不是由较大分子裂解产生的，并且初级翻译产物的大小不会因进入高尔基体或高尔基体后区室而改变。当诱导分泌时，45 kDa SBP而不是56或68 kDa SBP被释放到培养基中。当在诱导分泌时向培养基中加入45 kDa SBP的抗体时，抗体结合位点在细胞表面上显示为斑块。由于这些位点，当细胞在存在45 kDa SBP抗体和豚鼠补体的情况下刺激分泌时，细胞溶解。20分钟后抗体结合位点从细胞表面消失，此时在细胞内发现SBP抗体。这表明45-kDa SBP与5-HT一起包装在分泌囊泡中。由于胞吐作用，一些45 kDa SBP在分泌过程中丢失;然而，一部分45 kDa SBP仍然结合在分泌囊泡膜的腔表面。这种蛋白质暴露于环境介质作为胞吐的结果，但当囊泡膜在囊泡再循环期间被重新捕获时被重新内化。
Serotonin binding protein (SBP) is present in all neurectodermally derived cells that store serotonin (5‐HT). Three forms of SBP have been detected (68, 56, and 45 kDa), and antibodies to SBP that interfere with the binding of 5‐HT react with each of these proteins. The current experiments test two hypotheses: (a) that the 56‐ and 45‐kDa forms of SBP are produced by posttranslational cleavage of a 68‐kDa precursor molecule; and (b) that 45‐kDa SBP is a constituent of serotonergic secretory vesicles. Pulse‐chase experiments were carried out using medullary thyroid carcinoma cells as a model. These neurectodermally derived cells produce 5‐HT and all three forms of SBP. Following pulse labeling for 20 min withl‐[35S]methionine, the cells were incubated in the presence of an excess of unlabeledl‐methionine for 0, 30, 60, or 90 min at 37°C. Alternatively, the chase was performed under conditions (20°C, inhibition of ATP generation) that delay or stop transport of newly synthesized proteins from the rough endoplasmic reticulum through the Golgi apparatus. Following incubation, the cells were washed and solubilized, and SBP was immunoprecipitated. Radioactive proteins in the immunoprecipitate were electrophoretically resolved and quantified. Immediately after the pulse, each of the three forms of SBP was found to be labeled with35S. The relative proportions of35S‐labeled 68‐, 56‐, and 45‐kDa SBP remained the same at each interval of chase. These proportions were not changed when the chase was carried out at 20°C or under conditions that blocked the biosynthesis of ATP. These observations suggest that each form of SBP is a primary product of translation, that the smaller forms of SBP are not produced by cleavage from a larger molecule, and that the size of the primary products of translation is not altered by passage to the Golgi apparatus or a post‐Golgi compartment. When secretion was induced, 45‐kDa SBP, but not 56‐ or 68‐kDa SBP, was released to the medium. When antibodies to 45‐kDa SBP were added to the medium at the time secretion was induced, antibody binding sites appeared as patches on the cell surfaces. Because of these sites, cells were lysed when they were stimulated to secrete in the presence of antibodies to 45‐kDa SBP and guinea pig complement. Antibody binding sites disappeared from cell surfaces after 20 min, at which time antibodies to SBP were found inside the cells. It is suggested that 45‐kDa SBP is packaged with 5‐HT in secretory vesicles. Some 45‐kDa SBP is lost during secretion as a result of exocytosis; however, a fraction of the 45‐kDa SBP remains bound to the luminal surface of the membrane of secretory vesicles. This protein is exposed to the ambient medium as a consequence of exocytosis, but is reinternalized when the vesicular membrane is recaptured during vesicle recycling.