Characterization of ligand binding and processing by bombesin receptors in an insulin-secreting cell line.
Characterization of ligand binding and processing by bombesin receptors in an insulin-secreting cell line.
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DOI：
10.1042/bj2470731
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发表时间：
1987-11
期刊：
影响因子：
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通讯作者：
S. Swope;A. Schonbrunn
中科院分区：
文献类型：
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作者：
S. Swope;A. Schonbrunn
蛙皮素是一种十四肽，其通过分离的胰岛和HIT-T15细胞（仓鼠胰岛细胞的克隆系）在体内刺激胰岛素分泌。在本研究中，我们使用[125 I-Tyr 4]蛙皮素的特点在HIT-T15细胞蛙皮素受体。[125I-Tyr4]蛙皮素结合是时间和温度依赖性的：分别在37、22和4 ℃下45 min、90 min和10 h后出现最大结合。此后，细胞相关放射性在37 ℃和22 ℃下降，但在4 ℃没有下降。在4 ℃下测量的[125I-Tyr4]蛙皮素结合的Scatchard分析表明，HIT-T15细胞含有单一类别的结合位点（约等于85000个/细胞），表观Kd为0.9 +/-0.11 nM。结构无关的神经肽没有竞争[125I-Tyr4]铃蟾肽结合。然而，蛙皮素和四种蛙皮素类似物在抑制[125 I-Tyr 4]蛙皮素结合方面的相对效力与它们刺激胰岛素释放的能力相关。受体介导的处理[125 I-Tyr4]蛙皮素检查通过使用酸洗（0.2 M-乙酸/0.5 M-NaCl，pH 2.5）解离表面结合的肽从细胞。[125I-Tyr4]蛙皮素在4 ℃结合后，超过85%的细胞相关放射性可以通过酸释放。当温度升高至37 ℃时，结合的放射性迅速（t1/2小于3 min）转化为耐酸状态。这些结果表明，受体结合的[125I-Tyr4]蛙皮素是内在的温度依赖性的方式。事实上，整个配体-受体复合物似乎被内化，因为在37 ℃下用100 nM-蛙皮素预处理细胞90分钟，使[125 I-Tyr 4]蛙皮素的随后结合降低了90%。在37 ℃下结合孵育30 min后，通过从细胞中提取的物质的反相色谱法测定细胞相关放射性的化学性质。尽管70%的饱和结合放射性与完整的[125I-Tyr4]蛙皮素共洗脱，但90%的放射性随后从细胞中解离，以游离碘的形式进行色谱分析。至少有一些受体结合的[125 I-Tyr 4]蛙皮素的降解似乎发生在溶酶体中，因为氯喹增加了细胞积累的[125 I-Tyr 4]蛙皮素在37 ℃，并减缓释放的放射性。（400字处截断摘要）
Bombesin is a tetradecapeptide which stimulates insulin secretion in vivo by isolated islets and by HIT-T15 cells, a clonal line of hamster pancreatic-islet cells. In the present study we have used [125I-Tyr4]bombesin to characterize bombesin receptors in HIT-T15 cells. [125I-Tyr4]Bombesin binding was time- and temperature-dependent: maximum binding occurred after 45 min, 90 min and 10 h at 37, 22 and 4 degrees C respectively. Thereafter, cell-associated radioactivity declined at 37 degrees C and 22 degrees C but not at 4 degrees C. Scatchard analysis of [125I-Tyr4]bombesin binding measured at 4 degrees C showed that HIT-T15 cells contain a single class of binding sites (approximately equal to 85000/cell) with an apparent Kd of 0.9 +/- 0.11 nM. Structurally unrelated neuropeptides did not compete for [125I-Tyr4]bombesin binding. However, the relative potencies of bombesin and four bombesin analogues in inhibiting the binding of [125I-Tyr4]bombesin correlated with their ability to stimulate insulin release. Receptor-mediated processing of [125I-Tyr4]bombesin was examined by using an acid wash (0.2 M-acetic acid/0.5 M-NaCl, pH 2.5) to dissociate surface-bound peptide from the cells. Following [125I-Tyr4]bombesin binding at 4 degrees C, more than 85% of the cell-associated radioactivity could be released by acid. When the temperature was then increased to 37 degrees C, the bound radioactivity was rapidly (t1/2 less than 3 min) converted into an acid-resistant state. These results indicate that receptor-bound [125I-Tyr4]bombesin is internalized in a temperature-dependent manner. In fact, the entire ligand-receptor complex appeared to be internalized, since pretreatment of cells with 100 nM-bombesin for 90 min at 37 degrees C decreased the subsequent binding of [125I-Tyr4]bombesin by 90%. The chemical nature of the cell-associated radioactivity was determined by reverse-phase chromatography of the material extracted from cells after a 30 min binding incubation at 37 degrees C. Although 70% of the saturably bound radioactivity was co-eluted with intact [125I-Tyr4]bombesin 90% of the radioactivity subsequently dissociated from cells chromatographed as free iodide. At least some of the degradation of receptor-bound [125I-Tyr4]bombesin appeared to occur in lysosomes, since chloroquine increased the cellular accumulation of [125I-Tyr4]bombesin at 37 degrees C and slowed the release of radioactivity.(ABSTRACT TRUNCATED AT 400 WORDS)