Thermos K, Reisine T
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104.
Mol Pharmacol. 1988 Apr;33(4):370-7.
The functional and biochemical characteristics of somatostatin (somatotropin release-inhibiting factor) (SRIF) receptor subtypes were examined in the clonal pituitary cell lines AtT-20 and GH3. SRIF inhibits evoked calcium influx into each of these cell lines. The rank order of potencies of structural analogues of SRIF to inhibit calcium influx into GH3 versus AtT-20 cells was different. Inhibitory actions of SRIF on calcium influx desensitized in AtT-20 cells but not GH3 cells. The biochemical properties of the SRIF receptor subtypes in AtT-20 and GH3 cells were assessed by photoaffinity labeling of each receptor with the nonreducible SRIF analogue [125I]CGP 23996 and the photocrosslinking agent n-hydroxysuccinimidyl-4-azidobenzoate. The covalently labeled receptors in both cell lines had the same size, 55 +/- 5 kDa, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The covalent binding of [125I]CGP-23996 to GH3 and AtT-20 cell membranes was blocked by 1 microM SRIF, somatostatin 28, Trp8-SRIF and was GTP sensitive. Analysis of the labeled receptors in GH3 and AtT-20 cell membranes by two-dimensional polyacrylamide gel electrophoresis indicated that they were of similar charge (pI = 6-6.5) and that they comigrate when applied together. Proteolysis of the GH3 and AtT-20 cell SRIF receptors with Staphylococcus aureus V-8 and thermolysin revealed similar peptide maps. Pretreatment of AtT-20 cells with different stable SRIF analogues abolished the subsequent equilibrium or covalent labeling of the SRIF receptor with [125I]CGP-23996. Similar treatment of GH3 cells did not reduce the covalent labeling of the SRIF receptor by [125I]CGP 23996. These studies indicate that the functional characteristics of SRIF receptors in GH3 and AtT-20 cells are different. However, clear differences in the biochemical properties of these receptor subtypes were not observed. Subtle variations in the structure of the SRIF receptors may therefore be responsible for the functional differences.
在垂体克隆细胞系AtT - 20和GH3中研究了生长抑素（促生长激素释放抑制因子）（SRIF）受体亚型的功能和生化特性。SRIF抑制诱发的钙离子流入这些细胞系中的每一个。SRIF结构类似物抑制钙离子流入GH3细胞与AtT - 20细胞的效价顺序不同。SRIF对钙离子流入的抑制作用在AtT - 20细胞中会脱敏，但在GH3细胞中不会。通过用不可还原的SRIF类似物[125I]CGP 23996和光交联剂N - 羟基琥珀酰亚胺 - 4 - 叠氮苯甲酸对每个受体进行光亲和标记，评估了AtT - 20和GH3细胞中SRIF受体亚型的生化特性。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳评估，两个细胞系中经共价标记的受体大小相同，为55±5 kDa。1μM的SRIF、生长抑素28、色氨酸8 - SRIF可阻断[125I]CGP - 23996与GH3和AtT - 20细胞膜的共价结合，且其对GTP敏感。通过二维聚丙烯酰胺凝胶电泳分析GH3和AtT - 20细胞膜中的标记受体表明，它们的电荷相似（pI = 6 - 6.5），并且一起应用时会共迁移。用金黄色葡萄球菌V - 8和嗜热菌蛋白酶对GH3和AtT - 20细胞的SRIF受体进行蛋白水解，显示出相似的肽图。用不同的稳定SRIF类似物预处理AtT - 20细胞可消除随后[125I]CGP - 23996对SRIF受体的平衡或共价标记。对GH3细胞进行类似处理不会降低[125I]CGP 23996对SRIF受体的共价标记。这些研究表明，GH3和AtT - 20细胞中SRIF受体的功能特性不同。然而，未观察到这些受体亚型生化特性的明显差异。因此，SRIF受体结构的细微变化可能是功能差异的原因。
