Brown K D, Laurie M S, Littlewood C J, Blakeley D M, Corps A N
AFRC Institute of Animal Physiology, Babraham, Cambridge, U.K.
Biochem J. 1988 May 15;252(1):227-35. doi: 10.1042/bj2520227.
Bombesin and bombesin-related peptides such as gastrin-releasing peptide (GRP) stimulate DNA synthesis and proliferation of Swiss 3T3 cells in culture. We have used 125I-labelled [Tyr4]bombesin and 125I-labelled GRP to characterize and identify the receptors for these peptides on Swiss 3T3 cells. The binding of 125I-[Tyr4]bombesin, which retained full biological activity, was maximal between 20 and 30 min incubation at 37 degrees C, after which continued incubation led to a decline in cell-associated radioactivity. This decline was markedly slowed by the presence of lysosomal enzyme inhibitors. Specificity of the binding site was indicated by the competitive inhibition of binding by bombesin-related peptides, but not by unrelated peptides and growth factors. Scatchard analysis of binding data indicated a single class of high-affinity receptors. The calculated value for the dissociation constant (Kd) was 2.1 nM and each cell possesses approx. 240,000 receptors. Because [Tyr4]bombesin has no free amino group, 125I-GRP was used in chemical cross-linking studies. When disuccinimidyl suberate was used to covalently couple 125I-GRP to the cells, two major radiolabelled complexes were detected with molecular masses of approx. 80,000-85,000 and 140,000. The binding of 125I-[Tyr4]bombesin to the cells was pH-dependent with maximal binding at pH 6.5-7.5 and effectively no specific binding at pH values below 4.5. At 37 degrees C, cell-associated 125I-[Tyr4]bombesin quickly became resistant to removal by acidic buffers, suggesting its rapid transfer to an intracellular compartment. However, pre-incubation with unlabelled [Tyr4]bombesin did not induce down-regulation of bombesin receptors as measured by the subsequent binding of 125I-[Tyr4]bombesin. In contrast with the Swiss 3T3 cells, specific binding of 125I-[Tyr4]bombesin was not detectable in two cell lines which are biologically unresponsive to bombesin-related peptides.
蛙皮素及与蛙皮素相关的肽类，如胃泌素释放肽（GRP），可刺激培养的瑞士3T3细胞的DNA合成及增殖。我们已使用125I标记的[酪氨酸4]蛙皮素和125I标记的GRP来表征和鉴定瑞士3T3细胞上这些肽的受体。保留了全部生物活性的125I-[酪氨酸4]蛙皮素在37℃孵育20至30分钟时结合达到最大值，此后继续孵育导致细胞相关放射性下降。溶酶体酶抑制剂的存在显著减缓了这种下降。蛙皮素相关肽竞争性抑制结合，而非相关肽和生长因子则无此作用，这表明了结合位点的特异性。结合数据的Scatchard分析表明存在一类高亲和力受体。解离常数（Kd）的计算值为2.1 nM，每个细胞约有240,000个受体。由于[酪氨酸4]蛙皮素没有游离氨基，因此在化学交联研究中使用了125I-GRP。当使用辛二酸二琥珀酰亚胺酯将125I-GRP与细胞共价偶联时，检测到两种主要的放射性标记复合物，分子量约为80,000 - 85,000和140,000。125I-[酪氨酸4]蛙皮素与细胞的结合呈pH依赖性，在pH 6.5 - 7.5时结合最大，在pH值低于4.5时几乎没有特异性结合。在37℃时，细胞相关的125I-[酪氨酸4]蛙皮素很快变得对酸性缓冲液的去除具有抗性，这表明它迅速转移到细胞内区室。然而，用未标记的[酪氨酸4]蛙皮素预孵育并未如通过随后125I-[酪氨酸4]蛙皮素的结合所测量的那样诱导蛙皮素受体的下调。与瑞士3T3细胞相反，在两种对蛙皮素相关肽无生物学反应的细胞系中未检测到125I-[酪氨酸4]蛙皮素的特异性结合。
