DIRECT VISUALIZATION OF BINDING, AGGREGATION, AND INTERNALIZATION OF INSULIN AND EPIDERMAL GROWTH-FACTOR ON LIVING FIBROBLASTIC CELLS
DIRECT VISUALIZATION OF BINDING, AGGREGATION, AND INTERNALIZATION OF INSULIN AND EPIDERMAL GROWTH-FACTOR ON LIVING FIBROBLASTIC CELLS
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DOI：
10.1073/pnas.75.6.2659
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发表时间：
1978-01-01
影响因子：
11.1
通讯作者：
PASTAN, I
中科院分区：
文献类型：
--
作者：
SCHLESSINGER, J;SHECHTER, Y;PASTAN, I
我们详细研究了胰岛素和表皮生长因子的荧光衍生物与3 T3 [小鼠]成纤维细胞的结合。我们使用了2种胰岛素和表皮生长因子的荧光类似物：具有7至8个罗丹明分子的高荧光衍生物或具有单个罗丹明分子/1个胰岛素或表皮生长因子分子的荧光衍生物。这两种类型的类似物保留了大量的结合亲和力，通过放射受体测定和生物活性。用敏感的视频增强显微系统可视化用荧光类似物标记的细胞，该系统使我们能够直接观察荧光激素在表面上和活的成纤维细胞内的位置。我们发现，胰岛素和表皮生长因子最初都与细胞表面扩散结合，在4 ° C时，仍然分散。在23度的几分钟内。或37度。酶-受体复合物聚集成可容易地被胰蛋白酶去除而不能被过量的天然激素去除的斑块。最初在膜平面中移动的受体-受体复合物，由于受体聚集或内化，随后变得固定。在. apprx范围内37 ℃下30分钟，在细胞内发现许多标记的激素以跳跃方式在细胞质中移动的内吞囊泡的形式存在。的聚集和固定化的激素受体复合物可能是由于激素的相互作用在细胞膜上或激素诱导的构象变化的激素受体复合物。聚集和内化的激素受体复合物可能与某些方面的激素作用，激素降解，下调受体，或负协同激素结合。
We have studied in detail the binding of fluorescent derivatives of insulin and epidermal growth factor to 3T3 [mouse] fibroblasts. We have used 2 types of fluorescent analogues of insulin and epidermal growth factor: highly fluorescent derivatives which have 7 to 8 rhodamine molecules or fluorescent derivatives which have a single rhodamine molecule/1 molecule of insulin or epidermal growth factor. Both types of analogue retained substantial binding affinity as determined by radioreceptor assays and biological activity. The cells labeled with the fluorescent analogs were visualized with a sensitive video intensification microscopic system that enabled us to directly observe the location of the fluorescent hormone on the surface and within the living fibroblasts. We found that both insulin and epidermal growth factor initially bound diffusely to the cell surface and, at 4.degree., remained dispersed. Within a few minutes at 23.degree. or 37.degree. the hormone-receptor complexes aggregated into patches that could be readily removed by trypsin but not by excess native hormone. The hormone-receptor complexes, which were initially mobile in the plane of the membrane, become immobilized later as the consequence of the receptor aggregation or internalization. Within .apprx. 30 min at 37.degree., much of the labeled hormone was found within the cell in endocytic vesicles that moved about in the cytoplasm in a saltatory manner. The aggregation and immobilization of the hormone-receptor complexes could be due to either hormone-hormone interactions on the cell membrane or a hormone-induced conformational change in the hormone-receptor complex. Aggregation and internalization of hormone-receptor complexes could be associated with certain aspects of hormone action, hormone degradation, down regulation of receptors, or negative cooperativity of hormone binding.