Authier F, Di Guglielmo G M, Danielsen G M, Bergeron J J
Institut National de la Santé et de la Recherche Médicale U30, Hôpital Necker des Enfants Malades, 75015 Paris, France.
Biochem J. 1998 Jun 1;332 ( Pt 2)(Pt 2):421-30. doi: 10.1042/bj3320421.
Receptor-mediated endocytosis and subsequent endosomal proteolysis of [125I]TyrA14-[HisA8,HisB4,GluB10,HisB27]in sulin ([125I]TyrA14-H2 analogue), an insulin analogue exhibiting a high affinity for the insulin receptor, has been studied in liver parenchymal cells by quantitative subcellular fractionation and compared with that of wild-type [125I]TyrA14-insulin. Whereas the kinetics of uptake of the H2 analogue by liver was not different from that of insulin, the H2 analogue radioactivity after the 2 min peak declined significantly more slowly. A significant retention of the H2 analogue compared with insulin in both plasma membrane and endosomal fractions was observed and corresponded to decreased processing and dissociation of the H2 analogue. Cell-free endosomes preloaded in vivo with radiolabelled ligands and incubated in vitro processed insulin and extraluminally released insulin intermediates at a 2-3-fold higher rate than the H2 analogue. In vitro proteolysis of both non-radiolabelled and monoiodinated molecules by endosomal lysates showed a decreased response to the endosomal proteolytic machinery for the H2 analogue. However, in cross-linking and competition studies the H2 analogue exhibited an affinity for insulin-degrading enzyme identical with that of wild-type insulin. Brij-35-permeabilized endosomes revealed a 2-fold higher rate of dissociation of insulin from internalized receptors compared with the H2 analogue. After the administration of a saturating dose of both ligands, a rapid and reversible ligand-induced translocation of insulin receptor was observed, but without receptor loss. The H2 analogue induced a higher receptor concentration and tyrosine autophosphorylation of the receptor beta subunit in endosomes. Moreover, a prolonged temporal interaction of the in vivo injected H2 analogue with receptor was observed by direct binding assays performed on freshly prepared subcellular fractions. These results indicate that endosomal proteolysis for the H2 analogue is slowed as a result of an increased residence time of the analogue on the insulin receptor and a low affinity of endosomal acidic insulinase for the dissociated H2 molecule.
通过定量亚细胞分级分离法，在肝实质细胞中研究了[125I]TyrA14-[HisA8,HisB4,GluB10,HisB27]胰岛素（[125I]TyrA14-H2类似物）的受体介导的内吞作用及随后的内体蛋白水解，该胰岛素类似物对胰岛素受体具有高亲和力，并与野生型[125I]TyrA14-胰岛素进行了比较。虽然肝脏对H2类似物的摄取动力学与胰岛素无异，但2分钟峰值后的H2类似物放射性下降明显更慢。在质膜和内体组分中均观察到与胰岛素相比，H2类似物有显著保留，这与H2类似物加工和解离减少相对应。体内预先加载放射性标记配体并在体外孵育的无细胞内体对胰岛素和腔内释放的胰岛素中间体的加工速率比H2类似物高2 - 3倍。内体裂解物对非放射性标记和单碘化分子的体外蛋白水解显示，H2类似物对内体蛋白水解机制的反应降低。然而，在交联和竞争研究中，H2类似物对胰岛素降解酶的亲和力与野生型胰岛素相同。Brij - 35通透的内体显示，与H2类似物相比，胰岛素从内化受体解离的速率高2倍。给予两种配体的饱和剂量后，观察到快速且可逆的配体诱导的胰岛素受体易位，但无受体丢失。H2类似物在内体中诱导更高的受体浓度和受体β亚基的酪氨酸自磷酸化。此外，通过对新鲜制备的亚细胞组分进行直接结合测定，观察到体内注射的H2类似物与受体的相互作用时间延长。这些结果表明，H2类似物的内体蛋白水解减慢是由于该类似物在胰岛素受体上的停留时间增加以及内体酸性胰岛素酶对解离的H2分子亲和力较低所致。
