title: Sorting and recycling efficiency of apical insulin binding sites during endocytosis in proximal tubule cells.
abstract: Intracellular traffic and recycling of apical insulin binding sites are examined in isolated, perfused proximal tubules. The endocytic binding sites were specific as revealed by 90% reduction in 125I-labeled insulin binding by 10(-5) M insulin. The traffic was followed by developing a chemical cross-linking method to covalently label the binding sites. Only 3% of cross-linked insulin-gold was transported to the lysosomes, reflecting high sorting and recycling efficiency. Correspondingly, only 4% of cross-linked 125I-insulin was degraded, and only 5% of the electron microscopy-autoradiographic grains was associated with lysosomes. No label was transferred to the Golgi apparatus; thus neither lysosomes nor Golgi apparatus is involved in the recycling. In contrast, approximately 40% of non-cross-linked ligand was transferred to the lysosomes. Tubules first pretreated with cross-linker and then perfused with insulin-gold or 125I-labeled insulin-like growth factor I revealed lysosomal accumulation and degradation at control levels. Thus the cross-linker does not interfere with membrane or protein processing. The study also provides evidence for a vesicular transtubular transport because insulin-gold was transcytosed to the basolateral part of the cells and to the intracellular spaces (0.5%). In contrast cross-linked label was never observed in intercellular spaces, suggesting sorting of apical binding sites, a mechanism contributing to maintenance of cell polarity. In conclusion, traffic, sorting, and recycling of binding sites take place with high efficiency.
authors: Department of Cell Biology, University of Aarhus, Denmark.
authors: S
authors: S
authors: Nielsen
mesh_terms: D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D003432:Cross-Linking Reagents; D004705:Endocytosis; D005260:Female; D066298:In Vitro Techniques; D007328:Insulin / Q000031:analogs & derivatives / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000378:metabolism* / Q000648:ultrastructure; D008247:Lysosomes / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D015388:Organelles / Q000378:metabolism / Q000648:ultrastructure; D010477:Perfusion; D011817:Rabbits; D011972:Receptor, Insulin / Q000378:metabolism* / Q000648:ultrastructure; D013379:Substrate Specificity
chemical_list: D003432:Cross-Linking Reagents; D007328:Insulin; C008147:insulin, iodo-; D011972:Receptor, Insulin