Sorting and recycling efficiency of apical insulin binding sites during endocytosis in proximal tubule cells.
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.
Nielsen
S
S
Department of Cell Biology, University of Aarhus, Denmark.
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
D003432:Cross-Linking Reagents; D007328:Insulin; C008147:insulin, iodo-; D011972:Receptor, Insulin