Novel biphasic traffic of endocytosed EGF to recycling and degradative compartments in lacrimal gland acinar cells

• Published in: Journal of cellular physiology 2004-04, Vol.199 (1), p.108-125
• Main Authors: Xie, Jiansong, Qian, Limin, Wang, Yanru, Rose, Chadron M., Yang, Tao, Nakamura, Tamako, Hamm-Alvarez, Sarah F., Mircheff, Austin K.
• Format: Article
• Language: English
• Subjects: Animals; Blotting, Western; Cells, Cultured; Endocytosis - physiology; Epidermal Growth Factor - metabolism; Epidermal Growth Factor - pharmacokinetics; Epithelial Cells - metabolism; Female; Lacrimal Apparatus - cytology; Lacrimal Apparatus - physiology; Microscopy, Confocal; Protein Transport - physiology; Rabbits; Receptor, Epidermal Growth Factor - metabolism; Temperature; Time Factors
• ISSN: 0021-9541; 1097-4652
• DOI: 10.1002/jcp.10458

The purpose of this study was to delineate the traffic patterns of EGF and EGF receptors (EGFR) in primary cultured acinar epithelial cells from rabbit lacrimal glands. Uptake of [125I]‐EGF exhibited saturable and non‐saturable, temperature‐dependent components, suggesting both receptor‐mediated and fluid phase endocytosis. Accumulation of [125I] was time‐dependent over a 120‐min period, but the content of intact [125I]‐EGF decreased after reaching a maximum at 20 min. Analytical fractionation by sorbitol density gradient centrifugation and phase partitioning indicated that within 20 min at 37°C [125I] reached an early endosome, basal–lateral recycling endosome, pre‐lysosome, and lysosome. Small components of the label also appeared to reach the Golgi complex and trans‐Golgi network. Intact [125I]‐EGF initially accumulated in the recycling endosome; the content in the recycling endosome subsequently decreased, and by 120 min increased amounts of [125I]‐labeled degradation products appeared in the pre‐lysosomes and lysosomes. Confocal microscopy imaging of FITC‐EGF and LysoTrackerRed revealed FITC enriched in a dispersed system of non‐acidic compartments at 20 min and in acidic compartments at 120 min. Both confocal immunofluorescence microscopy and analytical fractionation indicated that the intracellular EGFR pool was much larger than the plasma membrane‐expressed pool at all times. Cells loaded with [125I]‐EGF released a mixture of intact EGF and [125I]‐labeled degradation products. The observations indicate that in lacrimal acinar cells, EGFR and EGF–EGFR complexes continually traffic between the plasma membranes and a system of endomembrane compartments; EGF‐stimulation generates time‐dependent signals that initially decrease, then increase, EGF–EGFR traffic to degradative compartments. J. Cell. Physiol. 199: 108–125, 2004© 2003 Wiley‐Liss, Inc.