[Analysis of tyrphostin AG1478 effect on behavior of internalized EGF receptor at different stages of endocytosis]

In the present work the effect of specific inhibitor of the receptor tyrosine kinase, tyrphostin AG1478, has been analyzed for behavior of internalized EGF receptor at different stages upon stimulation of endocytosis. It was found that tyrphostin addition in 30 min after endocytosis stimulation resulted in recycling of a significant portion of 125I-EGF onto cell surface. This portion was decreasing with time. EGF-receptor complexes, being recycled under action of AG1478, however, did not dissociate possibly because of tyrphostin ability to initiate receptor oligomerization in the absence of the ligand which can possibly affect dissociation constants. It was found that only a portion of EGF receptor localized in early endosomes was able to recycle upon TK inhibition. Addition of the inhibitor in 30 and 60 min after endocytosis stimulation resulted in decrease of labeled EGF degradation. At early stages internalized EGF-receptor complexes was blocked mostly in early endosomes, while at late stages their accumulation occured in incompletely matured late endosomes. These data speak in favor of late endocytic stage existence transition through which depends on the receptor TK. Besides, tyrphostin addition in 90 min after endocytosis led not to decrease, but on the contrary, to increase in degradation. That speaks about theexistence of the mechanisms providing a time window during which receptor TK can carry out the functions which are not connected directly with endocytosis.