[Ubiquitination of EGF receptors with C-terminal domain deletion and point mutations during endocytosis].

Analysis of ubiquitination of EGF receptor carrying different mutations of C-terminal domain was done. The mutants differed both by the set of major autophosphorylation sites that determine the way of interaction with ubiquitin-ligase c-Cbl, and by the presence of lysine residues which can be possible acceptor sites for ubiquitin. It was found that the receptor lacking tyrosine kinase activity due to lysine for phenylalanine substitution at ATP-binding site of tyrosine kinase (TK) domain (K721) failed to be ubiquitinated as well as the receptor without all binding sites for c-Cbl (CD165), while dynamics and pattern of ubiquitination of other deletion mutants was significantly different. The mutant lacking Grb2 binding sites but able to bind c-Cbl directly (CD123) was minimally ubiquitinated and only at early stages upon EGF endocytosis stimulation. At the same time, the receptor possessing all binding sites for Cbl but lacking C-terminal domain of 63 aminoacid residues (CD63) which contains two autophosphorylation sites (Y1148 and 1173) and 4 lysines, was less ubiquitinated and had more low-ubiquitinated forms comparing to the WT one. However, these lysines are not acceptor sites for ubiquitin since the full-size receptor lacking like CD63 the same major autophosphorylation sites underwent ubiquitination similar to the deletion mutant. Thus, C-terminal region of the EGF receptor, being not a substrate for ubiquitination per se, is involved in its regulation. It was also found that ubiquitination pattern at fast endocytosis differed from those at slow one. In the first case the total level of EGFR decreased dramatically as a result of efficient lysosomal degradation. The level of receptor-associated c-Cbl was practically the same, while the total intracellular c-Cbl dropped. Treatment of cells with proteasomal inhibition MG132 blocked the loss of Cbl only partially. In the second case, total amount of both EGF receptor and c-Cbl did not notably change that suggested recycling pathway for receptors even despite them beeng ubiquitinated.

[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.