[Effect of an vacuolar proton pump inhibitor bafilomycin A1 on the intracellular processing of markers for receptor-mediated and liquid phase endocytosis]. / Vliianie ingibitora vakuoliarnoi protonnoi pompy bafilomitsina A1 na vnutrikletochnyi protsessing markerov retseptor-oposredovannogo i zhidkofaznogo éndotsitoza.

By means of subcellular fractionation in density Percoll gradients, immunoblotting and immunofluorescense, the effect of BafA1 on endocytosis of EGF-receptor complexes and horse radish peroxidase (HRP) in A431, HER14 and HC11 cell lines was studied. It was shown that the pretreatment of all used cell lines with BafA1 completely inhibited EGF degradation, but did not interfere with the delivery of significant portion of EGF-receptor complexes to late endosomes and lysosomes and transition of the receptor to juxtranuclear region. At the same time, BafA1 was found to dramatically inhibit the delivery of fluid phase marker HRP to late endosomes of A431 cells. The BafA1 effect on endocytosis of high concentrations of EGF was similar to that on HRP endocytosis. Regulatory mechanisms of early-to-late endosomal compartment transition are discussed.

[Effect of nocodazole on endocytosis of epidermal growth factor receptor]. / Vliianie nokodazola na éndotsitoz retseptora épidermal'nogo faktora rosta.

During endocytosis EGF-receptor complexes are transported from early peripheral endosomes to late juxtranuclear-located endosomes to be then degraded in lysosomes. It is suggested that such a spatial organization of endosomal compartments is maintained by microtubule system and is necessary for lysosomal degradation of endocytosed cargo. In the present work, a study was made of the influence of Nocodazole, a microtubule depolymerizing agent, on endocytosis of fluid phase marker HRP and EGF entering the cell via receptor-mediated endocytosis. By subcellular fractionation in Percoll gradient it was shown that Nocodazole did not affect HRP internalization but stimulated its accumulation in a fraction sedimented together with late endosomes, thus preventing HRP delivery to lysosomes. On the contrary, Nocodazole exerted no influence on dynamics of compartmentalization and lysosomal degradation of EGF-receptor complexes. Moreover, no alterations were found in the functioning of a so well-known EGF-stimulated signal transduction pathway as MAP-kinase cascade. At the same time microtubule depolymerization dramatically changed the morphology of endosomal compartments abolishing juxtranuclear localization of late endosomes. Our data suggest that translocation of EGF-receptor complexes is not necessary for their normal lysosomal processing. Rab7, traditionally considered as a marker of late endosomes, has been found to demonstrate in Nocodazole-treated cells, in contrast to the control, a low extent of co-localization with endosomal structures. It could be supposed that the role of Rab7 is not so much to mediate early-to-late endosome transition as to maintain spatial organization of endosomal apparatus by mediating endosome-cytoskeleton interactions.

[The characteristics of protein p66--a substrate of the epidermal growth factor receptor]. / Kharakteristika belka p66--substrata retseptora épidermal'nogo faktora rosta.

The p66-kDa protein--a substrate of EGF receptor, is characterized. It is shown that the molecule of p66 is recognized by antibodies to phospholipase C gamma 1, includes conservative SH2--SH3 domains and lacks N-terminal part of PLC gamma 1. Protein p66 is associated to the EGF-R molecule on 992 tyrosine. Protein p66 binds to both membrane and internalized EGF-Rs and is redistributed with the receptors during receptor-mediated endocytosis. It is supposed that p66 may play a regulatory role in signal transduction.

[The effect of nocodazole on the redistribution of phosphoinositide-specific phospholipase C gamma 1 during mitogenic signal transduction in A-431 cells]. / Vliianie nokodazola na pereraspredelenie fosfoinozitid-spetsificheskooi fosfalipazy C gamma 1 v protsesse peredachi mitogennogo signala v kletkakh A-431.

Phosphoinositide-specific phospholipase C gamma 1 is associated with EGF receptor in A-431 cells after EGF treatment. It is shown that phospholipase C gamma 1 is co-localized with internalized receptors as well as with membrane ones during receptor-mediated endocytosis. Nocodazole is known as an inhibitor of microtubule assembly, thus leading to a complete disappearance of microtubule network. Nocodazole had no effect on phospholipase C gamma 1--EGF receptor association and co-localization, but the intracellular distribution of both the proteins differed dramatically. Phospholipase C gamma 1 and EGF receptor were localized in endosomes in the periphery of the cell. Besides, pretreatment of A-431 cells with nocodazole resulted in decreasing tyrosine phosphorylation of some proteins. These data suggest that the internalized receptor may serve as an additional starting point for triggering cell signalling.

[The association of a protein regulator of intracellular Rab7 membrane transport with endosomes containing an epidermal growth factor receptor with activated and inactivated tyrosine kinase]. / Assotsiatsiia belkovogo reguliatora vnutrikletochnogo membrannogo transporta Rab7 s éndosomami, soderzhashchimi retseptor épidermal'nogo faktora rosta s aktivirovannoi i neaktivirovannoi tirozinkinazoi.

By double indirect immunofluorescent microscopy, Rab7, traditionally considered as a late endosomal marker, has been demonstrated to colocalize with an internalized epidermal growth factor receptor (EGFR) possessing active and inactive tyrosine kinase (TK). The epidermal growth factor (EGF), which induces TK activity of EGFR, and monoclonal antibody Mab 108, which does not, have been exploited as ligands to stimulate endocytosis of EGFR. Colocalization between EGFR and Rab7 has been detected at both early (10 min) and delayed (60 and 120 min) endocytosis of EGFR, while it turned out to be much more obvious at the later ones. A comparison between EGFR-mediated and peroxidase fluid-phase endocytoses has revealed that Rab7 failed to be recruited on endosomal structures, containing peroxidase, even after 180 min endocytosis. Subcellular fractionation of endosomes containing 125I-EGF and 125I-Mab 108 in Percoll density gradients, in parallel with the analysis of ligand degradation, have verified the efficient transition of EGF-receptor complexes into the late endosomes and retention of Mab 108-receptor complexes within the early (sorting) endosomes. Taken together, the data cotained suggest that endogenous Rab7 is able to be recruited not only on late but also on maturating sorting EGFR-containing endosomes, thus mediating sorting along the EGFR endocytotic pathway.

[The dependence of the type of endocytosis of EGF receptor complexes on the basal level of tyrosine kinase activity in the epidermal growth factor receptor]. / Zavisimost' tipa éndotsitoza EFR-retseptornykh kompleksov ot bazal'nogo urovnia aktivnosti tirozinkinazy retseptora épidemal'nogo faktora rosta.

Two types of EGF-mediated endocytosis have been identified in A431 cells by the method of subcellular fractionation in Percoll density gradients. One ("slow") type of endocytosis is characterized by 125I-EGF retention in the fraction of light endosomes, while the other ("fast") type demonstrates efficient 125I-EGF transition into heavy endosomes and lysosomes. 32P-ATP phosphorylation assay of Percoll fractions, followed by alkaline treatment on the gels, has demonstrated that "slow" cells reveal an increased basal level of EGF-receptor tyrosine kinase (TK) activity compared to the "fast" cells. Pretreatment of "fast" cells with Mn2+, which was shown to induce TK stimulation without EGF (Mohammadi [correction of Muhammedi et al., 1993), caused a dramatic decrease in 125I-EGF transition to heavy endosomes and lysosomes. Analysis of tyrosine phosphorylation of the receptor, being performed in Mn(2+)-pretreated A431 cells, has confirmed the significant increase of 32P-incorporation into unoccupied EGFR. Taken together, our data suggest that sorting of internalized EGF-receptor complexes depends on the basal TK activity level of EGFR.

[The endocytosis of the epidermal growth factor (EGF) in EGF-dependent cells of the HC11 mouse mammary epithelium line]. / Endotsitoz épidermal'nogo faktora rosta (EFR) v EFR-zavisimykh kletkakh épieliia molochnoi zhelezy myshi linii HC11.

Mouse epithelial mammary cell line HC11, expressing about 30 thousands receptors for epidermal growth factor (EGF) per cell, is a physiological target for this growth factor. It is found that EGF behaves as a strong mitogen for HC11 cells: EGF (10 ng/ml) produced more than a 6-fold increase in 14C-thymidine incorporation into DNA of the cells, while 10% serum stimulated only a 2.5-fold increase, compared to control. It was shown that about 60-80% of surface-bound 125I-EGF were internalized within the first 5 min upon stimulation of endocytosis, regardless of cultivation conditions. However, the serum and EGF affected on later steps of endocytosis in different way. A prolonged presence of EGF in the growth medium before the experiment led lo a significant increase in transition of internalized 125I-EGF from light to heavy endosomes. At the same time, I 0 % serum caused a dramatic decrease if the rate of 125I-EGF degradation, which was resulted in accumulation of un-degraded growth factor in lysosomes. However, serum did not influence the earlier steps of endocytosis. A conclusion has been made that it is the efficiency of EGF transition from light to heavy endosomes (which means sorting of EGF-receptor complexes for degradative pathway) that is regulated by EGF-dependent manner in HC11 cells.

[The dependence of the endocytosis of the epidermal growth factor receptors on the degree of receptor occupancy]. / Zavisimost' éndotsitoza retseptorov épidermal'nogo faktora rosta ot stepeni zaniatosti retseptorov.

Two subpopulations of epidermal growth factor (EGF) receptor with different affinity to EGF have been demonstrated for many cell types. There are reasons to assume a key role of high-affinity receptors in stimulation of cell response to EGF. Nevertheless, characteristics of its action remain obscure. In the present work an attempt has been made to study internalization and intracellular compartmentalization of high-affinity receptors. For this purpose endocytosis of 125I-EGF in A431 cells was stimulated by low (less than 1 nM) EGF concentrations as well as after blocking of low-affinity binding sites with specific monoclonal antibodies 2E9. By subcellular fractionation in 17% Percoll gradient it was demonstrated that in both cases internalized 125I-EGF was found first in light, and then in heavy endosomes staying there for a long time. Effectiveness of 125I-EGF delivery to prelysosomal heavy endosomes as well as initial internalization rate is maximal at low EGF concentrations and is reduced dramatically with increasing of receptor occupancy. Monoclonal antibody Mab108 specifically recognizing high-affinity receptors were capable of stimulation of receptor internalization with initial rate higher than that of high EGF concentrations, but Mab108-receptor complexes were localized in light endosomes. Preincubation of the cells with low concentrations of EGF led to redistribution of considerable portion of 125I-Mab108 into heavy endosomes, which may be a result of high-affinity receptor tyrosine kinase activation. Our data confirm a hypothesis of TK involvement in regulation of both internalization and sorting of EGF-receptor complexes. Structural organization of high-affinity receptors is not sufficient for receptor targeting to degradation pathway.