Effect of EGF-Receptor Tyrosine Kinase Inhibitor on Rab5 Function During Endocytosis

Tyrosine autophosphorylation within the cytoplasmic tail of EGF-receptor is a key event, which in turn recruits several factors including Shc, Grb2 and Rin1 that are essential activities for receptor-mediated endocytosis and signaling. In this study, we demonstrated that treatment with AG1478, an EGF-receptor kinase inhibitor, blocked the formation of Rab5-positive endosomes as well as the activation of Rab5 upon addition of EGF. We also found that EGF-receptor catalytically inactive mutant failed to activate Rab5 upon EGF stimulation. Additionally, endosomal co-localization of Rab5 and EGF-receptor was inhibited by AG1478. Interestingly, AG1478 inhibitor did not block the formation of enlarged Rab5-positive endosomes in cells expressing Rab5 GTP hydrolysis defective mutant (Rab5:Q79L). AG1478 inhibitor also blocked the in vitroendosome fusion in a concentration-dependent manner, and more importantly, Rab5:Q79L mutant rescued it. Furthermore, addition of Rin1, a Rab5 guanine nucleotide exchange factor, partially restored endosome fusion in the presence of AG1478 inhibitor. Consistent with these observations, we also observed that Rin1 was unable to localize to membranes upon EGF-stimulation in the presence of AG1478 inhibitor. These results constitute first evidence that the enzymatic activity of a tyrosine kinase receptor is required endosome fusion via the activation of Rab5

Investigation of Atomic Level Patterns in Protein—Small Ligand Interactions

BACKGROUND: Shape complementarity and non-covalent interactions are believed to drive protein-ligand interaction. To date protein-protein, protein-DNA, and protein-RNA interactions were systematically investigated, which is in contrast to interactions with small ligands. We investigate the role of covalent and non-covalent bonds in protein-small ligand interactions using a comprehensive dataset of 2,320 complexes. METHODOLOGY AND PRINCIPAL FINDINGS: We show that protein-ligand interactions are governed by different forces for different ligand types, i.e., protein-organic compound interactions are governed by hydrogen bonds, van der Waals contacts, and covalent bonds; protein-metal ion interactions are dominated by electrostatic force and coordination bonds; protein-anion interactions are established with electrostatic force, hydrogen bonds, and van der Waals contacts; and protein-inorganic cluster interactions are driven by coordination bonds. We extracted several frequently occurring atomic-level patterns concerning these interactions. For instance, 73% of investigated covalent bonds were summarized with just three patterns in which bonds are formed between thiol of Cys and carbon or sulfur atoms of ligands, and nitrogen of Lys and carbon of ligands. Similar patterns were found for the coordination bonds. Hydrogen bonds occur in 67% of protein-organic compound complexes and 66% of them are formed between NH- group of protein residues and oxygen atom of ligands. We quantify relative abundance of specific interaction types and discuss their characteristic features. The extracted protein-organic compound patterns are shown to complement and improve a geometric approach for prediction of binding sites. CONCLUSIONS AND SIGNIFICANCE: We show that for a given type (group) of ligands and type of the interaction force, majority of protein-ligand interactions are repetitive and could be summarized with several simple atomic-level patterns. We summarize and analyze 10 frequently occurring interaction patterns that cover 56% of all considered complexes and we show a practical application for the patterns that concerns interactions with organic compounds

Diffusion of MMPs on the Surface of Collagen Fibrils: The Mobile Cell Surface – Collagen Substratum Interface

Remodeling of the extracellular matrix catalyzed by MMPs is central to morphogenetic phenomena during development and wound healing as well as in numerous pathologic conditions such as fibrosis and cancer. We have previously demonstrated that secreted MMP-2 is tethered to the cell surface and activated by MT1-MMP/TIMP-2-dependent mechanism. The resulting cell-surface collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 can initiate (MT1-MMP) and complete (MMP-2) degradation of an underlying collagen fibril. The following question remained: What is the mechanism of substrate recognition involving the two structures of relatively restricted mobility, the cell surface enzymatic complex and a collagen fibril embedded in the ECM? Here we demonstrate that all the components of the complex are capable of processive movement on a surface of the collagen fibril. The mechanism of MT1-MMP movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP) hydrolysis. It is similar to that of the MMP-1 Brownian ratchet we described earlier. In addition, both MMP-2 and MMP-9 as well as their respective complexes with TIMP-1 and -2 are capable of Brownian diffusion on the surface of native collagen fibrils without noticeable dissociation while the dimerization of MMP-9 renders the enzyme immobile. Most instructive is the finding that the inactivation of the enzymatic activity of MT1-MMP has a detectable negative effect on the cell force developed in miniaturized 3D tissue constructs. We propose that the collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 represents a Mobile Cell Surface – Collagen Substratum Interface. The biological implications of MT1-MMP acting as a molecular ratchet tethered to the cell surface in complex with MMP-2 suggest a new mechanism for the role of spatially regulated peri-cellular proteolysis in cell-matrix interactions

Rab5 and Epidermal Growth Factor Receptor During Endocytosis by Clathrin Dependent and Clathrin Independent Pathways

Endocytosis involves the formation of internal membranes from the plasma membrane lipid bilayer through which plasma membrane lipids, proteins and extracellular fluid get internalized inside the cell. Since endocytosis not only controls the uptake of molecules, but also regulates signal transduction, cell adhesion, migration and development, it is not surprising that a misregulation in the trafficking cascade may lead to various diseases ranging from neuro-degeneration and diabetes to cancer. The key ways in which molecules can get into the cell are through Clathrin Mediated Endocytosis, Non Clathrin Mediated Endocytosis and through Macropinocytosis. In essence, all of these pathways show a convergent structure as they commence with invagination of the plasma membrane, advance through various endosomal compartments that end at the lysosomes. Where they differ is in the coat that will comprise the budding vesicle and the GTPase involved in the pinching off of the vesicle. Epidermal Growth Factor Receptor (EGFR) is a well-established and best characterized member of the Receptor Tyrosine Kinase family, which is known to be involved in various cellular processes ranging from signaling to growth and proliferation. It is well known that aberrant activity of this receptor is involved in pathogenesis of numerous forms of cancer. This review will describe the current knowledge concerning EGFR, regulation of its uptake through clathrin and dynamin dependent/ independent endocytic pathways, as well as the subsequent implications in numerous disorders associated with aberrant trafficking of the aforementioned receptor

Inhibition of Rab5 Activation During Insulin Receptor-Mediated Endocytosis

Activation of receptor tyrosine kinases is a key feature in receptor signaling and membrane trafficking processes. In this study, we found thatthe insulin receptor tyrosine kinase activity is required for fusion between early endosomes. AG1024, a receptor tyrosine kinase inhibitor,blocked the in vitro endosome fusion in a concentration-dependent manner. We observed that Rab5: wild type partially rescued the fusionreaction, whereas Rab5: Q79L mutant fully rescued it. We also observed that treatment of cells with insulin receptor kinase inhibitorHNMPA-(AM)3 blocked the formation of Rab5-positive endosomes as well as the activation of Rab5 upon addition of insulin in intact cells.HNMPA-(AM)3 inhibitor also affected the endosomal co-localization of Rab5 and insulin receptor. However, the formation of Rab5: Q79Lmutant-positive endosomes were not affected by the HNMPA-(AM)3 inhibitor. In addition, HNMPA-(AM)3 inhibitor affected the associationof Rin1 to membrane upon insulin stimulation. Furthermore, Rin1 did not fully support endosome fusion in the presence of the AG1024inhibitor. These results constitute the first evidence that, at least in part, the enzymatic activity of insulin receptor is required for the fusionevents via the activation of Rab5