c-Cbl ubiquitinates the EGF receptor at the plasma membrane and remains receptor associated throughout the endocytic route

2001 ◽  
Vol 114 (11) ◽  
pp. 2167-2178 ◽  
Author(s):  
Annemieke A. de Melker ◽  
Gerda van der Horst ◽  
Jero Calafat ◽  
Hans Jansen ◽  
Jannie Borst
Keyword(s):  
Plasma Membrane ◽  
Tyrosine Kinase ◽  
Egf Receptor ◽  
Ring Finger ◽  
Endocytic Pathway ◽  
Immunogold Electron Microscopy ◽  
Coated Pits ◽  
Receptor System ◽  
Regulatory Capacity ◽  
Ubiquitin Conjugating Enzymes

Cbl family members have an evolutionarily conserved role in attenuating receptor tyrosine kinase function. Their negative regulatory capacity depends on a Ring finger domain that interacts with ubiquitin conjugating enzymes. Cbl molecules constitute a novel type of E3 or ubiquitin ligase family that is recruited to phosphotyrosine motifs. Ubiquitination of the receptor system is coupled to its downregulation, but it is unclear at which point in the endocytic pathway Cbl molecules come into play. Using low temperature and a dynamin mutant, we find that c-Cbl associates with and ubiquitinates the activated epidermal growth factor (EGF) receptor at the plasma membrane in the absence of endocytosis. With the aid of confocal microscopy and immunogold electron microscopy, we could demonstrate that c-Cbl associates with the EGF receptor at the plasma membrane prior to receptor recruitment into clathrin-coated pits and remains associated throughout the clathrin-mediated endocytic pathway. c-Cbl and the EGF receptor also colocalize in internal vesicles of multivesicular endosomes. Our data are consistent with a role for c-Cbl in clathrin-mediated endocytosis of tyrosine kinase receptors, as well as their intracellular sorting.

scholarly journals The R-SNARE Endobrevin/VAMP-8 Mediates Homotypic Fusion of Early Endosomes and Late Endosomes

2000 ◽  
Vol 11 (10) ◽  
pp. 3289-3298 ◽  
Author(s):  
Wolfram Antonin ◽  
Claudia Holroyd ◽  
Ritva Tikkanen ◽  
Stefan Höning ◽  
Reinhard Jahn
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Subcellular Fractionation ◽  
Immunogold Electron Microscopy ◽  
Fusion Reactions ◽  
Coated Pits ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Golgi Network

Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of thetrans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.

scholarly journals The Inhibitory Effect of ErbB2 on Epidermal Growth Factor-induced Formation of Clathrin-coated Pits Correlates with Retention of Epidermal Growth Factor Receptor-ErbB2 Oligomeric Complexes at the Plasma Membrane

2005 ◽  
Vol 16 (12) ◽  
pp. 5832-5842 ◽  
Author(s):  
Camilla Haslekås ◽  
Kamilla Breen ◽  
Ketil W. Pedersen ◽  
Lene E. Johannessen ◽  
Espen Stang ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Inhibitory Effect ◽  
Coated Pits ◽  
Transfected Cells ◽  
Epidermal Growth

By constructing stably transfected cells harboring the same amount of epidermal growth factor (EGF) receptor (EGFR), but with increasing overexpression of ErbB2, we have demonstrated that ErbB2 efficiently inhibits internalization of ligand-bound EGFR. Apparently, ErbB2 inhibits internalization of EGF-bound EGFR by constitutively driving EGFR-ErbB2 hetero/oligomerization. We have demonstrated that ErbB2 does not inhibit phosphorylation or ubiquitination of the EGFR. Our data further indicate that the endocytosis deficiency of ErbB2 and of EGFR-ErbB2 heterodimers/oligomers cannot be explained by anchoring of ErbB2 to PDZ-containing proteins such as Erbin. Instead, we demonstrate that in contrast to EGFR homodimers, which are capable of inducing new clathrin-coated pits in serum-starved cells upon incubation with EGF, clathrin-coated pits are not induced upon activation of EGFR-ErbB2 heterodimers/oligomers.

scholarly journals Isolation and preliminary characterization of the major membrane boundaries of the endocytic pathway in lymphocytes.

1990 ◽  
Vol 111 (5) ◽  
pp. 1811-1823 ◽  
Author(s):  
B D Beaumelle ◽  
A Gibson ◽  
C R Hopkins
Keyword(s):  
Plasma Membrane ◽  
Protein Composition ◽  
Endocytic Pathway ◽  
Coated Pits ◽  
Selective Labeling ◽  
Specific Proteins ◽  
T Lymphoma ◽  
Cellular Compartments ◽  
Density Shift

Plasma membrane, coated pits, endosomes, and lysosomes were isolated from a mouse T lymphoma cell line using a density shift protocol in which these compartments were selectively loaded with gold conjugates. The plasma membrane was prepared after selective labeling for 1 h at 2 degrees C with gold-ricin and gave a yield of 40% according to enzymatic and antigenic markers. Endosomes were obtained by loading the cells for 2 h at 22 degrees C with gold complexed to an antimouse transferrin receptor mAb. Coated pits were isolated using a similar procedure, but after an incubation at 10 degrees C, which allowed deep invagination of the pits but prevented internalization. The yield (calculated using the recovery of [125I]transferrin) was 32% for endosomes and 10% for coated pits. Finally lysosomes were prepared by loading the cells for 18 h at 37 degrees C with gold low density lipoproteins (LDLs) followed by a 3-h chase at 37 degrees C with LDL alone. The final lysosome yield (based on the recovery of lysosomal enzymes) was 16%. Studies of the protein composition of these cellular compartments on two-dimensional gels showed that while some major proteins are present throughout the pathway, specific proteins can be identified in each of the isolated fractions. The greatest change in the pattern of protein constituents seen along the pathway was between endosomal and lysosomal preparations.

scholarly journals Role for a P-type H+-ATPase in the acidification of the endocytic pathway of Trypanosoma cruzi

2005 ◽  
Vol 392 (3) ◽  
pp. 467-474 ◽  
Author(s):  
Mauricio Vieira ◽  
Peter Rohloff ◽  
Shuhong Luo ◽  
Narcisa L. Cunha-E-Silva ◽  
Wanderley De Souza ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Trypanosoma Cruzi ◽  
Subcellular Fractionation ◽  
Etiologic Agent ◽  
Proton Pumping ◽  
Endocytic Pathway ◽  
Immunogold Electron Microscopy ◽  
Intermediate Form ◽  
Confocal Immunofluorescence Microscopy ◽  
P Type

Previous studies in Trypanosoma cruzi, the etiologic agent of Chagas disease, have resulted in the cloning and sequencing of a pair of tandemly linked genes (TcHA1 and TcHA2) that encode P (phospho-intermediate form)-type H+-ATPases with homology to fungal and plant proton-pumping ATPases. In the present study, we demonstrate that these pumps are present in the plasma membrane and intracellular compartments of three different stages of T. cruzi. The main intracellular compartment containing these ATPases in epimastigotes was identified as the reservosome. This identification was achieved by immunofluorescence assays and immunoelectron microscopy showing their co-localization with cruzipain, and by subcellular fractionation and detection of their activity. ATP-dependent proton transport by isolated reservosomes was sensitive to vanadate and insensitive to bafilomycin A1, which is in agreement with the localization of P-type H+-ATPases in these organelles. Analysis by confocal immunofluorescence microscopy revealed that epitope–tagged TcHA1-Ty1 and TcHA2-Ty1 gene products are localized in the reservosomes, whereas the TcHA1-Ty1 gene product is additionally present in the plasma membrane. Immunogold electron microscopy showed the presence of the H+-ATPases in other compartments of the endocytic pathway such as the cytostome and endosomal vesicles, suggesting that in contrast with most cells investigated until now, the endocytic pathway of T. cruzi is acidified by a P-type H+-ATPase.

scholarly journals The paramyxovirus simian virus 5 hemagglutinin-neuraminidase glycoprotein, but not the fusion glycoprotein, is internalized via coated pits and enters the endocytic pathway.

1996 ◽  
Vol 7 (1) ◽  
pp. 155-172 ◽  
Author(s):  
G P Leser ◽  
K J Ector ◽  
R A Lamb
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Simian Virus ◽  
Endocytic Pathway ◽  
Coated Pits ◽  
Immunogold Staining ◽  
Simian Virus 5 ◽  
Fusion Glycoprotein ◽  
Infected Cells ◽  
Endosomal System

The hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of the paramyxovirus simian virus 5 (SV5) are expressed on the surface of virus-infected cells. Although the F protein was found to be expressed stably, the HN protein was internalized from the plasma membrane. HN protein lacks known internalization signals in its cytoplasmic domain that are common to many integral membrane proteins that are internalized via clathrin-coated pits. Thus, the cellular pathway of HN protein internalization was examined. Biochemical analysis indicated that HN was lost from the cell surface with a t1/2 of approximately 45-50 min and turned over with a t1/2 of approximately 2 h. Immunofluorescent analysis showed internalized SV5 HN in vesicle-like structures in a juxtanuclear pattern coincident with the localization of ovalbumin. In contrast the SV5 F glycoprotein and the HN glycoprotein of the highly related parainfluenza virus 3 (hPIV-3) were found only on the cell surface. Immunogold staining of HN on the surface of SV5-infected CV-1 cells and examination using electron microscopy, showed heavy surface labeling that gradually decreased with time. Concomitantly, gold particles were detected in the endosomal system and with increasing time, gold-labeled structures having the morphology of lysosomes were observed. On the plasma membrane approximately 5% of the gold-labeled HN was found in coated pits. The inhibition of the pinching-off of coated pits from the plasma membrane by cytosol acidification significantly reduced HN internalization. Internalized HN was co-localized with gold-conjugated transferrin, a marker for the early endosomal compartments, and with gold-conjugated bovine serum albumin, a marker for late endosomal compartments. Taken together, these data strongly suggest that the HN glycoprotein is internalized via clathrin-coated pits and delivered to the endocytic pathway.

scholarly journals The protein tyrosine kinase p56lck inhibits CD4 endocytosis by preventing entry of CD4 into coated pits

1992 ◽  
Vol 117 (2) ◽  
pp. 279-290 ◽  
Author(s):  
A Pelchen-Matthews ◽  
I Boulet ◽  
DR Littman ◽  
R Fagard ◽  
M Marsh
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Coated Pits ◽  
Protein Tyrosine ◽  
Src Gene ◽  
Fluid Phase Endocytosis ◽  
Nih 3T3

The lymphocyte glycoprotein CD4 is constitutively internalized and recycled in nonlymphoid cells, but is excluded from the endocytic pathway in lymphocytic cells (Pelchen-Matthews, A., J. E. Armes, G. Griffiths, and M. Marsh. 1991. J. Exp. Med. 173: 575-587). Inhibition of CD4 endocytosis is dependent on CD4 expressing an intact cytoplasmic domain and is only observed in cells where CD4 can interact with the protein tyrosine kinase p56lck, a member of the src gene family. We have expressed p56lck, p60c-src, or chimeras of the two proteins in CD4-transfected NIH-3T3 or HeLa cells. Immunoprecipitation of CD4 and in vitro kinase assays showed that p56lck and the lck/src chimera, which contains the NH2 terminus of p56lck, can associate with CD4. In contrast, p60c-src and the src/lck chimera, which has the NH2 terminus of p60c-src, do not associate with CD4. Endocytosis assays using radioiodinated anti-CD4 monoclonal antibodies demonstrated that coexpression of CD4 with p56lck, but not with p60c-src, inhibited CD4 endocytosis, and that the extent of the inhibition depended directly on the relative levels of CD4 and p56lck expressed. The uptake of mutant CD4 molecules which cannot interact with p56lck was not affected. Measurement of the fluid-phase endocytosis of HRP or the internalization of transferrin indicated that the effect of p56lck was specific for CD4, and did not extend to other receptor-mediated or fluid-phase endocytic processes. Immunogold labeling of CD4 at the cell surface and observation by electron microscopy demonstrated directly that p56lck inhibits CD4 endocytosis by preventing its entry into coated pits.

scholarly journals Association and Colocalization of Eps15 with Adaptor Protein-2 and Clathrin

1997 ◽  
Vol 136 (4) ◽  
pp. 811-821 ◽  
Author(s):  
Sanne van Delft ◽  
Christopher Schumacher ◽  
Willem Hage ◽  
Arie J. Verkleij ◽  
Paul M.P. van Bergen en Henegouwen
Keyword(s):  
Egf Receptor ◽  
Critical Role ◽  
Adaptor Protein ◽  
Coated Vesicles ◽  
Endocytic Pathway ◽  
Egf Receptors ◽  
Coated Pits ◽  
Early Endosomes ◽  
Endosomal Membranes ◽  
Cell Fractions

Eps15 has been identified as a substrate of the EGF receptor tyrosine kinase. In this report, we show that activation of the EGF receptor by either EGF or TGF-α results in phosphorylation of Eps15. Stimulation of cells with PDGF or insulin did not lead to Eps15 phosphorylation, suggesting that phosphorylation of Eps15 is a receptor-specific process. We demonstrate that Eps15 is constitutively associated with both α-adaptin and clathrin. Upon EGF stimulation, Eps15 and α-adaptin are recruited to the EGF receptor. Using a truncated EGF receptor mutant, we demonstrate that the regulatory domain of the cytoplasmic tail of the EGF receptor is essential for the binding of Eps15. Fractionation studies reveal that Eps15 is present in cell fractions enriched for plasma membrane and endosomal membranes. Immunofluorescence studies show that Eps15 colocalizes with adaptor protein-2 (AP-2) and partially with clathrin. No colocalization of Eps15 was observed with the early endosomal markers rab4 and rab5. These observations indicate that Eps15 is present in coated pits and coated vesicles of the clathrin-mediated endocytic pathway, but not in early endosomes. Neither AP-2 nor clathrin are required for the binding of Eps15 to coated pits or coated vesicles, since in membranes lacking AP-2 and clathrin, Eps15 still shows the same staining pattern. These findings suggest that Eps15 may play a critical role in the recruitment of active EGF receptors into coated pit regions before endocytosis of ligand-occupied EGF receptors.

scholarly journals Association with Membrane Protrusions Makes ErbB2 an Internalization-resistant Receptor

2004 ◽  
Vol 15 (4) ◽  
pp. 1557-1567 ◽  
Author(s):  
Anette M. Hommelgaard ◽  
Mads Lerdrup ◽  
Bo van Deurs
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Transferrin Receptor ◽  
Egf Receptor ◽  
Immunogold Labeling ◽  
Cross Linking ◽  
Coated Pits ◽  
Membrane Protrusions ◽  
Epidermal Growth ◽  
Bulk Membrane

In contrast to the epidermal growth factor (EGF) receptor, ErbB2 is known to remain at the plasma membrane after ligand binding and dimerization. However, why ErbB2 is not efficiently down-regulated has remained elusive. Basically, two possibilities exist: ErbB2 is internalization resistant or it is efficiently recycled after internalization. By a combination of confocal microscopy, immunogold labeling electron microscopy, and biochemical techniques we show that ErbB2 is preferentially associated with membrane protrusions. Moreover, it is efficiently excluded from clathrin-coated pits and is not seen in transferrin receptor-containing endosomes. This pattern is not changed after binding of EGF, heregulin, or herceptin. The exclusion from coated pits is so pronounced that it cannot just be explained by lack of an internalization signal. Although ErbB2 is a raft-associated protein, the localization of ErbB2 to protrusions is not a result of raft binding. Also, an intact actin cytoskeleton is not required for keeping ErbB2 away from coated pits. However, after efficient cross-linking, ErbB2 is removed from protrusions to occur on the bulk membrane, in coated pits, and in endosomes. These data show that ErbB2 is a remarkably internalization-resistant receptor and suggest that the mechanism underlying the firm association of ErbB2 with protrusions also is the reason for this resistance.

scholarly journals Sorting signals in the MHC class II invariant chain cytoplasmic tail and transmembrane region determine trafficking to an endocytic processing compartment.

1994 ◽  
Vol 126 (2) ◽  
pp. 317-330 ◽  
Author(s):  
C G Odorizzi ◽  
I S Trowbridge ◽  
L Xue ◽  
C R Hopkins ◽  
C D Davis ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mhc Class Ii ◽  
Cytoplasmic Tail ◽  
Class Ii ◽  
Endocytic Pathway ◽  
Invariant Chain ◽  
Transmembrane Region ◽  
Coated Pits ◽  
Endocytic Compartment ◽  
Sorting Signals

Targeting of MHC class II molecules to the endocytic compartment where they encounter processed antigen is determined by the invariant chain (Ii). By analysis of Ii-transferrin receptor (TR) chimera trafficking, we have identified sorting signals in the Ii cytoplasmic tail and transmembrane region that mediate this process. Two non-tyrosine-based sorting signals in the Ii cytoplasmic tail were identified that mediate localization to plasma membrane clathrin-coated pits and promote rapid endocytosis. Leu7 and Ile8 were required for the activity of the signal most distal to the cell membrane whereas Pro15 Met16 Leu17 were important for the membrane-proximal signal. The same or overlapping non-tyrosine-based sorting signals are essential for delivery of Ii-TR chimeras, either by an intracellular route or via the plasma membrane, to an endocytic compartment where they are rapidly degraded. The Ii transmembrane region is also required for efficient delivery to this endocytic processing compartment and contains a signal distinct from the Ii cytoplasmic tail. More than 80% of the Ii-TR chimera containing the Ii cytoplasmic tail and transmembrane region is delivered directly to the endocytic pathway by an intracellular route, implying that the Ii sorting signals are efficiently recognized by sorting machinery located in the trans-Golgi.

scholarly journals Functional reconstitutional of the human epidermal growth factor receptor system in Xenopus oocytes.

1990 ◽  
Vol 111 (4) ◽  
pp. 1661-1671 ◽  
Author(s):  
L K Opresko ◽  
H S Wiley
Keyword(s):  
Tyrosine Kinase ◽  
Intracellular Calcium ◽  
Oocyte Maturation ◽  
Mammalian Cells ◽  
Xenopus Oocytes ◽  
Kinase Activity ◽  
Egf Receptor ◽  
Biological Activities ◽  
Tyrosine Kinase Activity ◽  
Receptor System

We have expressed the human EGF receptor (hEGF-R) in Xenopus oocytes by injecting mRNA synthesized in vitro using SP6 vectors containing receptor cDNAs. Each oocyte could express over 1 x 10(10) receptors of a single affinity class and these were able to bind and rapidly internalize EGF. Occupancy resulted in receptor tyrosine autophosphorylation, downregulation, and release of intracellular calcium. Occupied receptors also rapidly induced meiotic maturation in stage VI oocytes. Receptors lacking tyrosine kinase activity bound EGF normally, but did not downregulate or induce any biological responses. The rate of oocyte maturation was proportional to hEGF-R occupancy and was significantly faster than progesterone-induced maturation at nanomolar EGF concentrations. Mutant hEGF-R truncated at residue 973 displayed identical phenotypes in both mammalian cells and oocytes in that they were defective in their ability to release intracellular calcium, undergo ligand induced internalization and receptor downregulation. However, these receptors were fully capable of inducing oocyte maturation. The remarkable retention of specific biological activities of different hEGF-R in the context of oocytes suggests that this receptor system interacts with generally available cellular components that have been conserved during evolution. In addition, it suggests that cell surface tyrosine kinase activity may play an important role in regulating resumption of the cell cycle.

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