scholarly journals Endocytosis of the ASGP receptor H1 is reduced by mutation of tyrosine-5 but still occurs via coated pits.

1991 ◽  
Vol 114 (3) ◽  
pp. 423-431 ◽  
Author(s):  
C Fuhrer ◽  
I Geffen ◽  
M Spiess
Keyword(s):  
Plasma Membrane ◽  
Lucifer Yellow ◽  
Fluid Phase ◽  
Membrane Receptors ◽  
Immunogold Localization ◽  
Wild Type ◽  
Coated Pits ◽  
Residual Rate ◽  
Tyrosine Residues ◽  
Plasma Membrane Receptors

The clustering of plasma membrane receptors in clathrin-coated pits depends on determinants within their cytoplasmic domains. In several cases, individual tyrosine residues were shown to be necessary for rapid internalization. We have mutated the single tyrosine at position 5 in the cytoplasmic domain of the major subunit H1 of the asialoglycoprotein receptor to alanine. Expressed in fibroblasts cells, the mutant protein was accumulated in the plasma membrane, and its rate of internalization was reduced by a factor of four. The residual rate of endocytosis, however, was still significantly higher than that of resident plasma membrane proteins. Upon acidification of the cytoplasm, which specifically inhibits the formation of clathrin-coated vesicles but not uptake of the fluid phase marker Lucifer yellow, residual endocytosis was blocked. By immunoelectron microscopy mutant H1 could be directly demonstrated in coated pits. The fraction of wild-type and mutant H1 present in coated pits as determined by immunogold localization correlated well with the respective rates of internalization. Thus, mutation of tyrosine-5 only partially inactivates recognition of H1 for incorporation into coated pits.

scholarly journals Plasma membrane glycosphingolipid signaling: a turning point

2021 ◽  
Author(s):  
Elena Chiricozzi
Keyword(s):  
Plasma Membrane ◽  
Chemical Synthesis ◽  
Bioinformatic Analysis ◽  
Membrane Receptors ◽  
Outer Side ◽  
Neuronal Homeostasis ◽  
Extracellular Stimuli ◽  
Conceptual Shift ◽  
Plasma Membrane Receptors ◽  
Key Events

AbstractPlasma membrane interaction is highly recognized as an essential step to start the intracellular events in response to extracellular stimuli. The ways in which these interactions take place are less clear and detailed. Over the last decade my research has focused on developing the understanding of the glycosphingolipids-protein interaction that occurs at cell surface. By using chemical synthesis and biochemical approaches we have characterized some fundamental interactions that are key events both in the immune response and in the maintenance of neuronal homeostasis. In particular, for the first time it has been demonstrated that a glycolipid, present on the outer side of the membrane, the long-chain lactosylceramide, is able to directly modulate a cytosolic protein. But the real conceptual change was the demonstration that the GM1 oligosaccharide chain is able, alone, to replicate numerous functions of GM1 ganglioside and to directly interact with plasma membrane receptors by activating specific cellular signaling. In this conceptual shift, the development and application of multidisciplinary techniques in the field of biochemistry, from chemical synthesis to bioinformatic analysis, as well as discussions with several national and international colleagues have played a key role.

Developmentally regulated expression of insulin-like growth factors by differentiated murine teratocarcinomas and extraembryonic mesoderm

Development ◽  
1986 ◽  
Vol 95 (1) ◽  
pp. 193-212
Author(s):  
John K. Heath ◽  
Wai-Kang Shi
Keyword(s):  
Plasma Membrane ◽  
Binding Protein ◽  
Membrane Receptors ◽  
Developmentally Regulated ◽  
Igf Receptors ◽  
Igf Binding ◽  
Plasma Membrane Receptors ◽  
Igf Binding Protein ◽  
Culture Supernatants ◽  
Insulin Like Growth Factors

The expression of plasma membrane receptors for insulin-like growth factors (IGFs) by PC13 embryonal carcinoma (EC) cells, and their immediate differentiated progeny PC13END was examined by binding radiolabelled IGF-I to cell monolayers. Both cell types express high-affinity IGF receptors, but the apparent number of unoccupied receptor sites falls by about 60% upon differentiation. Crosslinking studies reveal that both type 1 and type 2 IGF receptors are expressed by PC13EC cells. PC13END-cell-conditioned medium contains developmentally regulated, separable activities, one of which reacts directly with IGF-II, and the other with IGF for plasma membrane receptors. The former activity represents a soluble secreted IGF-binding protein. The latter activity is structurally and functionally similar to rat IGF-II. Polyclonal antibodies raised against purified rat IGF-II specifically recognize multiple forms of IGF in radiolabelled culture supernatants and material which closely resembles the soluble IGF-binding protein. Immunoprecipitation of radiolabelled culture supernatants with anti-rat IGF-II reveals that the differentiation of PC13EC cells is accompanied by the coexpression of IGF-like molecules and the soluble binding protein, and that IGF-like molecules are expressed by extraembryonic tissues of mesodermal origin in the early postimplantation mouse embryo. These findings show that IGF-like molecules are expressed in early mammalian development and may act in an autocrine fashion in vivo.

scholarly journals Acidification of the cytosol inhibits endocytosis from coated pits.

1987 ◽  
Vol 105 (2) ◽  
pp. 679-689 ◽  
Author(s):  
K Sandvig ◽  
S Olsnes ◽  
O W Petersen ◽  
B van Deurs
Keyword(s):  
Cell Surface ◽  
Lucifer Yellow ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Electron Microscopic ◽  
Coated Pits ◽  
Internal Ph ◽  
Microscopic Studies ◽  
Epidermal Growth ◽  
In Cells

Acidification of the cytosol of a number of different cell lines strongly reduced the endocytic uptake of transferrin and epidermal growth factor. The number of transferrin binding sites at the cell surface was increased in acidified cells. Electron microscopic studies showed that the number of coated pits at the cell surface was not reduced in cells with acidified cytosol. Experiments with transferrin-horseradish peroxidase conjugates and a monoclonal anti-transferrin receptor antibody demonstrated that transferrin receptors were present in approximately 75% of the coated pits both in control cells and in cells with acidified cytosol. The data therefore indicate that the reason for the reduced endocytic uptake of transferrin at internal pH less than 6.5 is an inhibition of the pinching off of coated vesicles. In contrast, acidification of the cytosol had only little effect on the uptake of ricin and the fluid phase marker lucifer yellow. Ricin endocytosed by cells with acidified cytosol exhibited full toxic effect on the cells. Although the pathway of this uptake in acidified cells remains uncertain, some coated pits may still be involved. However, the data are also consistent with the possibility that an alternative endocytic pathway involving smooth (uncoated) pits exists.

scholarly journals Murine fetal liver macrophages bind developing erythroblasts by a divalent cation-dependent hemagglutinin.

1988 ◽  
Vol 106 (3) ◽  
pp. 649-656 ◽  
Author(s):  
L Morris ◽  
P R Crocker ◽  
S Gordon
Keyword(s):  
Plasma Membrane ◽  
Divalent Cation ◽  
Plasma Membranes ◽  
Fetal Liver ◽  
Divalent Cations ◽  
Membrane Receptors ◽  
Erythroid Cells ◽  
Mammalian Development ◽  
Trypsin Treatment ◽  
Plasma Membrane Receptors

During mammalian development the fetal liver plays an important role in hematopoiesis. Studies with the macrophage (M phi)-specific mAb F4/80 have revealed an extensive network of M phi plasma membranes interspersed between developing erythroid cells in fetal liver. To investigate the interactions between erythroid cells and stromal M phi, we isolated hematopoietic cell clusters from embryonic day-14 murine fetal liver by collagenase digestion and adherence. Clusters of erythroid cells adhered to glass mainly via M phi, 94% of which bound 19 +/- 11 erythroblasts (Eb) per cell. Bound Eb proliferated vigorously on the surface of fetal liver M phi, with little evidence of ingestion. The M phi could be stripped of their associated Eb and the clusters then reconstituted by incubation with Eb in the presence of divalent cations. The interaction required less Ca++ than Mg++, 100 vs. 250 microM for half-maximal binding, and was mediated by a trypsin-sensitive hemagglutinin on the M phi surface. After trypsin treatment fetal liver M phi recovered the ability to bind Eb and this process could be selectively inhibited by cycloheximide. Inhibition tests showed that the Eb receptor differs from known M phi plasma membrane receptors and fetal liver M phi did not bind sheep erythrocytes, a ligand for a distinct M phi hemagglutinin. We propose that fetal liver M phi interact with developing erythroid cells by a novel nonphagocytic surface hemagglutinin which is specific for a ligand found on Eb and not on mature red cells.

scholarly journals Internalization of lectins in neuronal GERL.

1977 ◽  
Vol 73 (1) ◽  
pp. 1-13 ◽  
Author(s):  
N K Gonatas ◽  
S U Kim ◽  
A Stieber ◽  
S Avrameas
Keyword(s):  
Plasma Membrane ◽  
Membrane Receptors ◽  
Dorsal Root Ganglion Neurons ◽  
Cytochemical Study ◽  
Receptor Complexes ◽  
Mouse Dorsal Root Ganglion ◽  
Respiration Inhibitors ◽  
Ricin Agglutinin ◽  
Plasma Membrane Receptors ◽  
Ganglion Neurons

Conjugates of ricin agglutinin and phytohemagglutinin with horseradish peroxidase (HRP) were used for a cytochemical study of internalization of their plasma membrane "receptors" in cultured isolated mouse dorsal root ganglion neurons. Labeling of cells with lectin-HRP was done at 4 degrees C, and internalization was performed at 37 degrees C in a culture medium free of lectin-HRP. 15-20 min after incubation at 37 degrees C, lectin-HRP receptor complexes were seen in vesicles or tubules located near the plasma membrane. After 1-3 h at 37 degrees C, lectin-HRP-receptor complexes accumulated in vesicles and tubules corresponding to acid phosphatase-rich vesicles and tubules (GERL) at the trans aspect of the Golgi apparatus. A few coated vesicles and probably some dense bodies contained HRP after 3-6 h of incubation at 37 degrees C. Soluble HRP was not endocytosed under the conditions of this experiment or when it was present in the incubation medium at 37 degrees C. Internalization of lectin-HRP-receptor conjugates was decreased or inhibited by mitochondrial respiration inhibitors but not by cytochalasin B or colchicine. These studies indicate that lectin-labeled plasma membrane moieties of neurons are endocytosed primarily in elements of GERL.

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