Fas ligand is targeted to secretory lysosomes via a proline-rich domain in its cytoplasmic tail

2001 ◽  
Vol 114 (13) ◽  
pp. 2405-2416 ◽  
Author(s):  
Emma J. Blott ◽  
Giovanna Bossi ◽  
Richard Clark ◽  
Marketa Zvelebil ◽  
Gillian M. Griffiths
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Fas Ligand ◽  
Cytoplasmic Tail ◽  
Cell Types ◽  
Cell Surface Receptor ◽  
Cell Surface Expression ◽  
Rich Domain ◽  
Secretory Lysosomes ◽  
In Cells

Fas ligand (FasL) induces apoptosis through its cell surface receptor Fas. T lymphocytes and natural killer cells sort newly synthesised FasL to secretory lysosomes but, in cell types with conventional lysosomes, FasL appears directly on the plasma membrane. Here, we define a proline-rich domain (PRD) in the cytoplasmic tail of FasL that is responsible for sorting FasL to secretory lysosomes. Deletion of this PRD results in cell surface expression of FasL in cells with secretory lysosomes. Positively charged residues flanking the PRD are crucial to the sorting motif and changing the charge of these residues causes mis-sorting to the plasma membrane. In cells with conventional lysosomes, this motif is not recognised and FasL is expressed at the plasma membrane. The FasL PRD is not required for endocytosis in any cell type, as deletion mutants lacking this motif are endocytosed efficiently to the lysosomal compartment. Endogenous FasL cannot internalise extracellular antibody, demonstrating that FasL does not transit the plasma membrane en route to the secretory lysosomes. We propose that an interaction of the PRD of FasL with an SH3-domain-containing protein, enables direct sorting of FasL from the Golgi to secretory lysosomes.

Effect of aging on CD11b and CD69 surface expression by vesicular insertion in human polymorphonuclear leucocytes

1999 ◽  
Vol 97 (3) ◽  
pp. 323-329 ◽  
Author(s):  
J. M. NOBLE ◽  
G. A. FORD ◽  
T. H. THOMAS
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Receptor ◽  
Cell Surface Expression ◽  
Elderly Subjects ◽  
Polymorphonuclear Leucocytes ◽  
Cd11b Expression ◽  
Cd69 Expression ◽  
Vesicle Exocytosis

The exocytosis of intracellular vesicles is an important function of the plasma membrane, which is responsible for hormone secretion, cell surface expression of antigens, ion transporters and receptors, and intracellular and intercellular signalling. Human aging is associated with many physiological and cellular changes, many of which are due to alterations in plasma membrane functioning. Alterations in vesicle externalization with age could account for many of these changes. We investigated whether alterations in vesicle exocytosis occur with increasing age by flow-cytometric determination of CD11b and CD69 expression on the surface of human polymorphonuclear leucocytes (PMN) stimulated with phorbol myristate acetate (PMA), a tumour promoter which binds to and activates protein kinase C (PKC) directly, or with formyl-Met-Leu-Phe (fMLP), which activates PKC indirectly via interactions with a cell surface receptor and G-protein, and subsequent inositol phosphate hydrolysis. Following stimulation with PMA, a decrease in the proportion of PMN expressing CD69 at high levels was observed in elderly compared with young subjects (young, 55.3%; elderly, 43.9%; P = 0.01). No aging-related differences in the proportion of PMN expressing CD11b (young, 73.7%; elderly, 68.4%; P = 0.15), or in the number of molecules of CD69 or CD11b expressed per cell, were observed. Stimulation with fMLP or low PMA concentrations resulted in full CD11b expression but minimal CD69 expression in both young and elderly subjects. Cells which expressed CD69 had no CD11b expression, while those cells expressing CD11b had minimal CD69 expression. Thus the PMA-induced expression of CD11b and CD69 in human PMN represents two separate processes, only one of which is affected in aging. CD11b expression appears to require a lesser degree of PKC stimulation compared with that required for CD69 expression. The age-associated reduction in PMA-stimulated CD69 expression may occur either at or distal to PKC activation. Such a decrease may contribute to the age-associated impairments in PMN function that contribute, in turn, to immunosenescence.

Cellular Localization and Developmental Expression of Emp Correlates with Its Role in Erythroblastic Island Formation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 537-537
Author(s):  
Shivani Soni ◽  
Manjit Hanspal
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Cellular Localization ◽  
Cell Types ◽  
Developmental Expression ◽  
Cell Surface Expression ◽  
Erythroid Cells ◽  
Cellular Environment ◽  
Primary Mouse ◽  
Macrophage Precursors

Abstract In mammals, erythropoiesis takes place in distinct anatomic units called erythroblastic islands that are consisted of a central macrophage surrounded by erythroblasts of varying maturity. The attachment of erythroblasts to macrophages within these islands promotes terminal maturation and enucleation of erythroid cells. Among a few other adhesion molecules known to date, Emp (erythroblast macrophage protein) is involved in this association. Our recent gene targeting studies showed that in the absence of Emp, erythroblastic islands are not formed and erythroid cells do not undergo enucleation. In addition, Emp null macrophages do not fully mature. Information on the changes in the expression level and cellular localization of Emp in differentiating erythroid and macrophage cells is essential for understanding the function of Emp both in the formation of erythroblastic islands and in the development of these two cell types. Previously we showed that Emp is expressed in immature erythroid precursors but not in mature erythrocytes, consistent with the fact that mature red blood cells do not adhere. However, no information is available on the expression of Emp in macrophages. To address this issue, we have used primary mouse fetal liver macrophages cultured for various time periods. Our studies showed that while Emp was expressed in all stages of maturation, the localization pattern changed dramatically during maturation: in immature macrophages, a substantial fraction of Emp was intracellular, whereas in more mature cells, Emp was expressed largely at the plasma membrane. To determine if the membrane-associated Emp is exposed on the cell surface, we employed biotin labeling of surface proteins, and compared the fraction of total Emp that is accessible to the biotinylation reagent with the fraction that is inaccessible. Our data showed that approximately 5–20% of total cellular Emp was present on the surface of immature macrophage precursors compared to 60–70% in fully matured macrophages. Cell surface expression of Emp was further confirmed by live cell staining, without prior fixation, of macrophages at different stages of maturation. Intracellular pool of Emp was present largely in the nucleus where it co-localized with the nuclear matrix marker, the spliceosome assembly factor SC35. To examine the trafficking of newly synthesized Emp, we performed pulse-chase experiments in macrophages of varying maturity. We found that nascent Emp migrated intracellularly from the nucleus to the plasma membrane more efficiently in mature macrophages than in immature cells. Incubation of erythroid cells with macrophages in culture showed that erythroid cells attached to mature macrophages but not to immature macrophage precursors. Taken together, our data shows that the temporal and spatial expression of Emp in erythroid and myeloid cells correlates with its involvement in the attachment of these two cell types in mammalian erythropoiesis. Furthermore, localization of Emp at multiple sites within the cellular environment suggests functions not limited to cell attachment.

scholarly journals Domains of the TCR beta-chain required for early thymocyte development.

1996 ◽  
Vol 184 (5) ◽  
pp. 1833-1843 ◽  
Author(s):  
H Jacobs ◽  
J Iacomini ◽  
M van de Ven ◽  
S Tonegawa ◽  
A Berns
Keyword(s):  
Cell Surface ◽  
Cytoplasmic Tail ◽  
Cell Receptor ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Beta Chain ◽  
Thymocyte Development ◽  
Developmental Transition ◽  
T Cell Receptor Beta ◽  
Thymocyte Differentiation

The T cell receptor beta (TCR beta) chain controls the developmental transition from CD4-CD8- to CD4+8+thymocytes. We show that the extracellular constant region and the transmembrane region, but not the variable domain or cytoplasmic tail of the TCR beta chain are required for this differentiation step. TCR beta mutant chains lacking the cytoplasmic tail can be found at the cell surface both in functional TCR/CD3 complexes and in a GPI-anchored monomeric form indicating that the cytoplasmic tail of the TCR beta chain functions as an ER retention signal. The concordance between cell surface expression of the mutant chains as TCR/CD3 complexes and their capacity to mediate thymocyte differentiation supports the CD3 mediated feedback model in which preTCR/CD3 complexes control the developmental transition from CD4-CD8- to CD4+CD8+thymocytes.

scholarly journals Cell Surface Expression of Endosomal Toll-Like Receptors—A Necessity or a Superfluous Duplication?

2021 ◽  
Vol 11 ◽  
Author(s):  
Matylda Barbara Mielcarska ◽  
Magdalena Bossowska-Nowicka ◽  
Felix Ngosa Toka
Keyword(s):  
Immune Response ◽  
Cell Membrane ◽  
Cell Surface ◽  
Signaling Pathways ◽  
Distinct Group ◽  
Cell Types ◽  
Cysteine Proteases ◽  
Cell Surface Expression ◽  
Critical Event ◽  
Toll Like Receptors

Timely and precise delivery of the endosomal Toll-like receptors (TLRs) to the ligand recognition site is a critical event in mounting an effective antimicrobial immune response, however, the same TLRs should maintain the delicate balance of avoiding recognition of self-nucleic acids. Such sensing is widely known to start from endosomal compartments, but recently enough evidence has accumulated supporting the idea that TLR-mediated signaling pathways originating in the cell membrane may be engaged in various cells due to differential expression and distribution of the endosomal TLRs. Therefore, the presence of endosomal TLRs on the cell surface could benefit the host responses in certain cell types and/or organs. Although not fully understood why, TLR3, TLR7, and TLR9 may occur both in the cell membrane and intracellularly, and it seems that activation of the immune response can be initiated concurrently from these two sites in the cell. Furthermore, various forms of endosomal TLRs may be transported to the cell membrane, indicating that this may be a normal process orchestrated by cysteine proteases—cathepsins. Among the endosomal TLRs, TLR3 belongs to the evolutionary distinct group and engages a different protein adapter in the signaling cascade. The differently glycosylated forms of TLR3 are transported by UNC93B1 to the cell membrane, unlike TLR7, TLR8, and TLR9. The aim of this review is to reconcile various views on the cell surface positioning of endosomal TLRs and add perspective to the implication of such receptor localization on their function, with special attention to TLR3. Cell membrane-localized TLR3, TLR7, and TLR9 may contribute to endosomal TLR-mediated inflammatory signaling pathways. Dissecting this signaling axis may serve to better understand mechanisms influencing endosomal TLR-mediated inflammation, thus determine whether it is a necessity for immune response or simply a circumstantial superfluous duplication, with other consequences on immune response.

Thranbospondin Promotes Cell-and Platelet-Substratum Adhesion

1987 ◽  
Author(s):  
George P Tuszynski ◽  
Vicki L Rothman ◽  
Andrew Murphy ◽  
Katherine Siegler ◽  
Linda Smith ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cell Types ◽  
Preliminary Evidence ◽  
Human Platelets ◽  
Cell Surface Receptor ◽  
Binding Assays ◽  
Surface Receptor

Thrombospondin (TSP), isolated from human platelets, promotes the in vitro, calcium-specific adhesion of a variety of cells, including platelets, melanoma cells, muscle cells, endothelial cells, fibroblasts, and epithelial cells. The cell adhesion-promoting activity of TSP is species independent since human, bovine, pig, rat and mouse cells all adhered to TSP. Furthermore, the cell adhesion-promoting activity of TSP is specific and not due to a nonspecific protein effect or to contamination by fibronectin, vitronectin, or laminin. That is, neither bovine serum albumin nor TSP preparations treated with a monospecific anti-TSP antibody support cell adhesion. As analyzed by polyacrylamide-gel electrophoresis and specific antibody binding assays, the TSP preparations used in these studies contained no detectable fibronectin or laminin and less than 0.04% vitronectin. The cell surface receptor for TSP appears distinct frcm that of fibronectin since an antiserum that blocks cell adhesion to fibronectin has no effect on adhesion to TSP. In addition, The platelet cell surface receptor for TSP appears distinct, frcm that of fibrinogen since thrcmbasthenic platelets adhere to TSP as well as control platelets. Antibodies to the GPIIb-GPIIIa complex block platelet adhesion to fibrinogen but have no effect on adhesion to TSP. Initial characterization of the cell surface receptor for TSP shows it to be protein in nature since cells treated with trypsin fail to adhere to TSP. In summary, our results provide the first clear evidence that TSP specifically promotes cell-substratum adhesion of a variety of cell types independent of the animal species. Our preliminary evidence suggests that the cell-surface receptor(s) for TSP is protein and that it is distinct for the receptor for fibronectin and fibrinogen. Our data suggest that TSP may play a central role in normal adhesive events mediated by platelets and other cells, such as those involved in hemostasis and wound healing. In addition, TSP may be involved in pathological adhesive events mediated by platelets and tumor cells, such as those involved in cardiovascular disease and tumor cell metastasis.

scholarly journals Palmitoylation is not required for trafficking of human anion exchanger 1 to the cell surface

2004 ◽  
Vol 378 (3) ◽  
pp. 1015-1021 ◽  
Author(s):  
Joanne C. CHEUNG ◽  
Reinhart A. F. REITHMEIER
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Palmitic Acid ◽  
Anion Exchanger ◽  
Cell Surface Expression ◽  
Co2 Removal ◽  
Anion Exchanger 1 ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

AE1 (anion exchanger 1) is a glycoprotein found in the plasma membrane of erythrocytes, where it mediates the electroneutral exchange of chloride and bicarbonate, a process important in CO2 removal from tissues. It had been previously shown that human AE1 purified from erythrocytes is covalently modified at Cys-843 in the membrane domain with palmitic acid. In this study, the role of Cys-843 in human AE1 trafficking was investigated by expressing various AE1 and Cys-843Ala (C843A) mutant constructs in transiently transfected HEK-293 cells. The AE1 C843A mutant was expressed to a similar level to AE1. The rate of N-glycan conversion from high-mannose into complex form in a glycosylation mutant (N555) of AE1 C843A, and thus the rate of trafficking from the endoplasmic reticulum to the Golgi, were comparable with that of AE1 (N555). Like AE1, AE1 C843A could be biotinylated at the cell surface, indicating that a cysteine residue at position 843 is not required for cell-surface expression of the protein. The turnover rate of AE1 C843A was not significantly different from AE1. While other proteins could be palmitoylated, labelling of transiently transfected HEK-293 cells or COS7 cells with [3H]palmitic acid failed to produce any detectable AE1 palmitoylation. These results suggest that AE1 is not palmitoylated in HEK-293 or COS7 cells and can traffic to the plasma membrane.

scholarly journals Both Freshly Prepared and Frozen-Stored Amniotic Membrane Cells Express the Complement Inhibitor CD59

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Ágnes Füst ◽  
Éva Pállinger ◽  
Adrienn Stündl ◽  
Eszter Kovács ◽  
László Imre ◽  
...  
Keyword(s):  
Cell Surface ◽  
Amniotic Membrane ◽  
Ocular Surface ◽  
Cell Types ◽  
Surface Expression ◽  
Mesenchymal Cells ◽  
Cell Surface Expression ◽  
Complement Inhibitor ◽  
Becton Dickinson ◽  
Intracellular Expression

Amniotic membrane proved to be very effective tool in the treatment of a number of ocular surface diseases. The amniotic membrane, however, has to be stored before its transplantation onto the ocular surface followed by mandatory serologic control in order to exclude the transmission of certain viruses. Therefore it is most important to study if cryopreservation of the membrane affects cell surface expression of the molecules. We measured cell surface expression of CD59, a membrane-bound complement inhibitor on the cells of freshly prepared and cryopreserved amniotic membrane. Cells of amniotic membrane were separated mechanically. Epithelial and mesenchymal cells were identified by the intracellular expression of nanog and the cell surface ICAM1 positivity, respectively. Multicolor flow cytometric immunophenotyping was used for determination of the CD59 expression. CellQuest-Pro software program (Becton Dickinson) was used both for measurements and analysis. CD59-positive cells could be detected in all investigated samples and in all investigated cell types, although the expression level of CD59 differed. CD59 was expressed both on freshly prepared and frozen-stored samples. Higher level of CD59 was detected on ICAM1+ mesenchymal cells than on nanog+ epithelial cells. Our findings indicate that amniotic membranes maintain their complement inhibiting capacity after cryopreservation.

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