scholarly journals Internalization and degradation of macrophage Fc receptors during receptor-mediated phagocytosis.

1983 ◽  
Vol 96 (3) ◽  
pp. 887-895 ◽  
Author(s):  
I S Mellman ◽  
H Plutner ◽  
R M Steinman ◽  
J C Unkeless ◽  
Z A Cohn
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Rabbit Antiserum ◽  
Fc Receptors ◽  
Peritoneal Macrophages ◽  
Fc Receptor ◽  
Plasma Membrane Proteins ◽  
Coated Particles ◽  
Macrophage Receptors ◽  
Mouse Peritoneal Macrophages

Macrophage receptors for the Fc domain of immunoglobulin G (IgG) can mediate the efficient binding and phagocytosis of IgG-coated particles. After internalization, phagocytic vacuoles fuse with lysosomes, initiating the degradation of their contents. Using specific monoclonal and polyclonal antireceptor antibodies, we have now analyzed the internalization and fate of Fc receptors during the uptake of IgG-coated erythrocytes and erythrocyte ghosts by mouse peritoneal macrophages. Receptor-mediated phagocytosis led to the selective and largely irreversible removal of Fc receptors (greater than 50%) from the macrophage plasma membrane. The expression of several other plasma membrane proteins (including a receptor for complement), recognized by a series of antimacrophage monoclonal antibodies, was affected only slightly. Interiorized Fc receptors were rapidly and selectively degraded. This was demonstrated by a series of turnover studies in which Fc receptor was immunoprecipitated from lysates of 125I-labeled macrophages. These experiments were made possible by the development of a polyclonal rabbit antiserum, raised against isolated Fc receptor, which recognized the receptor even in the presence of bound ligand. In control cells, the receptor turned over with a t1/2 of approximately 10 h; after phagocytosis, greater than 50% of the receptors were degraded with a t1/2 of less than 2 h. The turnover of other unrelated plasma membrane proteins was unaffected (t1/2 of 18-23 h) under these conditions.

scholarly journals Studies on the mechanism of phagocytosis. I. Requirements for circumferential attachment of particle-bound ligands to specific receptors on the macrophage plasma membrane.

1975 ◽  
Vol 142 (5) ◽  
pp. 1263-1282 ◽  
Author(s):  
F M Griffin ◽  
J A Griffin ◽  
J E Leider ◽  
S C Silverstein
Keyword(s):  
Plasma Membrane ◽  
Sheep Erythrocyte ◽  
Peritoneal Macrophages ◽  
Membrane Receptors ◽  
Macrophage Receptors ◽  
Specific Receptors ◽  
Particle Ingestion ◽  
Plasma Membrane Receptors ◽  
Mouse Peritoneal Macrophages

These experiments were designed to evaluate the role of macrophage plasma membrane receptors for the third component of complement (C) and for the Fc portion of IgG in the ingestion phase of phagocytosis. Sheep erythrocyte (E) were coated with anti-E IgG [E(IgG)]; these E(IgG) were then attached to cultivated monolayers of mouse peritoneal macrophages under conditions which reversibly inhibit ingestion of E(IgG). The E(IgG)-macrophage complexes were further incubated under similar conditions with an antimacrophage IgG fraction which blocks Fc receptor-mediated ingestion but has no effect upon ingestion mediated by other phagocytic receptors. When these cultures were subsequently incubated under conditions optimal for particle ingestion, phagocytosis of the IgG-coated erythrocytes did not occur; the erythrocytes remained bound to the Fc receptors of the macrophage plasma membrane. To determine whether ligands must cover the entire surface of an attached particle to permit ingestion of that particle, C-coated E [E(IgM)C] were bound to the C receptors of thioglycollate-induced (activated) macrophages at 4 degrees C. E(IgM)C-macrophage complexes were then trypsinized at 4 degrees C, a procedure which resulted in cleavage of erythrocyte-bound C3b molecules to a form of C3 not recognized by the macrophage receptors for C3b. Under the conditions used, trypsin did not affect the attachment of E(IgM)C to the macrophage surface or the macrophage receptors for C3b. When these trypsin treated E(IgM)C-macrophage complexes were incubated at 37 degrees C, the bound E(IgM)C were not ingested; the erythrocytes remained attached to the macrophage plasma membrane via the macrophage's C receptors. These results indicate that attachment of a particle to specific receptors on the macrophage plasma membrane is not sufficient to trigger ingestion of that particle. Rather, ingestion requires the sequential, circumferential interaction of particle-bound ligands with specific plasma membrane receptors not involved in the initial attachment process.

scholarly journals MACROPHAGE-MELANOMA CELL HETEROKARYONS

1971 ◽  
Vol 134 (4) ◽  
pp. 947-962 ◽  
Author(s):  
Saimon Gordon ◽  
Zanvil Cohn
Keyword(s):  
Melanoma Cell ◽  
Peritoneal Macrophages ◽  
Red Cells ◽  
Similar Treatment ◽  
Coated Particles ◽  
Macrophage Receptors ◽  
Ehrlich Ascites ◽  
Surface Receptor ◽  
Plasma Membrane Receptor ◽  
Mouse Peritoneal Macrophages

Mouse peritoneal macrophages possess a specific plasma membrane receptor for antibody-coated particles. Sheep red cells coated with rabbit 7S antibody attach readily to the macrophage surface and are subsequently interiorized. The fusion of macrophage with nonphagocytic mouse melanoma cells produces heterokaryons in which the macrophage receptor is drastically altered. The receptor is present shortly after fusion and heterokaryons are actively phagocytic. The ability to bind and ingest red cells is, however, progressively lost over the next 12–24 hr and does not reappear thereafter. Exposure of heterokaryons to trypsin (1–100 µg/ml for 30 min at 37°C) results in the reappearance of initial receptor activity and the unmasking of the surface receptor. This property is again lost upon subsequent cultivation. The masking process takes place when cells are cultivated in the absence of IgG so that the adsorption of antibody from the medium is not responsible for this phenomenon. Inhibition of heterokaryon protein synthesis preserves phagocytic activity in a reversible fashion and prevents the masking of macrophage receptors. Inhibition of melanoma RNA synthesis before fusion is also able to block subsequent masking, but is ineffective if delayed until after fusion. Ultraviolet irradiation of the melanoma cell before fusion prevents subsequent masking, whereas similar treatment of the macrophage has no effect. Cells differ markedly in their ability to mask the macrophage phagocytic receptor after fusion. Ehrlich ascites tumor cells mask the receptor rapidly, primary chick fibroblasts minimally, and embryonic chick erythrocytes not at all.

scholarly journals Kinetics of intracellular transport and sorting of lysosomal membrane and plasma membrane proteins.

1987 ◽  
Vol 105 (3) ◽  
pp. 1227-1240 ◽  
Author(s):  
S A Green ◽  
K P Zimmer ◽  
G Griffiths ◽  
I Mellman
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Intracellular Transport ◽  
Rat Kidney ◽  
Fc Receptor ◽  
Lysosomal Membrane ◽  
Plasma Membrane Proteins ◽  
Kinetics Of

We have used monospecific antisera to two lysosomal membrane glycoproteins, lgp120 and a similar protein, lgp110, to compare the biosynthesis and intracellular transport of lysosomal membrane components, plasma membrane proteins, and lysosomal enzymes. In J774 cells and NRK cells, newly synthesized lysosomal membrane and plasma membrane proteins (the IgG1/IgG2b Fc receptor or influenza virus hemagglutinin) were transported through the Golgi apparatus (defined by acquisition of resistance to endo-beta-N-acetylglucosaminidase H) with the same kinetics (t1/2 = 11-14 min). In addition, immunoelectron microscopy of normal rat kidney cells showed that lgp120 and vesicular stomatitis virus G-protein were present in the same Golgi cisternae demonstrating that lysosomal and plasma membrane proteins were not sorted either before or during transport through the Golgi apparatus. To define the site at which sorting occurred, we compared the kinetics of transport of lysosomal and plasma membrane proteins and a lysosomal enzyme to their respective destinations. Newly synthesized proteins were detected in dense lysosomes (lgp's and beta-glucuronidase) or on the cell surface (Fc receptor or hemagglutinin) after the same lag period (20-25 min), and accumulated at their final destinations with similar kinetics (t1/2 = 30-45 min), suggesting that these two lgp's are not transported to the plasma membrane before reaching lysosomes. This was further supported by measurements of the transport of membrane-bound endocytic markers from the cell surface to lysosomes, which exhibited additional lag periods of 5-15 min and half-times of 1.5-2 h. The time required for transport of newly synthesized plasma membrane proteins to the cell surface, and for the transport of plasma membrane markers from the cell surface to lysosomes would appear too long to account for the rapid transport of lgp's from the Golgi apparatus to lysosomes. Thus, the observed kinetics suggest that lysosomal membrane proteins are sorted from plasma membrane proteins at a post-Golgi intracellular site, possibly the trans Golgi network, before their delivery to lysosomes.

scholarly journals Structural evidence for distinct IgG subclass-specific Fc receptors on mouse peritoneal macrophages

1980 ◽  
Vol 152 (5) ◽  
pp. 1147-1161 ◽  
Author(s):  
BC Lane ◽  
J Kan-Mitchell ◽  
MS Mitchell ◽  
SM Cooper
Keyword(s):  
Binding Proteins ◽  
Sodium Dodecyl ◽  
Fc Receptors ◽  
Peritoneal Macrophages ◽  
Fc Receptor ◽  
Rabbit Igg ◽  
Mouse Macrophages ◽  
Specific Proteins ◽  
Sepharose 4B ◽  
Mouse Peritoneal Macrophages

Membrane proteins which selectively bind to the Fc portion of IgG were identified in the Nonidet P-40 extracts of radiolabeled thioglycollate- elicited mouse peritoneal macrophages. Affinity columns of various IgG preparations coupled to Sepharose 4B were used to absorb the Fc-binding proteins. Analysis of the acetic acid or sodium dodecyl sulfate (SDS) eluates from aggregated human IgG or antigen-complexed rabbit IgG columns revealed two Fc(gamma)/-specific proteins with apparent 67,000 and 52,000 mol wt. These proteins were not detected in acid or SDS eluates from F(ab')(2) columns or in eluates from IgG column, over which were passed lysates of Fc receptor-negative cells. With the use of affinity columns that contained aggregated mouse myeloma proteins of different IgG subclasses, we found that the 67,000-dahon protein selectively binds to IgG2a, whereas the 52,000-dalton protein binds to IgG1 and IgG2b. Neither protein was found in SDS eluates from IgG3 columns. Trypsin treatment of the macrophages before detergent lysis removed the 67,000-dalton protein, although it leaves intact the 52,000-dalton protein. These results provide structural confirmation for the existence of separate Fc receptors on mouse macrophages and indicate that the two Fc-binding proteins identified in this study represent all or part of the trypsin- sensitive Fc receptor which binds IgG2a and the trypsin-resistant Fc receptor which binds IgG2b and IgG1.

scholarly journals Fc receptor-mediated phagocytosis occurs in macrophages at exceedingly low cytosolic Ca2+ levels.

1988 ◽  
Vol 106 (3) ◽  
pp. 657-666 ◽  
Author(s):  
F Di Virgilio ◽  
B C Meyer ◽  
S Greenberg ◽  
S C Silverstein
Keyword(s):  
Direct Evidence ◽  
Fc Receptors ◽  
Peritoneal Macrophages ◽  
Fc Receptor ◽  
Fura 2 ◽  
Monolayer Cultures ◽  
Low Concentrations ◽  
High Concentrations ◽  
Macrophage Populations ◽  
Mouse Peritoneal Macrophages

Cytosolic free Ca2+ ([Ca2+]i) homeostasis was investigated in mouse peritoneal macrophages and in the macrophage-like cell line J774. [Ca2+]i measurements were performed in both cells in suspension and cells in monolayers loaded with either quin2 or fura-2. Resting [Ca2+]i was 110-140 and 85-120 nM for cell suspensions and monolayers, respectively. There were no significant differences in [Ca2+]i between the two macrophage populations whether quin2 or fura-2 were used as Ca2+ indicators. Addition of heat-aggregated IgG, IgG-coated erythrocyte ghosts, or a rat monoclonal antibody (2.4G2) directed against mouse Fc receptor II induced a rise in [Ca2+]i. This [Ca2+]i increase was consistently observed in J774 and peritoneal macrophage suspensions and in J774 macrophage monolayers; in contrast it was observed inconsistently in peritoneal macrophages in monolayer cultures. The increase in [Ca2+]i induced by ligation of Fc receptors was inhibited totally in macrophages in suspension and by 80% in macrophages in monolayers by a short preincubation of macrophages with PMA; however, phagocytosis itself was unaffected. The effect of reducing cytosolic Ca2+ to very low concentrations on Fc receptor-mediated phagocytosis was also investigated. By incubating macrophages with high concentrations of quin2/AM in the absence of extracellular Ca2+, or by loading EGTA into the cytoplasm, the [Ca2+]i was buffered and clamped to 1-10 nM. Despite this, the phagocytosis of IgG-coated erythrocytes proceeded normally. These observations confirm the report of Young et al. (Young, J. D., S. S. Ko, and Z. A. Cohn. 1984. Proc. Natl. Acad. Sci. USA. 81:5430-5434) that ligation of Fc receptors causes Ca2+ mobilization in macrophages. However, these results confirm and extend the findings of McNeil et al. (McNeil, P. L., J. A. Swanson, S. D. Wright, S. C. Silverstein, and D. L. Taylor. 1986. J. Cell Biol. 102:1586-1592) that a rise in [Ca2+]i is not required for Fc receptor-mediated phagocytosis; and they provide direct evidence that Fc receptor-mediated phagocytosis occurs normally even at exceedingly low [Ca2+]i.

scholarly journals 2-Deoxyglucose selectively inhibits Fc and complement receptor-mediated phagocytosis in mouse peritoneal macrophages. I. Description of the inhibitory effect.

1976 ◽  
Vol 144 (6) ◽  
pp. 1465-1483 ◽  
Author(s):  
J Michl ◽  
D J Ohlbaum ◽  
S C Silverstein
Keyword(s):  
Particle Size ◽  
Plasma Membrane ◽  
Inhibitory Effect ◽  
Peritoneal Macrophages ◽  
Complement Receptor ◽  
Coated Particles ◽  
Mouse Peritoneal Macrophages ◽  
C3 Receptors ◽  
Glucose Analogue

Incubation of normal or thioglycollate-elicited mouse peritoneal macrophages with 2-deoxy-D-glucose (2-dG) inhibits the capacity of these macrophages to phagocytize IgG- or complement-coated particles via their Fc and C3 receptors. 2-dG has no inhibitory effect on the capacity of these macrophages to phagocytize latex or zymosan particles, which are ingested in the absence of specific opsonins, and it does not inhibit binding of IgG- or C3-coated particles to their respective receptors on the macrophage's plasma membrane. 2-dG exerts its inhibitory effect on the macrophage and not on the opsonized particle. The inhibition is independent of particle size, occurs within 15-30 min of addition of this glucose analogue to the medium at 37 degrees C, cannot be overcome by supra-agglutinating amounts of opsonizing antibody, and is completely reversible by substitution of 5.5 mM glucose for 50 mM 2-dG in the medium. Addition of equimolar amounts of glucose or mannose, but not of fructose, galactose, fucose, or glucosamine, to medium containing 50 mM 2-dG results in substantial reversal of the inhibitory effect of 2-dG on Fc and C3 receptor mediated phagocytosis.

Detergent fractionation with subsequent subtractive suppression hybridization as a tool for identifying genes coding for plasma membrane proteins

2009 ◽  
Vol 18 (6) ◽  
pp. 527-535 ◽  
Author(s):  
Andreas Lange ◽  
Claudia Kistler ◽  
Tanja B. Jutzi ◽  
Alexandr V. Bazhin ◽  
Claus Detlev Klemke ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Plasma Membrane Proteins ◽  
Subtractive Suppression Hybridization

scholarly journals pH-dependent Cargo Sorting from the Golgi

2011 ◽  
Vol 286 (12) ◽  
pp. 10058-10065 ◽  
Author(s):  
Chunjuan Huang ◽  
Amy Chang
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Atpase Activity ◽  
Secretory Pathway ◽  
Ph Dependence ◽  
Glucose Deprivation ◽  
Ph Homeostasis ◽  
Plasma Membrane Proteins ◽  
Cytosolic Ph ◽  
Cargo Sorting

The vacuolar proton-translocating ATPase (V-ATPase) plays a major role in organelle acidification and works together with other ion transporters to maintain pH homeostasis in eukaryotic cells. We analyzed a requirement for V-ATPase activity in protein trafficking in the yeast secretory pathway. Deficiency of V-ATPase activity caused by subunit deletion or glucose deprivation results in missorting of newly synthesized plasma membrane proteins Pma1 and Can1 directly from the Golgi to the vacuole. Vacuolar mislocalization of Pma1 is dependent on Gga adaptors although no Pma1 ubiquitination was detected. Proper cell surface targeting of Pma1 was rescued in V-ATPase-deficient cells by increasing the pH of the medium, suggesting that missorting is the result of aberrant cytosolic pH. In addition to mislocalization of the plasma membrane proteins, Golgi membrane proteins Kex2 and Vrg4 are also missorted to the vacuole upon loss of V-ATPase activity. Because the missorted cargos have distinct trafficking routes, we suggest a pH dependence for multiple cargo sorting events at the Golgi.

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