scholarly journals Binding of monomeric immunoglobulins to Fc receptors of mouse macrophages.

1975 ◽  
Vol 142 (6) ◽  
pp. 1520-1533 ◽  
Author(s):  
J C Unkeless ◽  
H N Eisen
Keyword(s):  
Dynamic Equilibrium ◽  
Primary Cultures ◽  
Fc Receptors ◽  
Peritoneal Macrophages ◽  
Dissociation Rate ◽  
Surface Receptors ◽  
Mouse Macrophages ◽  
Number Of Binding Sites ◽  
Dissociation Rate Constants ◽  
Mouse Myeloma

The binding properties of surface receptors of immunoglobulins on mouse macrophages were studied with mouse myeloma proteins and normal peritoneal macrophages, thioglycollate-stimulated macrophages, and a macrophage cell line, P388D1. Primary cultures of mouse embryo fibroblasts served as controls. IgG2a proteins were bound strongly;IgG2b was bound weakly (one-twentieth as well as IgG2a);IgM, IgA, and IgG1 were not bound significantly. The number of binding sites per cell for IgG2a was 4 X 10(5) for thioglycollate-stimulated cells and 1 X 10(5) for normal and P388D1 cells. Binding was exothermal: with decreasing temperature the equilibrium (association) constants increased and dissociation rate constants decreased (at 37degreesC the respective values were 2 X 10(7) M-1 and 0.26 min-1, the latter value corresponds to a half time for dissociation of 2.6 min). From the rapidity of association and dissociation, it appears that the surface of the macrophage is in a dynamic equilibrium with IgG2a molecules in the cell's immediate microenvironment. The receptors for IgG2a are clearly specific for determinants in the immunoglobulin constant domain: two IgG2a proteins with greatly different isoelectric points (determined by isoelectric focusing) were bound with the same affinity to the same receptors; moreover, the Fc fragment was bound and Fab fragments were not. The Fc receptors for IgG2a proteins were readily eliminated by exposing macrophages briefly to trypsin. The receptors were regenerated during subsequent cultivation in serum-free medium; regeneration was inhibited totally by cycloheximide and partially by actinomycin D.

scholarly journals Full characterization of GPCR monomer–dimer dynamic equilibrium by single molecule imaging

2011 ◽  
Vol 192 (3) ◽  
pp. 463-480 ◽  
Author(s):  
Rinshi S. Kasai ◽  
Kenichi G. N. Suzuki ◽  
Eric R. Prossnitz ◽  
Ikuko Koyama-Honda ◽  
Chieko Nakada ◽  
...  
Keyword(s):  
Equilibrium Constant ◽  
Single Molecule ◽  
Dynamic Equilibrium ◽  
Dissociation Rate ◽  
Formyl Peptide Receptor ◽  
Live Cells ◽  
Imaging Method ◽  
Full Characterization ◽  
Before And After ◽  
Dissociation Rate Constants

Receptor dimerization is important for many signaling pathways. However, the monomer–dimer equilibrium has never been fully characterized for any receptor with a 2D equilibrium constant as well as association/dissociation rate constants (termed super-quantification). Here, we determined the dynamic equilibrium for the N-formyl peptide receptor (FPR), a chemoattractant G protein–coupled receptor (GPCR), in live cells at 37°C by developing a single fluorescent-molecule imaging method. Both before and after liganding, the dimer–monomer 2D equilibrium is unchanged, giving an equilibrium constant of 3.6 copies/µm2, with a dissociation and 2D association rate constant of 11.0 s−1 and 3.1 copies/µm2s−1, respectively. At physiological expression levels of ∼2.1 receptor copies/µm2 (∼6,000 copies/cell), monomers continually convert into dimers every 150 ms, dimers dissociate into monomers in 91 ms, and at any moment, 2,500 and 3,500 receptor molecules participate in transient dimers and monomers, respectively. Not only do FPR dimers fall apart rapidly, but FPR monomers also convert into dimers very quickly.

scholarly journals A new Fc receptor on mouse macrophages binding IgG3.

1981 ◽  
Vol 153 (3) ◽  
pp. 514-519 ◽  
Author(s):  
B Diamond ◽  
D E Yelton
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Lines ◽  
Cytochalasin B ◽  
Fc Receptors ◽  
Peritoneal Macrophages ◽  
Fc Receptor ◽  
Igg Subclasses ◽  
Macrophage Cell ◽  
Sheep Erythrocytes ◽  
Mouse Macrophages

Monoclonal antibodies to sheep erythrocytes (SRBC) have proved useful in identifying two Fc receptors on mouse macrophages, one for IgG2a, and one for IgG1 and IgG2b. We have used monoclonal IgG3 anti-SRBC to identify a third Fc receptor on mouse macrophages which binds IgG3 uniquely. This receptor is present on primary resident and thioglycolate-induced peritoneal macrophages and on some macrophage cell lines. The binding of IgG3-coated SRBC is inhibited by aggregated byt not monomeric IgG3, and not by IgG1, IgG2a, and IgG2b aggregates. It is unaffected by treating the macrophages with trypsin or cytochalasin B and occurs at both 4 degrees and 37 degrees C. IgG3, like all other IgG subclasses, mediates phagocytosis. We have also generated a variant macrophage line which bears the receptors for IgG1 and IgG2b and for IgG2a, but not for IgG3.

scholarly journals Internalization and degradation of macrophage Fc receptors bound to polyvalent immune complexes.

1984 ◽  
Vol 98 (4) ◽  
pp. 1170-1177 ◽  
Author(s):  
I Mellman ◽  
H Plutner
Keyword(s):  
Immune Complexes ◽  
Fc Receptors ◽  
Fc Receptor ◽  
Surface Receptors ◽  
Molar Ratios ◽  
Mouse Macrophages ◽  
Immune Precipitation ◽  
Specificity Of Binding ◽  
Receptor Antibodies ◽  
Receptor Turnover

We have studied the Fc receptor-mediated pinocytosis of immunoglobulin G (IgG)-containing immune complexes by mouse macrophages. IgG complexes were formed from affinity-purified rabbit dinitrophenyl IgG and dinitrophenyl modified BSA at molar ratios of 2.5-10:1. Both the specificity of binding and the fate of internalized receptors were analyzed using monoclonal and polyclonal anti-Fc receptor antibodies. Based on the susceptibility of surface-bound ligand to release by proteolysis, we have found that at 37 degrees C, 125I-labeled IgG complexes were rapidly internalized (t1/2 less than 2 min) and delivered to lysosomes; acid-soluble 125I was detectable in the growth medium within 5-10 min of uptake. However, kinetic evidence indicated that Fc receptors were not efficiently re-used for multiple rounds of ligand uptake. Instead, macrophages that were exposed continuously to saturating concentrations of IgG complexes exhibited a selective and largely irreversible removal of Fc receptors from the plasma membrane. This loss of surface receptors correlated with an increased rate of receptor turnover, determined by immune precipitation of Fc receptors from 125I-labeled macrophages. Thus, in contrast to the results obtained in the accompanying paper (I. Mellman, H. Plutner, and P. Ukkonen, 1984, J. Cell Biol. 98:1163-1169) using a monovalent ligand, these data indicate that the interaction of Fc receptors with polyvalent complexes leads to the degradation of both ligand and receptor following 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 Site of binding of mouse IgG2b to the Fc receptor on mouse macrophages.

1979 ◽  
Vol 150 (3) ◽  
pp. 721-726 ◽  
Author(s):  
B Diamond ◽  
B K Birshtein ◽  
M D Scharff
Keyword(s):  
Heavy Chain ◽  
Fc Receptors ◽  
Myeloma Cell ◽  
Fc Receptor ◽  
Heavy Chains ◽  
Mouse Macrophages ◽  
Mouse Myeloma Cell Line ◽  
Ch2 Domain ◽  
A Site ◽  
Mouse Myeloma

Three mouse immunoglobulins with altered heavy chains have been used to study the specificity of the mouse IgG2b Fc receptor on mouse macrophages. These immunoglobulins were synthesized by variant clones derived from the MPC 11, IgG2b-producing mouse myeloma cell line. One variant, whose Fc receptor. A second variant, which makes a short heavy chain lacking the CH3 domain, binds specifically to the IgG2b Fc receptor. The third variant makes a hybrid IgG2b-IgG2a heavy chain whose CH3 domain is enterely IgG2a-like and binds to both IgG2a and IgG2b Fc receptors. These data suggest that the binding of mouse IgG2b immunoglobulins to the mouse macrophage Fc receptor involves a site within the CH2 domain and indicate that immunoglobulins with altered heavy chains are a useful tool to probe Fc receptors.

scholarly journals The presence of two Fc receptors on mouse macrophages: evidence from a variant cell line and differential trypsin sensitivity.

1977 ◽  
Vol 145 (4) ◽  
pp. 931-945 ◽  
Author(s):  
J C Unkeless
Keyword(s):  
Conformational Changes ◽  
Fc Receptors ◽  
Peritoneal Macrophages ◽  
Differential Sensitivity ◽  
Soluble Antigen ◽  
Variant Cell ◽  
Rabbit Igg ◽  
Mouse Macrophages ◽  
Antigen Antibody ◽  
Mouse Peritoneal Macrophages

A stable variant of a clone of the P388D1 macrophage line was isolated using four cycles of treatment with mouse IgG2a-rabbit anti-kappa complexes and rabbit complement. The variant had the same Ka and about the same number of sites per cell for IgG2a as the parent line. However, the variant had 10% as many binding sites for rabbit IgG in soluble antigen-antibody complexes, and the affinity of binding was threefold higher. This change in binding of complexes to cells of a cloned line without alternation of IgG2a binding provides evidence for the presence of two distinct Fc receptors. The two receptors could also be distiguished on the P388D1 line and on thioglycollate-induced mouse peritoneal macrophages by differential sensitivity to trypsinization. The receptors that bind monomeric IgG2a, sheep erythrocytes (SRBC) covalently bound with IgG2a or rabbit IgG using glutaraldehyde, and Sephadex beads coupled with IgG2a or rabbit IgG using cyanogen bromide activitation, is sensitive to trypsinization. The receptor that binds soluble rabbit antibody-antigen complexes, trinitrophenyl-SRBC and dinitrophenyl(DNP)-bovine serum albumin Sephadex beads coated with rabbit anti-DNP IgG is trypsin resitant, the observation that uncomplexed rabbit IgG oes not bind to the trypsin-resistant receptor, whereas the same IgG bound to its antigen does, suggests that conformational changes induced by the binding of ligand may be of consequence in macrophage function.

scholarly journals Introduction of B-chain-inactivated ricin into mouse macrophages and rat Kupffer cells via their membrane Fc receptors.

1976 ◽  
Vol 143 (6) ◽  
pp. 1464-1474 ◽  
Author(s):  
K Refsnes ◽  
A C Munthe-Kaas
Keyword(s):  
Protein Synthesis ◽  
Cell Membrane ◽  
Kupffer Cells ◽  
Fc Receptors ◽  
Peritoneal Macrophages ◽  
Cellular Protein ◽  
Membrane Receptors ◽  
Soluble Antigen ◽  
Surface Receptors ◽  
Cellular Protein Synthesis

Experiments have been made to test whether the toxic lectin ricin can be bound to and introduced into cells by some other mechanism than via its B chain, the natural binding moiety of the toxin, without its toxic effect being neutralized. Complexes consisting of ricin and antibodies specifically directed against ricin B chain were incubated with mouse peritoneal macrophages and rat Kupffer cells, which are known to possess surface receptors for the Fc portion of the immunoglobulin molecule. After incubation for 26 h, cellular protein synthesis, as measured by incorporation of labeled leucine into acid-insoluble material, was completely inhibited. HeLa cells, which do not possess Fc receptors, were unaffected by the complex. The effect of the complex on protein synthesis of macrophages was prevented by soluble antigen-antibody complexes, but not by the presence of lactose which prevents attachment of the ricin B chain to the cell membrane. The [ricin-antiricin B] complex was attached to red cells, and the resulting complex was incubated with rat Kupffer cells. Cellular protein synthesis ceased after 6 h, and phase contrast microscopy studies showed that the complexes were taken up by the Kupffer cells. The data indicate that ricin, when present in the complex with antiricin B, can be introduced into cells through cell membrane receptors other than the B chain receptor, in this case the Fc receptor, and that the internalized toxin retains a least part of its activity.

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