Interaction of antibodies with Fc receptors in substrate-supported planar membranes measured by total internal reflection fluorescence microscopy

Poglitsch, Claudia L.; Thompson, Nancy L.

doi:10.1021/bi00453a033

Cited by 50 publications

(34 citation statements)

References 30 publications

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“…The measured values of K,,, for the five monoclonal IgGl antibodies with FcyRII on 5774 cell surfaces (Table I) are consistent with previous measurements for mouse myeloma IgGl on P388Di cells using competitive radioimmunoassays with 1251-labelled, cross-linked polyclonal rabbit IgG (3 x IO5 M-', O'C) [ll]. The K,,, values are also consistent with previous immunofluorescence measurements for polyclonal IgG and monoclonal IgGl on planar model membranes constructed from isolated 5774 membrane fragments (l-5 x lo5 M-', 25°C) [18] or from purified and reconstituted FcyRII (3-9 x 10' M-', 25°C) [20].…”

Section: Discussionsupporting

confidence: 90%

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Association constants of anti‐hapten monoclonal IgGI with mouse FcγRII in the presence and absence of hapten

Sumner

1993

FEBS Letters

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The binding of five different mouse monoclonal anti‐dinitrophenyl (DNP) IgGI monomers, in the presence and absence of DNP‐glycine, to FcγRII on the mouse macrophage‐like cell line J774 has been investigated. Membrane association constants were estimated using a competitive radioimmunoassay in which the ability of the IgGl monomers to block the binding of 125I‐labelled Fabs of the anti‐FcγRII monoclonal antibody 2.4G2 was monitored. The IgGl‐FcγRII association constants ranged from (8 ± 1) × 104 M−1 to (6 ± 2) × 105 M−1. No large differences in the IgGl‐FcγRII association constants were measured for different IgG1 antibodies or for any single IgG1 antibody in the presence and absence of DNP‐glycine.

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Section: Discussionsupporting

confidence: 90%

“…The K,,, values are also consistent with previous immunofluorescence measurements for polyclonal IgG and monoclonal IgGl on planar model membranes constructed from isolated 5774 membrane fragments (l-5 x lo5 M-', 25°C) [18] or from purified and reconstituted FcyRII (3-9 x 10' M-', 25°C) [20].…”

Section: Discussionsupporting

confidence: 88%

Association constants of anti‐hapten monoclonal IgGI with mouse FcγRII in the presence and absence of hapten

Sumner

1993

FEBS Letters

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“…Polyclonal anti-MBP antibodies were obtained commercially (New England Biolabs). The monoclonal antibodies 1B4C14, which bind both mFcyRJIb1 and mFcyRIIb2 cytoplasmic regions (Green et al, 1985), were isolated from hybridoma supernatants by using affinity chromatography with the mouse anti-(rat K light chain) antibody MAR18.5 (Poglitsch & Thompson, 1990). Horseradish peroxidase-conjugated whole molecule anti-(rat IgG) or anti-(rabbit IgG) was obtained commercially (Sigma Chemical Co., St. Louis, Missouri).…”

Section: Antibodiesmentioning

confidence: 99%

Design, synthesis, expression, and characterization of the genes for mouse FcγRIIb1 and FcγRIIb2 cytoplasmic regions

1997

Self Cite

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The cytoplasmic regions of the mouse low-affhity FcyRII isoforms, mFcyRIIb1, and mFcyRIIb2, play a key role in signal transduction by mediating different cellular functions. mFcyRIIbl has a 94-residue cytoplasmic region, whereas mFcyRIIb2 has a 47-residue cytoplasmic region. Genes encoding the cytoplasmic regions of mFcyRIIbl (bl-94) and mFcyRIIb2 (b2-47) were designed, synthesized, and expressed as fusion proteins in Escherichia coli. A sequencespecific protease, thrombin, was used to release the bl-94 peptide, which was purified by using HPLC. The b2-47 peptide was synthesized chemically. CD spectropolarimetry was employed to examine the secondary structures of bl-94 and b2-47. These studies were conducted in aqueous solution, in mixtures of water and trifluoroethanol or methanol, and as a function of temperature. The results indicate that the bl-94 and b2-47 structures are sensitive functions of the solvent environment, and that nonaqueous solvents induce significant a-helical structure. , 1991;Fridman et al., 1992;Hulett & Hogarth, 1994;Indik et al., 1995). In mouse and human, there are at least three Fcy receptors, denoted FcyRI, FcyRII, and FcyRIII. The present work concerns mouse FcyRII (mFcyRII). This receptor is found on numerous cell types, including lymphocytes, neutrophils, macrophages, eosinophils, platelets, mast cells, and monocytes; and is thought to participate in a number of biological processes, including endocytosis, phagocytosis, Reprint requests to: Gary J. Pielak or Nancy L. Thompson, Department of Chemistry, Campus Box 3290, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290; e-mail: gary-pielak@unc.edu or nlt@unc.edu.Abbreviarions: bl-94, 94-amino acid cytoplasmic region of mFcyRIIbl with a methionine and six histidine residues at the C-terminus; b2-47, 47-amino acid cytoplasmic region of mFcyRIIb2; DHFF, dihydrofolate reductase; IgG, immunoglobulin G; IF'TG, iso-propyl-/%D-thiogalactoside; ITAM, immune receptor tyrosine-based activation motif; ITIM, immune receptor tyrosine-based inhibitor motif; MBP, maltose-binding protein; MBPbl-94, MBP fusion with thrombin cleavage site and bl-94; MBP-DHFFb2-47, MBP-DHFR fusion with thrombin cleavage site, b2-47, and a methionine and six histidine residues at the C-terminus; NTA, nitrilotriacetic acid; pLC47, plasmid containing the MBP-DHFR-b2-47 gene; pLC94, plasmid containing the MBP-bl-94 gene; pMAL-c2, plasmid containing the MBP gene; TFE, trifluoroethanol; 1B4C14, monoclonal antibody against bl-94 and b2-47.

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“…34 The technique has also been used by other laboratories to study the binding of prothrombin to planar phospholipid membranes 35 and the binding of immunoglobulin (Ig) G to a mouse IgG receptor reconstituted in substrate-supported planar membranes. 36 ' 37 Other roles for TIRFS technology include the examination of protein adsorption at solid-liquid interfaces 38 and the adsorption of proteins on polymer films. 39 Under the experimental conditions chosen, there is selective excitation of bound fluorescent probes at the interface without significant excitation of fluorescent molecules in the bulk solution.…”

Section: The Assembly Of the Prothrombinase Complex On Adherent Platementioning

confidence: 99%

The assembly of the prothrombinase complex on adherent platelets.

Swords

Mann

1993

Arterioscler Thromb

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Prothrombinase complex assembly, in real time, on platelets adherent to immobilized von Willebrand Factor (vWf) was examined by total internal reflection fluorescence spectroscopy (TIRFS). Electron microscopy showed that the platelets adhered to vWf in a largely unactivated state and could be activated by thrombin. Antibody binding to glycoprotein (GP) Ib and functional GPIIb-IIIa receptor molecules on adherent platelet membranes monitored by TIRFS also indicated that platelets adhered in a largely unactivated state. Maximal expression of the receptor form of GPIIb-IIIa detected by antibody binding was seen only after thrombin stimulation of the adherent platelets. Antibody binding to GPIb was detected on adherent platelets. A reduction in antibody binding was observed after thrombin stimulation of the platelets, indicating a change in GPIb as a consequence of thrombin stimulation of the platelets. The binding of the protein components of the prothrombinase complex to adherent and thrombin-stimulated adherent platelets was then studied individually. Factor Va bound to adherent and thrombin-stimulated adherent platelets with an estimated K a of 58 nmoI/L. Minimal factor Xa binding was observed on adherent platelets before thrombin stimulation. Factor Xa binding was, however, readily observed on thrombin-stimulated adherent platelets. This factor Xa binding was not saturable, and no K d value could be estimated. Direct measurement of prothrombinase complex assembly was demonstrated by using an energy transfer phenomenon between fluorescein-labeled factor Va and rhodamine-labeled factor Xa. Prothrombinase complex assembly was observed on both adherent and thrombin-stimulated adherent platelets. The estimated K A for the factor Va/factor Xa interaction was 4 nmol/L. TIRFS demonstrated that adherent platelets have the ability to support prothrombinase complex assembly, as shown by a direct energy transfer reaction between fluorescently labeled factors Va and Xa. (Arterioscler Thromb.

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Interaction of antibodies with Fc receptors in substrate-supported planar membranes measured by total internal reflection fluorescence microscopy

Cited by 50 publications

References 30 publications

Association constants of anti‐hapten monoclonal IgGI with mouse FcγRII in the presence and absence of hapten

Association constants of anti‐hapten monoclonal IgGI with mouse FcγRII in the presence and absence of hapten

Design, synthesis, expression, and characterization of the genes for mouse FcγRIIb1 and FcγRIIb2 cytoplasmic regions

The assembly of the prothrombinase complex on adherent platelets.

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