Interaction of membrane-bound Fc gamma receptors with the cytoskeletal matrix.

Binding of ligands to cell surface receptors may induce an interaction of the receptors with the cell cytoskeleton. This interaction may decrease the solubility of the receptors in nonionic detergents. We studied effect of binding of various 125I-labeled immunoglobulin ligands to Fc gamma receptors on guinea pig peritoneal macrophages and human placental syncytiotrophoblast plasma membranes on an interaction of these receptors with the cytoskeletal matrix. The receptor-cytoskeleton association was evaluated by measurement radioactivity of bound ligands in pellets and supernatants obtained after lysis of cells or membranes in a nonionic detergent NP-40. Binding of soluble immune complexes or crosslinking of IgG bound induces much stronger insolubilization of the receptors than binding of monomeric or aggregated IgG. It shows that the interaction of the receptors with the cytoskeletal matrix strongly depends on the degree of cross-linking of the Fc gamma receptors by ligands bound. The observed effects were IgG Fc region-specific. Isolated, purified putative Fc gamma receptors from guinea pig peritoneal macrophages and from human placental syncytiotrophoblast plasma membranes do not interact with free G or F actin. We also studied association of the guinea pig peritoneal macrophage Fc gamma receptor with the cytoskeleton, before and after shedding of macrophage membrane proteins. The results obtained showed that the macrophages have only one class of Fc gamma receptors interacting with the cytoskeletal matrix. Effect of a cytoskeleton-destabilizing buffer and DNAse I on release of the receptors from the cytoskeleton suggests that insolubilization of ligand-Fc gamma R complexes was caused, at least partially, by an interaction of the receptors with actin filaments in the cytoskeleton. The results presented in this paper suggest that the cytoskeleton might play a role in transmission of signals from Fc gamma receptors to the cells. They underline the role of immune complexes as physiological ligands for Fc receptors and correlate well with activation of cells via their Fc receptors (e.g. superoxide burst) observed by other authors after treatment of the cells with immune complexes, but not with monomeric or aggregated IgG.

A mild method for the purification of guinea pig peritoneal macrophage Fc gamma receptors. Affinity chromatography and elution of the receptor with reducing agents.

A method for the purification of Fc gamma receptors from guinea pig peritoneal macrophages using mild conditions is described. The method is based on the observation that reduction and alkylation of IgG disulfide bonds partially or completely abrogate their binding to Fc gamma receptors. Cell lysates were directly applied to Sepharose-IgG or Sepharose-TNP-Ab(IgG) and the specifically bound Fc gamma receptor was eluted from adsorbents by incubation with reducing agents (2-mercaptoethanol or dithiothreitol). Alternatively, cell lysates were first treated with IgG and then applied to Protein A-Sepharose and the receptor was eluted with reducing agents. Yields of the purified Fc gamma receptor preparations and their activities were considerably higher than when the receptor was eluted from affinity chromatography gels with acetic acid or other acidic buffers or chaotropic agents. The best results were obtained when Fc gamma receptor-IgG complexes were applied to Protein A-Sepharose. No significant difference in the subunit structure was observed using SDS-PAGE when receptor preparations obtained by elution with reducing agents were compared with preparations obtained by elution with acidic buffers.

Human placental membrane receptor for IgG. Purification of the receptor and its subunit structure.

The method of purification of the human placental Fc receptor to an active form is described. The FcR was purified from the glycoprotein fraction of the placental membranes by immunoprecipitation and chromatography on DEAE-cellulose. The purified FcR corresponded to 1.5-2% of the protein present in the crude glycoprotein fraction (PGP) and showed the tendency to aggregate. In the presence of 1% SDS, 4 M urea or 5 M guanidine- HCl the placental FcR dissociated into subunits of molecular weight of 60,000-65,000. The 60,000-65,000 dalton glycoprotein subunits regarded as monomers of FcR are composed of two chains of molecular weight 25,000-30,000, linked by disulphide bonds. The subunits, after removal of dissociating agents, displayed IgG binding activity.

Immunoglobulins of colostrum. V. Localization of structural differences between serum and colostral ovine and bovine IgG1 and IgG2 immunoglobulins.

Studies on localization of structural differences between ovine and bovine serum and colostral IgG immunoglobulins are described. Comparison of heterogeneity, susceptibility to proteolytic enzymes, peptide maps, amino acid compositions, and antigenic properties of immunoglobulins and their Fab and Fc fragments and H and L chains showed that structural differences are localized in the Fc region. The strongest differences were found in case of IgG2. It was also shown that no Fc fragments could be obtained from bovine serm IgG2 and ovine serum and colostral IgG2 due to their susceptibility to papain and trypsin. The results obtained confirmed our suggestion that colostral IgG2 are locally synthesized in mammary glands, whereas colostral IgG1 might be a mixed population of molecules locally synthesized and transferred from the serum.

Localization of structural differences between serum and colostral ovine and bovine IgG1 and IgG2 immunoglobulins.

Physicochemical and serological studies on immunoglobulins, their Fab and Fc fragments, L and H chains, showed that the strongest differences occurred in case of IgG2, and that the differences were localized in the Fc region.