Expression and characterization of a truncated murine Fc gamma receptor.
We have isolated a recombinant secreted Fc gamma R beta molecule by deletion of the transmembrane and cytoplasmic domains encoding sequence from a Fc gamma R beta 1 cDNA clone, and insertion of the truncated cDNA into a eukaryotic expression vector, pcEXV-3. To express and amplify the production of the truncated Fc gamma R beta molecule, we transfected the truncated cDNA plasmid into a dihydrofolate reductase-minus CHO cell line along with a dhfr minigene, and amplified the gene products with methotrexate. The resulting cell line secretes 2-3 micrograms/ml/24 h of truncated Fc gamma R beta, which can be readily purified by affinity chromatography on IgG-Sepharose. The truncated Fc gamma R beta has a Mr of 31-33,000 on SDS-PAGE and is glycosylated. N-glycosidase F cleavage reduces the Mr to 19,000, consistent with the size of the truncated product, 176 amino acid residues. There are two disulfide bonds in the protein. Binding of immune complexes formed between DNP20BSA and anti-DNP mAbs reveals better binding of IgG1 aggregates than that of IgG2b and IgG2a aggregates. The binding of the immune complexes was somewhat better at more acidic pH, in contrast to previous experiments with binding of purified Fc gamma R to immune complex-coated beads. We were surprised to observe that the truncated Fc gamma R beta did not react with the anti-Fc gamma R mAb 6B7C. Previous work had shown that 6B7C reacts with Fc gamma R on immunoblots, fails to bind to the surface of resting B cells and peritoneal macrophages, but does bind to macrophage cell lines and LPS-stimulated B cells. We show, by binding of mAb 6B7C to a peptide conjugate, that the 6B7C epitope lies within residues 169-183 of the intact Fc gamma R beta, which is just outside the plasma membrane. The availability of the truncated Fc gamma R beta in microgram quantities should facilitate further analysis of structure and function of these receptors.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000936:Antigen-Antibody Complex / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics; D000939:Epitopes / Q000276:immunology; D005260:Female; D005347:Fibroblasts; D006031:Glycosylation; D051379:Mice; D010053:Ovary; D011499:Protein Processing, Post-Translational; D011961:Receptors, Fc / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D011961:Receptors, Fc; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004247:DNA