We have purified the human lymphocyte Fc receptor specific for IgE (Fcc receptor) and its soluble form by using the anti-Fcc receptor monoclonal antibody H107. Using an oligonucleotide probe corresponding to the partial amino acid sequence of the soluble Fce receptor related to IgE binding factor, we cloned, sequenced, and expressed a cDNA for the receptor. The Fcc receptor has 321 amino acid residues with no NH2-terminal signal sequence. The receptor was separated into two domains by a putative 24-amino acid residue transmembrane region located near the NH2-terminal end. The Fce receptor showed a marked homology with animal lectins including human and rat asialoglycoprotein receptors, chicken hepatic lectin, and rat mannose binding proteins. Tresyl-Sepharose (Pharmacia) at 5 mg/1 ml of beads (10). Soluble FCE receptor was purified from the conditioned medium of RPMI 8866 cells using H107-Sepharose after absorption with the human gamma globulinSepharose. The FcE receptor was purified as described (8). The cell lysate of RPMI 8866 cells in 0.5% Nonidet P-40 was absorbed with bovine serum albumin-, human gamma globulin-, and DNase I-conjugated Sepharose, and then incubated with the H107-Sepharose. After extensive washing with a high-salt buffer (10 mM sodium phosphate, pH 7.5/0.5 M NaCl/0.1% Nonidet P-40), soluble FCE receptor and FcE receptors were eluted from H107-Sepharose with an elution buffer (0.1 M acetic acid, pH 4.0/0.5 M NaCl/0.1% Nonidet P-40). Affinity-purified, soluble FCE receptor was further fractionated on Superose column with Fast Protein Liquid Chromatography (FPLC; Pharmacia), to elucidate its IgE binding activity. NaDodSO4/PAGE and Protein Amino Acid Sequence. The method of NaDodSO4/PAGE is described elsewhere (8). For immunoblots, nonreducing 13% polyacrylamide gels were used. After electrotransfer to a nitrocellulose filter, the membrane, blocked with 3% (wt/vol) bovine serum albumin, was incubated with or without H107 mAb (1 ,g/ml) and then with goat anti-mouse IgG [F(ab')2] conjugated to horseradish peroxidase (Tago). The antibody-bound bands were visualized with 3,3'-diaminobenzidine tetrahydrochloride and H202. For amino acid sequencing, both affinity-purified, soluble FcE receptor and FcE receptor were further purified by reverse-phase HPLC (using a Synchropak RP-P C18 column) (Beckman) with a linear gradient of 2-propanol with Abbreviations: FC£ receptor, Fc receptor for IgE; IgE-BF, IgE binding factor; mAb, monoclonal antibody. tTo whom reprint requests should be addressed.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.