Immunoprecipitation of the Solubilized Membrane Receptor for IgE of Human Cultured Lymphoblastoid Cells

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Abstract
The putative IgE Fc receptor (Fc.epsilon.) of cultured human lymphoblastoid cells was characterized using a goat anti-receptor antiserum. The antiserum was prepared against NP-40-solubilized cell components of Wil-2WT cells which bound to an IgE-Sepharose-4B immunoadsorbent column. The antiserum specifically inhibited binding of 125I-labeled IgE to Fc.epsilon.-positive RPMI-8866 lymphoblastoid cells. Absorption of the antiserum with FC.epsilon.-negative Raji and Molt-4 cells and with IgE and IgG did not change this inhibitory activity. Antiserum extensively absorbed with Raji and Molt-4 cells precipitated 7% of the radioactivity of lysates of 125I-lactoperoxidase-labeled RPMI-8866 cells but only 0.5-1.8% of that of Raji and Molt-4 cells. Autoradiography of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses demonstrated 2 major labeled peptides of 86,000 and 47,000 MW in reduced immunoprecipitates from RPMI-8866 but not from Raji or Molt-4 cell lysates. As determined by Sepharose-6B gel filtration, the approximate MW of the solubilized radiolabeled membrane component that reacted with the antiserum was 250,000 solubilized in NP-40 and 125,000 in NP-40-4 M urea. Both the 250,000 and 125,000 MW material consisted of the 86,000 and 47,000 peptides. Evidently the anti-receptor antiserum inhibited binding of 125I-labeled IgE to Fc.epsilon.-receptor-positive lymphoblastoid cells. The receptor may consist of 2 non-covalently linked polypeptides that remain associated in NP-40 and NP-40-4 M urea.