Biosynthesis of the human C3b/C4b receptor during differentiation of the HL-60 cell line. Identification and characterization of a precursor molecule.

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Abstract

The C3b receptor (C3bR) of the human promyelocytic leukemia cell line (HL-60) was induced by incubating these cells with dimethylsulfoxide (DMSO) or retinoic acid. A majority of differentiated (DMSO- or retinoic acid-treated) but not undifferentiated cells formed rosettes with C3b-coated erythrocytes and were morphologically mature granulocytes. HL-60 cells were surface- or biosynthetically labeled and then solubilized in 1% Nonidet P-40 in the presence of multiple protease inhibitors. The C3bR was isolated either by immunoprecipitation with anti-C3bR antibodies or by affinity chromatography with hemolytically inactive components in which the internal thioester bond within the alpha-chain was cleaved (iC3)- or iC4-Sepharose. Autoradiographs of NaDodSO4-polyacrylamide gels indicated that the surface-labeled C3bR on the differentiated cells had an Mr of 210,000 (nonreduced) or 240,000 (reducing conditions). The bulk (approximately 85%) of the radiolabeled material that was isolated from biosynthetically labeled cells co-migrated with the surface-labeled band. A small fraction (approximately 15%) of the biosynthetically labeled material that was isolated by affinity chromatography or immunoprecipitation had an Mr of 188,000, which did not correspond to any surface-labeled band. This putative precursor molecule was characterized by pulse-chase experiments and by analysis of its carbohydrate. In pulse-chase (15-min pulse) studies of differentiated cells, only the 188,000-mol wt molecule was detected at 0 h. By 2 h, greater than 80% of counts had chased from the 188,000 to the 210,000-mol wt molecule. Treatment of these two molecules with endoglycosidases indicated that the 188,000-mol wt molecule possessed high mannose oligosaccharides, while the mature C3bR had complex oligosaccharides. We conclude from these data that the 188,000-mol wt molecule is a precursor of the C3b receptor of HL-60 cells. Other experiments indicated that the half-maximal time for newly synthesized receptor to attain an Mr of 210,000 was 45 min, and that the t1/2 for the disappearance of the receptor on the surface of differentiated HL-60 cells in tissue culture was approximately 10 h. The ability to observe the induction of the C3b receptor as the HL-60 cell line differentiates is an instructive model system to study the biosynthesis of a human integral membrane receptor glycoprotein.

Authors

J P Atkinson, E A Jones