Translation of the human C3b/C4b receptor mRNA in a cell-free system and by Xenopus oocytes.
The C3b/C4b complement receptor (CR1) is a large, single-chain integral membrane glycoprotein present on erythrocytes, leukocytes, glomerular podocytes, and splenic dendritic-reticular cells that mediates the binding of complement-coated particles and immune complexes. CR1 is unusual in that it is polymorphic in size with the four allelic variants having molecular weights of 190,000, 220,000, 250,000, and 280,000 (SDS-PAGE, reducing conditions). The in vitro translation of the common (Mr 220,000) allelic variant CR1 has been achieved by using mRNA in lysates of rabbit reticulocytes and in Xenopus oocytes. HL-60, a promyelocytic human leukemic cell line, was treated with DMSO to induce differentiation and synthesis of CR1. Poly(A+) RNA was purified from these cells by column chromatography on oligo(dT)-cellulose. In the rabbit reticulocyte system, no CR1 was detected unless the translation mixture was denatured. In the presence of methylmercuric hydroxide, the CR1 translation product, unlike most translation products, had the same molecular weight in gel electrophoresis as the high-mannose-containing pro-CR1 and was 15-20K larger than nonglycosylated CR1. This suggests that a cotranslational modification of CR1 structure occurs, probably involving a proteolytic cleavage event. When poly(A+) RNA was translated in Xenopus oocytes, CR1 could be detected by treatment of oocytes with anti-CR1 monoclonal antibody followed by fluorescein-conjugated goat anti-mouse IgG. CR1 was diffusely distributed but preferentially localized to the vegetal surface. The molecular weight of this product, identified in immunoprecipitates of lysates of [35S]methionine-labeled oocytes, was identical with that of CR1 of HL-60.
Kumar
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V
Howard Hughes Medical Institute Laboratories, Washington University School of Medicine, St. Louis, Missouri 63110.
Farries
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T
Swierkoz
J
J
Atkinson
J P
JP
D000818:Animals; D002460:Cell Line; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D007700:Kinetics; D009865:Oocytes / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D014982:Xenopus laevis
D010980:Platelet Membrane Glycoproteins; D012333:RNA, Messenger