title: Binding and internalization of recombinant human erythropoietin in murine erythroid precursor cells.
abstract: Erythropoietin (EPO) biosynthetically labelled with [35S]cysteine was produced from Chinese hamster ovary (CHO) cells containing amplified copies of human EPO cDNA. The glycosylated recombinant [35S]EPO, purified to virtual radiochemical homogeneity, was biologically active. We studied the interaction of this labeled recombinant EPO with erythroid precursor cells from mice made anemic with phenylhydrazine. The [35S]-labeled molecule bound to erythroid precursors in a time- and temperature-dependent manner. The binding was specific for EPO, and neither insulin, transferrin, epidermal growth factor, nor multiplication stimulating activity could compete for EPO binding sites. In the presence of 0.2% sodium azide, which blocks 80% to 90% of internalization, the recombinant molecule bound with an apparent Kd of 750 pmol/L and 100 to 200 binding sites per cell at 37 degrees C. Asialo-EPO was a more effective competitor than sialated EPO for the available binding sites. Thus, the enhanced biological specific activity of asialo-EPO could result from its enhanced binding affinity. We also studied recombinant human EPO labeled with 125I and found that it also bound to the erythroid cells in a saturable and specific manner. After 90 minutes of incubation at 37 degrees C, most of the bound [35S]EPO was internalized, whereas most of the [125I]EPO remained on the cell surface. The reduced internalization of the iodinated molecule could account for the previously reported functional deficit associated with iodination.
mesh_terms: D000818:Animals; D001386:Azides / Q000494:pharmacology; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D004900:Erythroblasts / Q000378:metabolism*; D004920:Erythropoiesis; D004921:Erythropoietin / Q000378:metabolism*; D005260:Female; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D007457:Iodine Radioisotopes; D051379:Mice; D008815:Mice, Inbred Strains; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism*; D019810:Sodium Azide; D013329:Structure-Activity Relationship; D013696:Temperature; D013997:Time Factors
chemical_list: D001386:Azides; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D004921:Erythropoietin; D019810:Sodium Azide