Isolation of the full-length murine erythropoietin receptor using a baculovirus expression system.
The full-length murine erythropoietin receptor was expressed in Spodoptera frugiperda (Sf9) cells using a recombinant baculovirus vector. Erythropoietin receptor protein production was maximal 48 hours after infection, as determined by metabolic labeling and immunoblotting; receptor protein varied in molecular mass from 62 to 76 kD. Erythropoietin receptors produced in Sf9 cells could be solubilized using CHAPS in a form capable of binding erythropoietin, and the solubilized receptor bound to immobilized Concanavalin A (Con A) and wheat germ agglutinin, as well as to immobilized recombinant human erythropoietin. Analysis of the distribution of erythropoietin receptors in Sf9 plasma membrane and cytosol fractions using lectin affinity chromatography revealed that membrane-bound receptor had a higher apparent molecular mass and contained the bulk of receptors that bound to wheat germ agglutinin. The receptor was purified by sequential affinity chromatography on Con A-Sepharose and immobilized erythropoietin. Erythropoietin receptors expressed in Sf9 cells were inserted into the plasma membrane in the correct orientation, bound 125I-erythropoietin with a single affinity (kD, 330 pmol/L), and were internalized after ligand binding. However, kD varied inversely with the number of cell surface receptors. Solubilized erythropoietin receptors in whole-cell lysates and isolated plasma membranes exhibited high-affinity binding, with kD values of 92 and 57 pmol/L, respectively. Erythropoietin bound to the surface of infected Sf9 cells could be cross-linked to two proteins with molecular masses of 90 and 65 kD using the homobifunctional cross-linker, disuccinimidyl suberate (DSS). Similar results were obtained with solubilized receptors in whole-cell lysates, and both proteins could be immunoprecipitated by an antiserum to the erythropoietin receptor carboxyl-terminal domain.
Spivak
J L
JL
Division of Hematology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Avedissian
L S
LS
Pierce
J H
JH
Williams
D
D
Hankins
W D
WD
Jensen
R A
RA
D000818:Animals; D002460:Cell Line; D003432:Cross-Linking Reagents; D018076:DNA, Complementary / Q000235:genetics; D004921:Erythropoietin / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D006031:Glycosylation; D006801:Humans; D008565:Membrane Proteins / Q000302:isolation & purification; D051379:Mice / Q000235:genetics*; D008970:Molecular Weight; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D018411:Spodoptera; D013388:Succinimides
D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D008565:Membrane Proteins; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D013388:Succinimides; D004921:Erythropoietin; C019358:disuccinimidyl suberate