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Isolation of the full-length murine erythropoietin receptor using a
baculovirus expression system
JL Spivak, LS Avedissian, JH Pierce, D Williams, WD Hankins and RA Jensen
Division of Hematology, Johns Hopkins University School of Medicine,
Baltimore, MD 21205, USA.
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.
Volume 87,
Issue 3,
pp. 926-937,
02/01/1996
Copyright © 1996 by The American Society of Hematology
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