ISOLATION OF THE FULL-LENGTH MURINE ERYTHROPOIETIN RECEPTOR USING A BACULOVIRUS EXPRESSION SYSTEM

The full-length murine erythropoietin receptor was expressed in Spodop tera frugiperda (Sf9) cells using a recombinant baculovirus vector. Er ythropoietin receptor protein production was maximal 48 hours after in fection, as determined by metabolic labeling and immunoblotting; recep tor 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 b ound to immobilized Concanavalin A (Con A) and wheat germ agglutinin, as well as to immobilized recombinant human erythropoietin. Analysis o f the distribution of erythropoietin receptors in Sf9 plasma membrane and cytosol fractions using lectin affinity chromatography revealed th at membrane-bound receptor had a higher apparent molecular mass and co ntained 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 exp ressed in Sf9 cells were inserted into the plasma membrane in the corr ect orientation, bound I-125-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. Solubiliz ed 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, disuccinimid yl suberate (DSS). Similar results were obtained with solubilized rece ptors in whole-cell lysates, and both proteins could be immunoprecipit ated by an antiserum to the erythropoietin receptor carboxyl-terminal domain. (C) 1996 by The American Society of Hematology.