| First Author | Spivak JL | Year | 1993 |
| Journal | Blood | Volume | 82 |
| Pages | 288a (Abstr.) | Mgi Jnum | J:30270 |
| Mgi Id | MGI:77909 | Citation | Spivak JL, et al. (1993) Isolation of the full length murine erythropoietin receptor (EPO-R) using a baculovirus expression system. Blood 82:288a (Abstr.) |
| abstractText | Full text of Abstract: ISOLATION OF THE FULL LENGTH MURINE ERYTHROPOIETIN RECEPTOR (EPO-R) USING A BACULOVIRUS EXPRESSION SYSTEM. J.L. Spivak, L.S. Avedissian*, R.A. Jensen*, D. Williams*, W.D. Hankins and J. Pierce*. Division of Hematology, Johns Hopkins University School of Medicine, Baltimore, MD and The National Cancer Institute, Bethesda, MD. Erythropoiesis is regulated by erythropoietin (EPO) but although the genes for EPO and the EPO-R have been molecularly cloned, the nature of the receptor-ligand interaction is not fully understood. Attempts to resolve this issue have been hindered by the low number of surface receptors expressed by normal or transformed erythroid progenitor cells. To address this problem, we used a heterologous eukaryotic protein expression system which employs a recombinant baculovirus vector and the insect cell line Sf9. A full length cDNA for the murine EPO-R was cloned into the unique Bam HI site of the baculovirus vector pVL941 and recombinant virus was obtained by cotransfection of Sf9 cells with wild-type baculovirus DNA and the pVL941 construct using calcium phosphate coprecipitation. Sf9 cells infected with the recombinant baculovirus produced EPO-R maximally after 48 hours as determined by metabolic labeling. Solubilized EPO-R in whole cell lysates bound to both immobilized Con A and wheat germ agglutinin as well as to immobilized erythropoietin and could be purified by sequential affinity chromatography. The EPO-R expressed in Sf9 cells was inserted into the plasma membrane in the correct orientation, bound 125-I-EPO with a Kd of 3.6 nM and was internalized after ligand binding. By contrast, solubilized EPO-R in whole cell lysates exhibited both high and low affinity binding to 125-I-EPO with Kd values of 0.65 nM and 18.5 nM respectively. 125-I-EPO bound to the surface of infected Sf9 cells could be crosslinked with DSS to two proteins with molecular masses of 90 and 65 kDa. Similar results were also obtained with solubilized EPO-R in whole cell lysates. These two EPO binding proteins appear to represent EPO-R which have been differentially processed. The baculovirus expression system using Sf9 cells provides a means of obtaining large quantities of biologically active, full length EPO-R as well as a model system for studying EPO-R metabolism. |
