LIGAND-BINDING KINETICS OF A SOLUBLE FULL-LENGTH MURINE ERYTHROPOIETIN RECEPTOR

The full-length murine erythropoietin receptor was expressed in Sf9 ce lls using a baculovirus vector. Erythropoietin receptors in solubilize d Sf9 cell lysates bound erythropoietin with high affinity (92 pM) Ery thropoietin receptor-I-125-labeled erythropoietin association and diss ociation kinetics using solubilized Sf9 cell lysates revealed a ka of 0.16 nM(-2) min(-1) and a kd of 0.00055 min(-1) giving an observed KD of 3.45 pM. The erythropoietin receptor was partially purified from Sf 9 cell lysates by chromatography on Con A Sepharose. When erythropoiet in receptors were crosslinked to I-125-labeled erythropoietin and anal yzed by SDS-7.5% PAGE protein complexes of 90 and 125 kDa were observe d with receptors in solubilized lysate, and 170 and 190 kDa with the p artially purified receptors. (C) 1995 Academic Press, Inc.