Ligand binding kinetics of a soluble full length murine erythropoietin receptor

The full-length murine erythropoietin receptor was expressed in Sf9 cells using a baculovirus vector. Erythropoietin receptors in solubilized Sf9 cell lysates bound erythropoietin with high affinity (92 pM) Erythropoietin receptor-¹²⁵I-labeled erythropoietin association and dissociation 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 Sf9 cell lysates by chromatography on Con A Sepharose. When erythropoietin receptors were crosslinked to ¹²⁵I-labeled erythropoietin and analyzed by SDS-7.5% PAGE protein complexes of 90 and 125 kDa were observed with receptors in solubilized lysate, and 170 and 190 kDa with the partially purified receptors.