A High-Throughput Assay to Identify Compounds That Can Induce Dimerization of the Erythropoietin Receptor

Erythropoietin induces dimerization of the erythropoietin receptor on the surface of erythroid progenitor cells, promoting the differentiation of these cells into mature red blood cells. To facilitate screening of large chemical collections for identification of compounds that can dimerize erythropoietin receptor, we have developed a novel, high-throughput in vitro assay to detect compounds that can cause dimerization of the erythropoietin receptor in solution. To develop this assay, amino acid sequences corresponding to the extracellular domain of erythropoietin receptor were expressed in Escherichia coli as erythropoietin-binding protein (rEBP). A modified version of this protein (33P-rEBP) containing a protein kinase A substrate site incorporated into the rEBP was also expressed in E. coli and labeled in vitro using protein kinase A and [γ-33P]ATP. An erythropoietin mimetic peptide (EMP-1), that induces dimerization of rEBP in solution was used to demonstrate dimerization of 33P-rEBP and rEBP in a 96-well microtiter plate format. EMP-1 induced dimerization of rEBP in this assay with an EC50 of ∼245 nM and had a maximal effect at 0.5–2 μM and required the presence of rEBP immobilized on the plate capable of binding EMP-1. EMP-1-induced dimerization of 33P-rEBP and rEBP was reversed by excess unlabeled rEBP and was not masked by complex mixtures such as whole cell fungal extracts. These data demonstrate the ability of 33P-rEBP to dimerize with rEBP in vitro in a format that is fully compatible with high-throughput screening.