Human EPOR Reporter Assay System

This assay utilizes proprietary human cells that provide constitutive expression of the Human Erythropoietin Receptor (EPOR).EPOR is a member of the class I cytokine receptor superfamily. Erythropoietin (EPO), the physiological activator of EPOR, is a glycoprotein that regulates erythropoiesis. EPO, produced by the adult kidneys, activates the cell-surface EPOR to initiate signal transduction through JAK2/STAT5, and other pathways including AKT and ERK1/2. The activation of these various pathways may culminate in the activation of cytosolic targets, or in the activation of specific transcription factors and the induction of their specific target genes.JAK2 dependent phosphorylation and activation of the transcription factor STAT5 is a prominent outcome of EPOR activation, and it is the signaling pathway exploited by the reporter cells in this assay kit. Specifically, INDIGO's Reporter Cells contain the luciferase gene functionally linked to an engineered minimal promoter sequence with upstream tandem STAT5 genetic response element (GRE) sequences. Erythropoietin activates EPOR in a dose-dependent manner, thereby triggering the JAK2/STAT signal transduction cascade. Activated STAT5 binds to its consensus GREs to initiate the formation of a complete transcription complex that drives expression of the Luc reporter gene. Therefore, quantifying changes in luciferase activity from peptide-, drug-, or antibody-treated reporter cells relative to that of 'untreated' cells provides a sensitive, dose-dependent surrogate measure of changes in the activity of EPOR.The clinical use of recombinant human EPO has led to the successful treatment of anemia associated with conditions ranging from chronic kidney disease, chemotherapy treatment for cancer, and surgery-associated blood loss. However, side effects including hypertension, increased risk for stroke, venous thromboembolism, and death, are also risks for patients treated with hEPO and other biosimilars. This provides an opportunity for the development of additional erythropoiesis stimulating agents (ESAs) which lack these adverse effects. Additionally, though erythroid progenitor cells express the highest levels of EPOR, it is also expressed on non-erythroid cells, including neural cells, myoblasts, and adipocytes, indicative of important of physiological functions of EPO outside its essential role in erythropoiesis. Accordingly, the primary application of this reporter assay is to screen test materials for any functional activity, either agonistic or inhibitory, that they may exert against the human EPOR. Kits are offered in different assay formats to accommodate researchers' needs: 3x 32, 1x 96, and 1x 384 assay formats for screening small numbers of test compounds, as well as custom bulk reagents for HTS applications. Assay systems are all inclusive, providing reporter cells, optimized growth media, media for diluting test compounds, a positive-control agonist, luciferase detection reagent, a cell culture-ready assay plate, and a detailed protocol. All kits are shipped on dry ice.Reporter Cells are transiently transfected and prepared as frozen stocks using INDIGO's proprietary CryoMite(TM) process. This cryo-preservation method allows for the immediate dispensing of healthy, division-competent reporter cells into assay plates. There is no need for cumbersome intermediate treatment steps such as spin-and-rinse of cells, viability determinations, or cell titer adjustments prior to assay setup.INDIGO's assay kits provide the convenience of an all-inclusive cell-based assay system. In addition to EPOR Reporter Cells, provided are two optimized media for use in recovering the cryopreserved cells and for diluting test samples, the physiological activator Erythropoietin), Luciferase Detection Reagents, and a cell culture-ready assay plate. Protein function: Receptor for erythropoietin. Mediates erythropoietin-induced erythroblast proliferation and differentiation. Upon EPO stimulation, EPOR dimerizes triggering the JAK2/STAT5 signaling cascade. In some cell types, can also activate STAT1 and STAT3. May also activate the LYN tyrosine kinase. [The UniProt Consortium]