We previously reported the isolation and sequencing of the genomic and cDNA genes for the human erythropoietin receptor (hEpoR) and the development of a receptor transcript phenotype (RTP) assay based on the polymerase chain reaction (PCR). In this report we will discuss how these accomplishments have enabled us 1) to explore the role of the human EpoR in viability, proliferation, differentiation and signal transduction in erythropoiesis and 2) to begin to define sequences responsible for the appropriate regulation of the human EpoR. In the first project, both the normal and a mutationally activated cDNA gene for the human EpoR were cloned into expression vectors and electroporated into an interleukin-3 (IL-3) dependent murine myeloid progenitor cell line, 32D, which does not otherwise express the EpoR. Our analysis of these transformants indicate that both the wild type and mutant human EpoR can functionally substitute for the IL-3 receptor and support viability, proliferation and tyrosine phosphorylation in 32D cells. Transformants expressing the human EpoR do not exhibit overt erythroid differentiation nor are they inhibited from myeloid differentiation. In the second project we have begun to test if tissue and lineage specific transcription is appropriately regulated by sequences in the 5' flanking region of the human EpoR gene. We have made a mammalian expression vector which fuses the 5' flanking region of the human EpoR to the reporter gene beta-galactosidase and begun to study its regulation in different lineages of tissue culture cells.
