The erythropoietin receptor mediates pro-erythroblast production; can promote erythrocytosis; and can cytoprotect select non-hematopoietic tissues. For certain key aspects of EPOR biology, knowledge nonetheless remains limited. This includes regulated EPOR trafficking, which we presently investigate using a novel panel of specific rabbit monoclonal anti-EPOR antibodies. In exponentially growing EPO-dependent UT7epo cells, a single high-Mr species was detected at low levels, and EPOR turnover appeared to predominate. The limiting of EPO led to the accumulation of two high Mr EPOR forms, EPOR-68K and EPOR-70K. EPO exposure rapidly converted an indicated cell surface EPOR-70K form to an EPOR-72K tyrosine-phosphorylated species (and to lower Mr fragments). In contrast, EPOR-68K was not rapidly modulated by EPO, and proved to comprise an intracellular, endoglycosidase-sensitive EPOR pool. At the cell surface, EPOR repopulation also was regulated and interestingly was accelerated in response to higher-dose EPO exposure. Constitutive EPOR turnover was observed, but as comparably limited ligand-independent trafficking. EPOR expression in primary erythroid progenitors also was analyzed. Flow cytometry and western blotting each unexpectedly revealed peak EPOR expression at a GPAhigh late erythroblast stage. These studies provide new insight into dynamic, EPO dose-dependent EPOR trafficking, together with an observed sustained expression of the EPOR within maturing primary human erythroblasts.
Leu:Affymax, Inc.: Employment. Mortensen:Affymax, Inc.: Employment. Young:Affymax, Inc.: Employment. Schatz:Affymax, Inc.: Employment. Green:Affymax, Inc.: Employment.
Asterisk with author names denotes non-ASH members.