Dynamic Ligand Modulation of EPO Receptor Pools, and Dysregulation by Polycythemia-Associated EPOR Alleles

Abstract

Erythropoietin (EPO) and its cell surface receptor (EPOR) are essential for erythropoiesis; can modulate non-erythroid target tissues; and have been reported to affect the progression of certain cancers. Basic studies of EPOR expression and trafficking, however, have been hindered by low-level EPOR occurrence, and the limited specificity of anti-EPOR antibodies. Consequently, these aspects of EPOR biology are not well defined, nor are actions of polycythemia- associated mutated EPOR alleles. Using novel rabbit monoclonal antibodies to intracellular, PY- activated and extracellular EPOR domains, the following properties of the endogenous hEPOR in erythroid progenitors first are unambiguously defined. 1) High- Mr EPOR forms become obviously expressed only when EPO is limited. 2) EPOR-68K plus -70K species sequentially accumulate, and EPOR-70K comprises an apparent cell surface EPOR population. 3) Brefeldin A, N-glycanase and associated analyses point to EPOR-68K as a core-glycosylated intracellular EPOR pool (of modest size). 4) In contrast to recent reports, EPOR inward trafficking is shown (in UT7epo cells, and primary proerythroblasts) to be sharply ligand-dependent. Beyond this, when C-terminal truncated hEPOR-T mutant alleles as harbored by polycythemia patients are co-expressed with the wild-type EPOR in EPO-dependent erythroid progenitors, several specific events become altered. First, EPOR-T alleles are persistently activated upon EPO- challenge, yet are also subject to apparent turn-over (to low-Mr EPOR products). Furthermore, during exponential cell growth EPOR-T species become both over-represented, and hyper-activated. Interestingly, EPOR-T expression also results in an EPO dose-dependent loss of endogenous wild-type EPOR's (and, therefore, a squelching of EPOR C-terminal- mediated negative feedback effects). New knowledge concerning regulated EPOR expression and trafficking therefore is provided, together with new insight into mechanisms via which mutated EPOR-T polycythemia alleles dysregulate the erythron. Notably, specific new tools also are characterized for studies of EPOR expression, activation, action and metabolism.

Stage-specific functional roles of integrins in murine erythropoiesis

“…Considering the previously reported four-fold increase of Epo-R levels from late erythroid progenitor cells to the early erythroblast BFU-e progeny [13], this pattern of ablation in our mice likely reflects more efficient Cre activation and consequent integrin ablation beyond the CD71 hi TER119 lo level. These data are consistent with maintenance of functional EpoR in late (hGlycophorinA hi ) erythroblasts [14]. Earlier progenitors, i.e., BFU-e/CFU-e, present in kit + CD71 hi populations, were only partially ablated (Suppl.…”

Immunosuppressive Effects of Erythropoietin on Human Alloreactive T Cells

“…Previous work by others showed that anti-CD3/anti-CD28 upregulates the Sp1 transcription factor known to positively regulate EPO-R transcription, 22 further supporting this concept. Whether TCR signaling alters trafficking of EPO-R to the T-cell surface, which has been reported in erythroid cells, 23 remains untested.…”

A dimeric peptide with erythropoiesis-stimulating activity uniquely affects erythropoietin receptor ligation and cell surface expression

“…UT7epo cells and Baf3-hEPOR cells were cultured as per Singh et al (Singh, et al 2012), and Boehm et al (Boehm, et al 2014). Towards establishing dose equivalencies for peginesatide and rHu-EPO, cells were plated (1.5 × 10 5 cells/mL) and cultured in either peginesatide or rHu-EPO at varied concentrations.…”

“…Assays of cell surface EPOR expression were performed using antibody EC-c38.5 as described previously (Singh, et al 2012). Briefly, cells in antibody binding buffer (ABB) were incubated for 10 minutes at 4°C with 0.3μg/mL Fc blocker (Stem Cell Technologies, cat #01470) and then with hIgG (Jackson Immunoresearch, cat #009-000-003) (1μg per 0.2mL assay).…”