title: 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.
authors.affiliation: Center of Excellence in Stem Cell Biology and Regenerative Medicine, Maine Medical Center Research Institute, Scarborough, Maine, United States of America.
authors.forename: Seema
authors.initials: S
authors.lastname: Singh
authors.forename: Rakesh
authors.initials: R
authors.lastname: Verma
authors.forename: Anamika
authors.initials: A
authors.lastname: Pradeep
authors.forename: Karen
authors.initials: K
authors.lastname: Leu
authors.forename: R Bruce
authors.initials: RB
authors.lastname: Mortensen
authors.forename: Peter R
authors.initials: PR
authors.lastname: Young
authors.forename: Miho
authors.initials: M
authors.lastname: Oyasu
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Schatz
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Green
authors.forename: Don M
authors.initials: DM
authors.lastname: Wojchowski
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D020126:Brefeldin A / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D004705:Endocytosis / Q000187:drug effects; D015672:Erythroid Precursor Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D004921:Erythropoietin / Q000494:pharmacology; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008024:Ligands; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D011086:Polycythemia / Q000235:genetics*; D021381:Protein Transport / Q000187:drug effects; D017467:Receptors, Erythropoietin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D008024:Ligands; D017467:Receptors, Erythropoietin; D004921:Erythropoietin; D020126:Brefeldin A