Central biological properties of hematopoietic growth factor receptors include regulated expression, cell surface residency and internalization features. These can be diverse, and are important to understand within contexts of defining cytokine target tissues and cells; possible effects of cytokine dosing on receptor up- and/or down- modulation; distinct routes engaged by growth factor agonists, mimetics or antagonists; and possible dysregulation of such receptor properties due to mutation or skewed expression. Among HGFR's, EPOR complexes present a clinically relevant system that continues to serve as an important paradigm. To illustrate, the EPOR was first to be directly associated with Janus kinase action 
; the first HGF-R to be understood to transduce trans-membrane conformational signaling events 
; and among the first to be defined as a target of SOCS plus SHIP negative regulators 
. Recently, new impetus to further advance an understanding of EPOR properties has been brought to bear. This includes the emergence of new EPOR agonists 
as well as apparent EPOR- cytoprotective effects on vascular 
, retinal 
and pancreatic beta- cells 
. This is being met with continued new discoveries, including reports on Lnk protein 
and transferrin receptor-2 
as novel transducers of EPOR action in primary proerythroblasts. With a goal of better understanding basic EPOR properties, the present study focuses first on defining the nature of distinct EPOR forms, their interrelatedness, and an unresolved question of the extent to which EPOR expression at the cell surface is subject to ligand independent- vs
By developing a novel panel of rabbit monoclonal antibodies specific to the hEPOR, and through analyses of the endogenous EPOR expression in an EPO- dependent human erythroid progenitor cell model, we first show that an EPOR-68K form corresponds to a core- glycosylated apparent intracellular EPOR pool; and that this pool increases in levels when EPO is limiting. Upon further sustained limiting of EPO, an EPOR-70K form is generated, which (unlike EPOR-68K) is indicated to then rapidly convert upon EPO exposure to an activated EPOR-72K species. (Interestingly, quantitative RT-PCR analyses of EPOR
transcript levels rule out up-modulation at this level as a potential underlying mechanism) (Figure S8
). Here, novel information generated is several-fold. The finding that expression levels of intracellular and apparent cell surface EPOR forms flux in ligand-dependent fashions first is insightful. Specifically, this is unlike the majority of growth factor receptors which typically maintain at least moderate pools of mature cell surface receptor forms during ligand- exposure. In studies of cells and tissues that have been exposed to EPO, lack of insight into these EPOR properties can unwittingly skew interpretations (for example, regarding the presence vs
absence of EPOR's among candidate target cells). For apparent intracellular species, interesting questions also are raised as to what specific post-translational features may distinguish EPOR-68K from EPOR-70K forms (and which might possibly affect cell surface translocation). In addition, certain prior studies of the EPOR in Friend virus transformed MEL cells 
, or non-erythroid cells ectopically EPOR forms 
have implicated the occurrence of large pools of intracellular EPOR's. This may relate to viral gp55 plus EPOR interactions and/or consequences of EPOR overexpression – High-level intracellular EPOR pools presently have not been observed in UT7epo erythroid progenitor cells (under any conditions).
A second major aspect of the present studies addresses the extent to which inward trafficking of EPOR's might be ligand -independent vs
-dependent. In particular, recent studies by Becker and co-workers 
have suggested that ligand-independent EPOR trafficking may be a prime property of the EPOR system, and that this property may allow for linear integration of EPOR signals over a broad range of ligand concentrations. In contrast, the present studies of endogenous EPOR internalization in EPO- dependent UT7epo human erythroid progenitor cells strongly argue for sharply ligand- dependent EPOR expression (and turnover) routes. This finding is based on both flow cytometry, and western blot analyses of mature EPOR forms. Furthermore, this is to the extent that cells when cultured in EPO express only nominal levels of cell surface EPOR's. Conversely, when EPO becomes limiting, endogenous EPOR forms become substantially up-modulated at the cell surface. This basic aspect of EPOR trafficking has been a matter of controversy, and is non-trivial for considerations of ligand pharmacokinetics, and responses of EPOR's and target cells to physiological 1000-fold fluxes in EPO levels 
. Factors that may contribute to apparently disparate results and/or interpretations for ligand-modulated EPOR trafficking merit brief discussion. One may involve effects of EPOR epitope- tagging as frequently employed to overcome limiting features of available EPOR antibodies. Our laboratory was among the first to employ this approach for the EPOR in the form of an N-terminal HAI tag 
, and recently we have alternatively incorporated a FLAG tag at the hEPOR's C-terminus. Unfortunately, each modification compromises EPOR biological activity (i.e., EPO-dependenrt cell growth) several- fold as assessed in BaF3, FDCW2 and/or UT7epo cells (data not shown). This complication provided impetus for our development of new EPOR antibodies with improved properties. A second may relate to possibly altered trafficking if/when EPOR's are ectopically over-expressed. Third, analyses of EPOR trafficking in non-erythroid or non-hematopoietic cells also may be affected in unpredictable ways by heterologous factors.
The present studies also consider cell surface expression, activation and internalization properties of representative C-terminal truncated EPOR forms that have been described among PFCP patients 
. Each form presently studied harbors a translational stop mutation within exon-8. One, G5881T, gives rise to an EPOR-T392 form 
; and the other, G1251T, to an EPOR-T374 form 
. In addition, each lacks 7 of 9 cytoplasmic phosphotyrosine motifs for signal transduction factor recruitment 
as well as the majority of lysine sites for potential ubiquitination. Also lacking is a proposed cytoplasmic binding motif for a BTRC E3 ubiquitin ligase 
. One hypothesis advanced for enhanced functional attributes of EPOR-T alleles has involved a prediction that such truncated receptor forms might be substantially compromised in internalization capacities (and consequently may reside persistently on the surface of erythroid progenitors) 
. Recent studies of mutated EPOR alleles in transfected gamma-2A and BaF3 cells also are consistent with this notion 
. As presently studied in EPO-dependent erythroid progenitors, however, internalization rates for truncated EPOR-T forms were attenuated by only ~25% (see supporting data, Figure S9
). In exponentially growing erythroid progenitors, it nonetheless was apparent that truncated EPOR-T392 and EPOR-T374 forms accumulate as mature species at levels greater than observed for the wt-EPOR. One interpretation of these results is that hyperallelic activities of truncated EPOR forms may depend on attenuated internalization (by speculation), together with possibly attenuated transit through endosomes and/or proteosomes. In addition, these defects are exerted most strikingly under physiological conditions of exponential growth. By analogy to recent findings in the JAK kinase- linked IL7R and IL5R systems, HGF receptor activation may involve (or depend upon) early endosome entry 
. If this proves to be the case for the EPOR, then attenuated EPOR-T endosomal transit might also contribute to sustained activation. Consistent with this interpretation is an observed sustained activation of PY-EPOR-T forms (together with JAK2).
An additional observation that merits discussion concerns effects of truncated EPOR alleles on the expression of the endogenous EPO receptor. (Notably, truncated EPOR alleles typically are co-expressed together with a normal EPOR allele in primary familial and congenital polycythemia patients) 
. In particular, when cells were maintained in EPO, EPOR-T expression resulted in obvious decreases in endogenous EPOR levels. Several mechanisms might mediate this effect. First, when heterodimerized with truncated EPOR forms, the endogenous EPOR may be co-internalized but more readily processed (via C-terminal sub-domains) upon entering endosomes. Second, during outward trafficking, JAK2 chaperone effects, and/or EPOR processing may be affected. In support of this notion, EPOR-T effects on endogenous EPOR levels appear to also include decreases in EPOR-68K forms. Via either (or both) route(s), decreases in levels of wild-type EPOR's in erythroid progenitors of polycythemia patients translate to an increased escape of EPOR-JAK2 complexes from negative-feedback factors (as recruited via EPOR C-terminal domains). For other HGFR's that occur as truncated alleles (e.g., MPL, GCSFR) 
it should be of significant interest to discover possible parallels in dysregulation in likewise clinically meaningful contexts.
A final notable advancement of the present study involves properties and utilities of our novel anti-EPOR antibodies per se. Antibody IC-c1.1 exhibits high specificity (and sensitivity) in western blotting; and antibody EC-c38.5 is proposed to possess uniquely advantageous properties for flow cytometry. Specific immunoprecipitation of EPOR's by antibodies IC-c1.1, IC-c34.11 and EC-c38.5 also is illustrated. In addition, promising utility of ICD antibody IC-c1.1 in IHC is demonstrated. Together, our new EPOR antibody panel therefore represents a valuable new tool set for investigations of the intriguing and clinically important properties of the EPOR.