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

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.

促红细胞生成素（Erythropoietin, EPO）与其细胞表面受体红细胞生成素受体（EPOR）是红细胞生成（erythropoiesis）过程的必需调控因子；它们可调控非红细胞靶组织，且据报道会影响部分癌症的进展。然而，由于EPOR内源表达水平极低，且抗EPOR抗体的特异性有限，EPOR的表达与细胞内转运的基础研究长期受到阻碍。因此，EPOR生物学的诸多关键环节尚未得到明确阐释，与真性红细胞增多症（polycythemia）相关的突变型EPOR等位基因的功能也未被清晰阐明。本研究使用针对EPOR胞内结构域、磷酸酪氨酸激活结构域及胞外结构域的新型兔单克隆抗体，首次明确阐释了红细胞祖细胞中内源性人EPOR（hEPOR）的以下特性：1）高分子量EPOR亚型仅在EPO匮乏时才会显著表达；2）EPOR-68K与EPOR-70K亚型会依次积累，且EPOR-70K对应细胞表面的EPOR群体；3）布雷菲德菌素A（Brefeldin A）、N-糖苷酶（N-glycanase）及相关分析结果显示，EPOR-68K是一个规模适中的核心糖基化胞内EPOR储备库；4）与近期研究报道相悖，本研究在UT7epo细胞与原代成红细胞（proerythroblasts）中证实，EPOR的内向转运严格依赖配体EPO。在此基础上，当将真性红细胞增多症患者携带的C端截短型hEPOR-T突变等位基因与野生型EPOR在EPO依赖型红细胞祖细胞中共表达时，若干特异性事件发生了改变：其一，EPOR-T等位基因在EPO刺激后会持续激活，但同时也会被降解为低分子量的EPOR产物；其二，在细胞指数生长期，EPOR-T亚型的占比会升高且呈现过度激活状态；其三，EPOR-T的表达还会导致内源性野生型EPOR的数量呈EPO剂量依赖性减少，进而削弱EPOR C端介导的负反馈调控效应。本研究不仅为EPOR表达与转运的调控机制提供了全新的认知，还为阐明突变型EPOR-T等位基因失调红细胞系统（erythron）稳态的分子机制提供了新视角。值得注意的是，本研究同时开发了可用于EPOR表达、激活、功能及代谢研究的新型特异性研究工具。