Oxygen-dependent Regulation of Erythropoietin Receptor Turnover and Signaling

Abstract

von Hippel-Lindau (VHL) disease is a rare familial cancer predisposition syndrome caused by a loss or mutation in a single gene, VHL, but it exhibits a wide phenotypic variability that can be categorized into distinct subtypes. The phenotypic variability has been largely argued to be attributable to the extent of deregulation of the ␣ subunit of hypoxia-inducible factor ␣, a well established target of VHL E3 ubiquitin ligase, ECV (Elongins/ Cul2/VHL). Here, we show that erythropoietin receptor (EPOR) is hydroxylated on proline 419 and 426 via prolyl hydroxylase 3. EPOR hydroxylation is required for binding to the ␤ domain of VHL and polyubiquitylation via ECV, leading to increased EPOR turnover. In addition, several type-specific VHL diseasecausing mutants, including those that have retained proper binding and regulation of hypoxia-inducible factor ␣, showed a severe defect in binding prolyl hydroxylated EPOR peptides. These results identify EPOR as the second bona fide hydroxylation-dependent substrate of VHL that potentially influences oxygen homeostasis and contributes to the complex genotypephenotype correlation in VHL disease.von Hippel-Lindau (VHL) 2 disease is a rare familial cancer predisposition syndrome characterized by the development of tumors in several organ systems, including the central nervous system and retinal hemangioblastoma (HAB), which represent the two cardinal features of VHL disease, as well as endolymphatic sac tumor of the inner ear, pancreatic cystadenoma, benign cystadenoma of epididymis, adnexal papillary cystadenoma of probable mesonephric origin, clear-cell renal cell carcinoma (RCC), paragangliomas, and pheochromocytoma (PHEO) (1). Some VHL patients also develop polycythemia with or without the other stigmata of VHL disease. VHL disease is subcategorized into type 1, characterized by HAB and RCC without PHEO; type 2A, characterized by PHEO and HAB without RCC; type 2B, characterized by PHEO and HAB with RCC; type 2C, characterized by exclusive development of PHEO; and type 3, which is characterized by the development of polycythemia without an increased cancer predisposition (2). Notably, despite the phenotypic heterogeneity, VHL disease is caused by a mutation or loss of a single gene, VHL.VHL is the substrate recognizing subunit of a multiprotein E3 ubiquitin ligase ECV (Elongins BC/Cul2/VHL) that targets the ␣ subunit of hypoxia-inducible factor (HIF␣) for ubiquitylation under normal oxygen tension (3). HIF␣ is hydroxylated on conserved prolines within the oxygen-dependent degradation domain (ODD) by a family of prolyl hydroxylase enzymes (PHD1-3) in the presence of 2-oxoglutarate, iron, and oxygen (4 -7). VHL binds exclusively to prolyl hydroxylated HIF␣. Thus, under hypoxia, the unmodified HIF␣ escapes the recognition by VHL and heterodimerizes with the constitutively expressed HIF␤ to form an active transcription factor, which transactivates numerous hypoxia-inducible genes involved in various adaptive processes to hypoxia such as anaerobic metabolism, angiogenesis, and er...

Genotype-Phenotype Relations of the von Hippel-Lindau Tumor Suppressor Inferred from a Large-Scale Analysis of Disease Mutations and Interactors

“…Based on these data, we suggest that the pathogenic assessment of mutations affecting this area should also include other putative pVHL hydroxylated-substrates beyond the sole HIF-1/2α, e.g. SPRY2 (37), ADRB2 (38), EPOR (39). Eleven interactors are found to be affected by surface C mutations.…”

New Insights into Protein Hydroxylation and Its Important Role in Human Diseases

“…[103] showed for the first time that EPOR protein levels are regulated by PHD3/EglN3 -mediated prolyl hydroxylation on Pro419 and Pro426 residues (Fig. 6A).…”

The pVHL neglected functions, a tale of hypoxia-dependent and -independent regulations in cancer

“…Recently, another example of hydroxy-degron was described by Ohh and co-workers for the erythropoietin receptor (EPOR) [ 22 ]. EPOR is hydroxylated on Pro419 and Pro426 via PHD3, the resulting degrons have no sequence identity to LxxLAP motif, while localize within a proline-rich region ( figure 2 ).…”

“…Indeed, the authors reported examples of pVHL mutants that retain proper binding and regulation of HIF-1 α while showing severe defect in binding EPOR. In particular, pVHL mutations classified as promoting VHL disease sub-phenotype 2C, showed a marked defect in binding to hydroxylated EPOR [ 22 ]. These findings suggest at least one novel molecular framework to explain the wide phenotypic variability in VHL disease.…”