title: Erythrocytosis-associated HIF-2alpha mutations demonstrate a critical role for residues C-terminal to the hydroxylacceptor proline.
abstract: A classic physiologic response to hypoxia in humans is the up-regulation of the ERYTHROPOIETIN (EPO) gene, which is the central regulator of red blood cell mass. The EPO gene, in turn, is activated by hypoxia inducible factor (HIF). HIF is a transcription factor consisting of an alpha subunit (HIF-alpha) and a beta subunit (HIF-beta). Under normoxic conditions, prolyl hydroxylase domain protein (PHD, also known as HIF prolyl hydroxylase and egg laying-defective nine protein) site specifically hydroxylates HIF-alpha in a conserved LXXLAP motif (where underlining indicates the hydroxylacceptor proline). This provides a recognition motif for the von Hippel Lindau protein, a component of an E3 ubiquitin ligase complex that targets hydroxylated HIF-alpha for degradation. Under hypoxic conditions, this inherently oxygen-dependent modification is arrested, thereby stabilizing HIF-alpha and allowing it to activate the EPO gene. We previously identified and characterized an erythrocytosis-associated HIF2A mutation, G537W. More recently, we reported two additional erythrocytosis-associated HIF2A mutations, G537R and M535V. Here, we describe the functional characterization of these two mutants as well as a third novel erythrocytosis-associated mutation, P534L. These mutations affect residues C-terminal to the LXXLAP motif. We find that all result in impaired degradation and thus aberrant stabilization of HIF-2alpha. However, each exhibits a distinct profile with respect to their effects on PHD2 binding and von Hippel Lindau interaction. These findings reinforce the importance of HIF-2alpha in human EPO regulation, demonstrate heterogeneity of functional defects arising from these mutations, and point to a critical role for residues C-terminal to the LXXLAP motif in HIF-alpha.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Paul W
authors.initials: PW
authors.lastname: Furlow
authors.forename: Melanie J
authors.initials: MJ
authors.lastname: Percy
authors.forename: Scott
authors.initials: S
authors.lastname: Sutherland
authors.forename: Charlene
authors.initials: C
authors.lastname: Bierl
authors.forename: Mary Frances
authors.initials: MF
authors.lastname: McMullin
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Master
authors.forename: Terence R J
authors.initials: TR
authors.lastname: Lappin
authors.forename: Frank S
authors.initials: FS
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D051792:Basic Helix-Loop-Helix Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D002460:Cell Line; D006801:Humans; D006900:Hydroxylation; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011086:Polycythemia / Q000235:genetics / Q000378:metabolism*; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics / Q000378:metabolism; D011392:Proline / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D016415:Sequence Alignment; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000378:metabolism; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D011393:Procollagen-Proline Dioxygenase; D011392:Proline; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D000099162:Endothelial PAS Domain-Containing Protein 1