Erythrocytosis-associated HIF-2α 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 α subunit (HIF-α) and a β subunit (HIF-β). Under normoxic conditions, prolyl hydroxylase domain protein (PHD, also known as HIF prolyl hydroxylase and egg laying-defective nine protein) site specifically hydroxylates HIF-α 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-α for degradation. Under hypoxic conditions, this inherently oxygen-dependent modification is arrested, thereby stabilizing HIF-α 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-2α. 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-2α 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-α. 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 α subunit (HIF-α) and a β subunit (HIF-β). Under normoxic conditions, prolyl hydroxylase domain protein (PHD, also known as HIF prolyl hydroxylase and egg laying-defective nine protein) site specifically hydroxylates HIF-α 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-α for degradation. Under hypoxic conditions, this inherently oxygen-dependent modification is arrested, thereby stabilizing HIF-α 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-2α. 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-2α 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-α. Gene transcriptional responses to hypoxia in mammals are in large part mediated by hypoxia inducible factor (HIF) 2The abbreviations used are: HIF, hypoxia inducible factor; EPO, erythropoietin, Hyp, hydroxylproline; PHD, prolyl hydroxylase domain protein; VHL, von Hippel Lindau protein; GST, glutathione S-transferase. (1Semenza G.L. Science. 2007; 318: 62-64Crossref PubMed Scopus (562) Google Scholar). Although the core mechanism, that is, PHD-induced hydroxylation of HIF-α leading to VHL-mediated degradation, is relatively simple, an additional layer of complexity is provided by the multiplicity of isoforms at different levels of this pathway (2Schofield C.J. Ratcliffe P.J. Nat. Rev. Mol. Cell Biol. 2004; 5: 343-354Crossref PubMed Scopus (1620) Google Scholar, 3Kaelin W.G. Annu. Rev. Pathol. 2007; 2: 145-173Crossref PubMed Scopus (274) Google Scholar). Thus, there are three HIF-α isoforms (HIF-1α, HIF-2α, and HIF-3α) and three PHD isoforms (PHD1, PHD2, and PHD3) (1Semenza G.L. Science. 2007; 318: 62-64Crossref PubMed Scopus (562) Google Scholar, 2Schofield C.J. Ratcliffe P.J. Nat. Rev. Mol. Cell Biol. 2004; 5: 343-354Crossref PubMed Scopus (1620) Google Scholar, 3Kaelin W.G. Annu. Rev. Pathol. 2007; 2: 145-173Crossref PubMed Scopus (274) Google Scholar, 4Gordan J.D. Simon M.C. Curr. Opin. Genet. Dev. 2007; 17: 71-77Crossref PubMed Scopus (376) Google Scholar). The role of these isoforms in oxygen sensing is an area of active investigation. HIF binds to the promoters and enhancers of a broad range of genes involved in cellular, local, and systemic responses to hypoxia. They include genes that facilitate glucose uptake, enhance glycolysis, inhibit the Krebs cycle, promote angiogenesis, and augment red blood cell production (4Gordan J.D. Simon M.C. Curr. Opin. Genet. Dev. 2007; 17: 71-77Crossref PubMed Scopus (376) Google Scholar, 5Chowdhury R. Hardy A. Schofield C.J. Chem. Soc. Rev. 2008; 37: 1308-1319Crossref PubMed Scopus (89) Google Scholar, 6Semenza G.L. Annu. Rev. Cell Dev. Biol. 1999; 15: 551-578Crossref PubMed Scopus (1676) Google Scholar). The latter is mediated by EPO, a glycoprotein that is the product of the EPO gene, which is considered a paradigm of oxygen-regulated gene expression because of its high dynamic range of inducibility and the central importance of this gene for oxygen delivery to tissues (7Stockmann C. Fandrey J. Clin. Exp. Pharmacol. Physiol. 2006; 33: 968-979Crossref PubMed Scopus (128) Google Scholar, 8Jelkmann W. Eur. J. Haematol. 2007; 78: 183-205Crossref PubMed Scopus (287) Google Scholar). Aside from addressing the fundamental biologic interest in oxygen-dependent regulation of EPO, this pathway has also provided a framework for understanding certain rare forms of erythrocytosis in which there is abnormally increased red blood cell production due to aberrant up-regulation of EPO (9Lee F.S. Percy M.J. McMullin M.F. Cell Cycle. 2006; 5: 941-945Crossref PubMed Scopus (11) Google Scholar, 10Percy M.J. Lee F.S. Haematologica. 2008; 93: 963-967Crossref PubMed Scopus (19) Google Scholar, 11Gordeuk V.R. Stockton D.W. Prchal J.T. Haematologica. 2005; 90: 109-116PubMed Google Scholar, 12Lee F.S. Blood Rev. 2008; 22: 321-332Crossref PubMed Scopus (34) Google Scholar). The first gene of the oxygen-sensing pathway in which erythrocytosis-associated mutations were identified was the VHL gene (13Ang S.O. Chen H. Hirota K. Gordeuk V.R. Jelinek J. Guan Y. Liu E. Sergueeva A.I. Miasnikova G.Y. Mole D. Maxwell P.H. Stockton D.W. Semenza G.L. Prchal J.T. Nat. Genet. 2002; 32: 614-621Crossref PubMed Scopus (430) Google Scholar), followed by mutations in the PHD2 gene (14Percy M.J. Zhao Q. Flores A. Harrison C. Lappin T.R. Maxwell P.H. McMullin M.F. Lee F.S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 654-659Crossref PubMed Scopus (261) Google Scholar). Subsequently, we described a mutation of the HIF2A gene in a family with hereditary erythrocytosis (15Percy M.J. Furlow P.W. Lucas G.S. Li X. Lappin T.R. McMullin M.F. Lee F.S. N. Engl. J. Med. 2008; 358: 162-168Crossref PubMed Scopus (244) Google Scholar). This mutation, G537W, affects a residue in close proximity to the primary prolyl hydroxylation site in HIF-2α, Pro-531. Functional studies indicated that it impairs both the ability of PHD2 to bind to and hydroxylate HIF-2α, as well as the subsequent capacity of VHL to recognize it. We more recently reported two additional erythrocytosis-associated HIF2A mutations, G537R and M535V (16Percy M.J. Beer P.A. Campbell G. Dekker A.W. Green A.R. Oscier D. Rainey M.G. van Wijk R. Wood M. Lappin T.R. McMullin M.F. Lee F.S. Blood. 2008; 111: 5400-5402Crossref PubMed Scopus (100) Google Scholar). One of these mutations, G537R, has subsequently been independently identified in other patients with erythrocytosis (17Perrotta S. Della Ragione F. N. Engl. J. Med. 2008; 358 (author reply 1966–1967): 1966PubMed Google Scholar, 18Gale D.P. Harten S.K. Reid C.D. Tuddenham E.G. Maxwell P.H. Blood. 2008; 112: 919-921Crossref PubMed Scopus (120) Google Scholar). The existence of these new mutations raises questions as to whether they lead to stabilization of HIF-2α, and if so, whether this is due to altered interactions with PHD2 and/or VHL. Such altered interactions might also raise the possibility of dominant negative mechanisms. Our analysis of these mutations as well as a third novel erythrocytosis-associated HIF2A mutation, P534L (19Percy M.J. Furlow P.W. Beer P.A. Campbell G. Dekker A.W. Green A.R. Li X. Sutherland S. Oscier D. Rainey M.G. Rivera E. van Wijk R. Wood M. Lappin T.R.J. McMullin M.F. Lee F.S. Blood. 2008; 112 (abstr.): 481Crossref Google Scholar), reveals that whereas all mutations result in the aberrant stabilization of HIF-2α, they do so in ways that are not entirely identical. Moreover, whereas initial attention on HIF prolyl hydroxylation focused on a highly conserved LXXLAP motif containing residues N-terminal to the hydroxylacceptor proline, these results actually highlight a critical role for residues that are in fact C-terminal to it. Patients and Mutational Analysis of the HIF2A Gene-Individuals with inappropriately normal or elevated serum Epo levels were selected from a registry of idiopathic erythrocytosis patients for analysis of HIF2A (14Percy M.J. Zhao Q. Flores A. Harrison C. Lappin T.R. Maxwell P.H. McMullin M.F. Lee F.S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 654-659Crossref PubMed Scopus (261) Google Scholar, 20Percy M.J. McMullin M.F. Jowitt S.N. Potter M. Treacy M. Watson W.H. Lappin T.R. Blood. 2003; 102: 1097-1099Crossref PubMed Scopus (83) Google Scholar). Previous investigations revealed the absence of defects in the VHL and PHD2 genes. Exon 12 of HIF2A was amplified by PCR and sequenced as described previously (15Percy M.J. Furlow P.W. Lucas G.S. Li X. Lappin T.R. McMullin M.F. Lee F.S. N. Engl. J. Med. 2008; 358: 162-168Crossref PubMed Scopus (244) Google Scholar). Plasmids-The pcDNA5/FRT/TO-3xFLAG-hHIF-2α wild type and P531A plasmids have been described previously (15Percy M.J. Furlow P.W. Lucas G.S. Li X. Lappin T.R. McMullin M.F. Lee F.S. N. Engl. J. Med. 2008; 358: 162-168Crossref PubMed Scopus (244) Google Scholar). Corresponding constructs for the P534L, M535V, and G537R mutations were prepared as follows. First, a 1.6-kb PCR product was obtained using IMAGE Consortium clone 6305604 as a template and the following primers: 5′ GTA CGG ATC CAG ATG ACA GCT GAC AAG GAG-3′ and 5′-GTA CGC GGC CGC ATG CGC TAG CGT CTC CAA GTC CAG CTC-3′. The PCR product was digested with BamHI and NotI and subcloned into the BamHI/NotI site of pcDNA5/FRT/TO-3xFLAG to yield pcDNA5/FRT/TO-3xFLAG-hHIF-2α-N. Then, 1.0-kb PCR products were obtained using the same IMAGE clone as a template, the primer 5′-GTC AGC GGC CGC TCA GGT GGC CTG GTC CAG-3′, and one of the following primers: P534L, 5′-GAG ACG CTA GCA CCC TAT ATC CTC ATG GAC GGG GAA GAC TTC CAG-3′; M535V, 5′-GAG ACG CTA GCA CCC TAT ATC CCG GTG GAC GGG GAA GAC TTC CAG C-3′; or G537R, 5′-GAG ACG CTA GCA CCC TAT ATC CCG ATG GAC GAA GAC TTC CAG CTA The PCR products were digested with and subcloned into the site of The 1.0-kb from of the plasmids were subcloned into the site of to yield pcDNA5/FRT/TO-3xFLAG-hHIF-2α P534L, M535V, and G537R, has been described previously (15Percy M.J. Furlow P.W. Lucas G.S. Li X. Lappin T.R. McMullin M.F. Lee F.S. N. Engl. J. Med. 2008; 358: 162-168Crossref PubMed Scopus (244) Google Scholar). constructs with were prepared by into the site of of the following TCA GTA CCC CGG TCA ATG AGC CAG ACG GGG AAG CCG and GCT CGG GGC GGC TCA CCC CGT TAT CTG GTG GCT CCG GGG P534L, TCA GTA CCC CGG TCA ATG AGC TCA ACG GGG AAG CCG and GCT CGG GGC GGC TCA CCC CGT TAT GTG GTG GCT CCG GGG M535V, TCA GTA CCC CGG TCA ATG AGC CCG ACG GGG AAG CCG and GCT CGG GGC GGC TCA CCC CGT CGG TAT GTG GTG GCT CCG GGG G537R, TCA GTA CCC CGG TCA ATG AGC ACA GGG AAG CCG and GCT CGG GGC GGC TCA CCC TAT GTG GTG GCT CCG GGG constructs were prepared in an using of the following HIF-2α TCA AGC AGC CCG AGC TAG CCC AGC CTC CCC CCG GGG ACG TCA CTC ATC and GCT CGT CCC CGG GGG GCT GGG CTA GCT CGG GCT GCT TCA GTA CCC CGG TCA ATG AGC CCC CCG GGG ACG TCA CTC ATC and GCT CGT CCC CGG GGG GTG GTG GCT CCG GGG TCA AGC AGC CCG AGC TAG CCC AGC CTC ACG GGG AAG CCG and GCT CGG GGC GGC TCA CCC CGT TAT GCT GGG CTA GCT CGG GCT GCT constructs were by and binding or hydroxylase was as previously described J. Zhao Q. Lee F.S. J. Biol. Chem. 2002; PubMed Scopus Google and at or from of using and into for the subsequent in were prepared using the and in was prepared using and was from as described previously Gene PubMed Scopus Google Scholar). or wild P534L, M535V, or were obtained from The following were also by HIF-2α HIF-2α HIF-2α HIF-2α and N-terminal PHD2 on of was with in as described J. Zhao Q. Lee F.S. J. Biol. Chem. 2002; PubMed Scopus Google Scholar). The were with and the to and analysis on an on of was and for by the of and R. PubMed Scopus Google as by S. 2007; PubMed Scopus Google Scholar). were in a of of with and of in the absence or of wild type or were for at in with with The and on the was by in the absence of HIF were from that in its to HIF hydroxylase on of was with containing and at in a of of the same with and in the absence or of of were in with and to with an analysis was on a at high with were to VHL was prepared by on with in the of and and the was with of The was with in as described previously in the absence or of The were with and the to and Cell and of cell in which the is into a were using to the wild type and P531A have been previously described (15Percy M.J. Furlow P.W. Lucas G.S. Li X. Lappin T.R. McMullin M.F. Lee F.S. N. Engl. J. Med. 2008; 358: 162-168Crossref PubMed Scopus (244) Google Scholar). These were in with and HIF-2α expression was by with for protein was at a of was in an were in containing and were at for at were by to the of protein were by and to The was with containing and were with were with followed by was as a for was from cell with for using to the was prepared using the PCR analysis was using Green and an PCR gene levels were to that of Green with the of were as CTA CAA and CTC CGG CGG AAG and GGC GAA GCT and GGT GCA ACA and CGG CAG AAG analysis for each of revealed The of the have been previously described J. D. Flores A. Zhao Q. Lee F.S. J. 2007; PubMed Scopus Google Scholar). was using the and as the cell were with F. Zhao Q. Lee F.S. Google and the using and with for were to hypoxia were with and by at for at were using a and a and analysis of were used for were considered and functional characterization of the initial erythrocytosis-associated mutation in the HIF2A gene (15Percy M.J. Furlow P.W. Lucas G.S. Li X. Lappin T.R. McMullin M.F. Lee F.S. N. Engl. J. Med. 2008; 358: 162-168Crossref PubMed Scopus (244) Google Scholar), we subsequently reported on two additional HIF2A mutations, M535V and G537R (16Percy M.J. Beer P.A. Campbell G. Dekker A.W. Green A.R. Oscier D. Rainey M.G. van Wijk R. Wood M. Lappin T.R. McMullin M.F. Lee F.S. Blood. 2008; 111: 5400-5402Crossref PubMed Scopus (100) Google Scholar). this we describe the functional characterization of these latter two HIF-α mutants as well as a third novel P534L (19Percy M.J. Furlow P.W. Beer P.A. Campbell G. Dekker A.W. Green A.R. Li X. Sutherland S. Oscier D. Rainey M.G. Rivera E. van Wijk R. Wood M. Lappin T.R.J. McMullin M.F. Lee F.S. Blood. 2008; 112 (abstr.): 481Crossref Google Scholar). We binding in which we PHD2 with in wild type or and we the PHD2 and for binding with wild type the mutants all more and The of P534L, M535V, and G537R were and of that of wild type in all binding to PHD2 might in we PHD2 with wild type or in the of and the as a of HIF prolyl hydroxylase with the PHD2 binding we find that the mutations result in of to to PHD2 wild type the PHD2 P534L, M535V, and G537R HIF-2α was and in all The an of whether the mutations we PHD2 with wild P534L, M535V, or G537R and the of hydroxylation by Under in which the wild type was hydroxylated all three were hydroxylated so at both the and to effects on PHD2 binding and these mutations also affect subsequent recognition by VHL. this we the capacity of wild type and to with hydroxylated for binding to in VHL (15Percy M.J. Furlow P.W. Lucas G.S. Li X. Lappin T.R. McMullin M.F. Lee F.S. N. Engl. J. Med. 2008; 358: 162-168Crossref PubMed Scopus (244) Google Scholar). hydroxylated VHL, whereas the not and wild type for VHL as by binding to We find that the P534L and the G537R are in for VHL binding and with The M535V in is as the wild type This is by results to wild type at both high with and with We that P534L, M535V, and HIF-2α The constructs for the HIF-2α mutants into a site in this cell so as to cell a of two for each of these three mutants revealed levels of protein as by with with wild type HIF-2α from the same we find that the protein levels for each is cell a P531A HIF-2α also levels of protein wild as reported previously (15Percy M.J. Furlow P.W. Lucas G.S. Li X. Lappin T.R. McMullin M.F. Lee F.S. N. Engl. J. Med. 2008; 358: 162-168Crossref PubMed Scopus (244) Google Scholar). subsequent we selected a clone from each of P534L, M535V, and G537R PCR for the the that the wild P534L, M535V, and G537R levels are all the of these in more we protein by of with and the protein by using a wild type HIF-2α is of each We find that P534L, M535V, and G537R mutants of HIF-2α mutants are each more wild type HIF-2α three of not as as the P531A hydroxylation of We by the expression of the HIF genes gene and factor C.J. Simon M.C. Mol. Biol. 2003; PubMed Scopus Google in the wild type and HIF-2α The expression of M535V and G537R HIF-2α, as well as P531A HIF-2α, increased levels from the gene as with wild type HIF-2α. The expression of P534L and G537R HIF-2α, as well as P531A HIF-2α, increased levels from the and genes as with wild type and The levels of a gene the different cell We also the of each HIF gene in this the of all three genes are with all of the HIF-2α mutants that obtained with wild type HIF-2α. The levels of HIF genes by the HIF-2α mutants is with increased transcriptional this we the cell with a hypoxia response gene Under normoxic conditions, and with the we find that gene is increased with all three mutants as with wild hypoxic conditions, which to wild type HIF-2α, to levels of gene that are to that of wild three of the mutations affect residues C-terminal to the primary site of hydroxylation in HIF-2α, a role for these The site of hydroxylation in HIF-2α, is in this HIF-2α and HIF-2α it at all three of the residues that are in erythrocytosis patients by this we prepared in HIF-2α to the site of We find that it binds more to PHD2 the from the primary site of hydroxylation and We prepared a containing the N-terminal residues from the primary site of hydroxylation and C-terminal residues from the site of hydroxylation and find that it also binds more and in a with the N-terminal residues from the site of hydroxylation and C-terminal residues from the primary site of hydroxylation binds with to PHD2 other we also the ability of to the primary and of hydroxylation to bind to VHL this we used the previously described in a hydroxylated containing the primary site of HIF-2α hydroxylation binds with high to VHL, as by its capacity to inhibit VHL binding to hydroxylated with The site of hydroxylation binds with to VHL a containing the C-terminal residues from the primary site of hydroxylation and N-terminal residues from the site binds with to VHL and more that of the with the These reinforce the importance of residues C-terminal to the hydroxylacceptor in HIF-2α, both in the with PHD2 and in the with VHL. the initial and characterization of an HIF2A mutation (15Percy M.J. Furlow P.W. Lucas G.S. Li X. Lappin T.R. McMullin M.F. Lee F.S. N. Engl. J. Med. 2008; 358: 162-168Crossref PubMed Scopus (244) Google Scholar), we have subsequently identified three additional distinct HIF2A mutations (16Percy M.J. Beer P.A. Campbell G. Dekker A.W. Green A.R. Oscier D. Rainey M.G. van Wijk R. Wood M. Lappin T.R. McMullin M.F. Lee F.S. Blood. 2008; 111: 5400-5402Crossref PubMed Scopus (100) Google Scholar, M.J. Furlow P.W. Beer P.A. Campbell G. Dekker A.W. Green A.R. Li X. Sutherland S. Oscier D. Rainey M.G. Rivera E. van Wijk R. Wood M. Lappin T.R.J. McMullin M.F. Lee F.S. Blood. 2008; 112 (abstr.): 481Crossref Google Scholar). The characterization of these mutations provides new and into this First, all three mutations lead to degradation and stabilization of HIF-2α. all a of negative for increased binding to PHD2 leading to impaired hydroxylation of the wild type HIF-2α, or increased binding to VHL leading to impaired degradation of wild type HIF-2α, are not by the of the mutants was to have increased for PHD2 or VHL, and in there were in whereas all mutations to HIF-2α, they do so in ways that are not identical. Thus, whereas the P534L and G537R mutations both binding to PHD2 and VHL, we find that the M535V mutation impairs binding to the of a to VHL, to HIF-2α is not a residue with VHL, and its from VHL K. Schofield C.J. Maxwell P.H. Ratcliffe P.J. 2002; PubMed Scopus Google Scholar, H. M. F. W.G. Science. 2002; PubMed Scopus Google Scholar). to HIF-2α forms a van with VHL. which is not conserved in is C-terminal to a residue that to which also a van with VHL K. Schofield C.J. Maxwell P.H. Ratcliffe P.J. 2002; PubMed Scopus Google Scholar, H. M. F. W.G. Science. 2002; PubMed Scopus Google Scholar). This that the M535V mutation is effects on PHD2 binding and This in that of the is to and is with the fact that mutations in PHD2 a distinct of erythrocytosis M.J. Lee F.S. Haematologica. 2008; 93: 963-967Crossref PubMed Scopus (19) Google Scholar, 12Lee F.S. Blood Rev. 2008; 22: 321-332Crossref PubMed Scopus (34) Google Scholar, M.J. Zhao Q. Flores A. Harrison C. Lappin T.R. Maxwell P.H. McMullin M.F. Lee F.S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 654-659Crossref PubMed Scopus (261) Google Scholar, M. K. M. H. C. Blood Mol. 2008; PubMed Scopus Google Scholar, M.J. Furlow P.W. Beer P.A. Lappin T.R. McMullin M.F. Lee F.S. Blood. 2007; PubMed Scopus Google Scholar). of the is to erythrocytosis is independently by the existence of erythrocytosis-associated VHL mutations M.J. Lee F.S. Haematologica. 2008; 93: 963-967Crossref PubMed Scopus (19) Google Scholar, S.O. Chen H. Hirota K. Gordeuk V.R. Jelinek J. Guan Y. Liu E. Sergueeva A.I. Miasnikova G.Y. Mole D. Maxwell P.H. Stockton D.W. Semenza G.L. Prchal J.T. Nat. Genet. 2002; 32: 614-621Crossref PubMed Scopus (430) Google Scholar, 20Percy M.J. McMullin M.F. Jowitt S.N. Potter M. Treacy M. Watson W.H. Lappin T.R. Blood. 2003; 102: 1097-1099Crossref PubMed Scopus (83) Google Scholar, H. K. N. U. R. E. Haematologica. 2005; 90: Google Scholar, S. M. C. A. F. F. G. Ragione Blood. 2006; PubMed Scopus Google Scholar, Y. K. Guan Y. Liu E. J. H. Prchal Prchal J.T. J. Genet. 2003; PubMed Scopus (100) Google Scholar). in turn, raises the possibility that there might to identified HIF2A mutations that the of HIF-2α with VHL, not with A third result of these studies is that all erythrocytosis-associated HIF2A mutations identified to affect residues C-terminal to the hydroxylacceptor This is of an because of the attention on in HIF-α focused on residues in a highly conserved LXXLAP motif (where underlining indicates the hydroxylacceptor that N-terminal to the hydroxylacceptor Mole J. A. M. Schofield C.J. Maxwell P.H. Ratcliffe P.J. Science. PubMed Scopus Google Scholar, M. K. H. W. J. M. A. W.G. Science. PubMed Scopus Google Scholar, F. Zhao Q. Lee F.S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). C-terminal to the are well these mutations highlight the fact that they are This is by the that the primary and of hydroxylation of HIF-2α bind with high to PHD2 and to VHL in a that with the as to residues in this of interest to the of HIF-2α to these studies reinforce the importance of prolyl hydroxylation as the modification the HIF pathway in response to in oxygen the role of the primary site of hydroxylation in HIF-2α in its protein and the importance of HIF-2α in the of EPO and in We the have patients with erythrocytosis from the and to registry and provided