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Interaction of Hemojuvelin with Neogenin Results in Iron Accumulation in Human Embryonic Kidney 293 Cells

2005; Elsevier BV; Volume: 280; Issue: 40 Linguagem: Inglês

10.1074/jbc.m506207200

ISSN

1083-351X

Autores

An‐Sheng Zhang, Anthony P. West, Anne E. Wyman, Pamela J. Björkman, Caroline Enns,

Tópico(s)

Erythropoietin and Anemia Treatment

Resumo

Type 2 hereditary hemochromatosis (HH) or juvenile hemochromatosis is an early onset, genetically heterogeneous, autosomal recessive disorder of iron overload. Type 2A HH is caused by mutations in the recently cloned hemojuvelin gene (HJV; also called HFE2) (Papanikolaou, G., Samuels, M. E., Ludwig, E. H., MacDonald, M. L., Franchini, P. L., Dube, M. P., Andres, L., MacFarlane, J., Sakellaropoulos, N., Politou, M., Nemeth, E., Thompson, J., Risler, J. K., Zaborowska, C., Babakaiff, R., Radomski, C. C., Pape, T. D., Davidas, O., Christakis, J., Brissot, P., Lockitch, G., Ganz, T., Hayden, M. R., and Goldberg, Y. P. (2004) Nat. Genet. 36, 77–82), whereas Type 2B HH is caused by mutations in hepcidin. HJV is highly expressed in both skeletal muscle and liver. Mutations in HJV are implicated in the majority of diagnosed juvenile hemochromatosis patients. In this study, we stably transfected HJV cDNA into human embryonic kidney 293 cells and characterized the processing of HJV and its effect on iron homeostasis. Our results indicate that HJV is a glycosylphosphatidylinositol-linked protein and undergoes a partial autocatalytic cleavage during its intracellular processing. HJV co-immunoprecipitated with neogenin, a receptor involved in a variety of cellular signaling processes. It did not interact with the closely related receptor DCC (deleted in Colon Cancer). In addition, the HJV G320V mutant implicated in Type 2A HH did not co-immunoprecipitate with neogenin. Immunoblot analysis of ferritin levels and transferrin-55Fe accumulation studies indicated that the HJV-induced increase in intracellular iron levels in human embryonic kidney 293 cells is dependent on the presence of neogenin in the cells, thus linking these two proteins to intracellular iron homeostasis. Type 2 hereditary hemochromatosis (HH) or juvenile hemochromatosis is an early onset, genetically heterogeneous, autosomal recessive disorder of iron overload. Type 2A HH is caused by mutations in the recently cloned hemojuvelin gene (HJV; also called HFE2) (Papanikolaou, G., Samuels, M. E., Ludwig, E. H., MacDonald, M. L., Franchini, P. L., Dube, M. P., Andres, L., MacFarlane, J., Sakellaropoulos, N., Politou, M., Nemeth, E., Thompson, J., Risler, J. K., Zaborowska, C., Babakaiff, R., Radomski, C. C., Pape, T. D., Davidas, O., Christakis, J., Brissot, P., Lockitch, G., Ganz, T., Hayden, M. R., and Goldberg, Y. P. (2004) Nat. Genet. 36, 77–82), whereas Type 2B HH is caused by mutations in hepcidin. HJV is highly expressed in both skeletal muscle and liver. Mutations in HJV are implicated in the majority of diagnosed juvenile hemochromatosis patients. In this study, we stably transfected HJV cDNA into human embryonic kidney 293 cells and characterized the processing of HJV and its effect on iron homeostasis. Our results indicate that HJV is a glycosylphosphatidylinositol-linked protein and undergoes a partial autocatalytic cleavage during its intracellular processing. HJV co-immunoprecipitated with neogenin, a receptor involved in a variety of cellular signaling processes. It did not interact with the closely related receptor DCC (deleted in Colon Cancer). In addition, the HJV G320V mutant implicated in Type 2A HH did not co-immunoprecipitate with neogenin. Immunoblot analysis of ferritin levels and transferrin-55Fe accumulation studies indicated that the HJV-induced increase in intracellular iron levels in human embryonic kidney 293 cells is dependent on the presence of neogenin in the cells, thus linking these two proteins to intracellular iron homeostasis. Hereditary hemochromatosis (HH) 2The abbreviations used are: HH, hereditary hemochromatosis; HJV, hemojuvelin; JH, juvenile hemochromatosis; FPN, ferroportin; RGM, repulsive guidance molecule; m, mouse; DCC, deleted in Colon Cancer; HEK293, human embryonic kidney 293; Tf, transferrin; sHJV, soluble hemojuvelin; TfR, transferrin receptor; PI-PLC, phosphatidylinositol-specific phospholipase C; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; siRNA, small interfering RNA; GPI, glycosylphosphatidylinositol.2The abbreviations used are: HH, hereditary hemochromatosis; HJV, hemojuvelin; JH, juvenile hemochromatosis; FPN, ferroportin; RGM, repulsive guidance molecule; m, mouse; DCC, deleted in Colon Cancer; HEK293, human embryonic kidney 293; Tf, transferrin; sHJV, soluble hemojuvelin; TfR, transferrin receptor; PI-PLC, phosphatidylinositol-specific phospholipase C; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; siRNA, small interfering RNA; GPI, glycosylphosphatidylinositol. includes a heterogeneous group of inherited iron overload diseases resulting from mutations in at least the following five genes: HFE, hemojuvelin (HJV; also called HFE2), hepcidin (HAMP), transferrin receptor-2, and ferroportin (FPN; also called SLC40A1) (1Hentze M.W. Muckenthaler M.U. Andrews N.C. Cell. 2004; 117: 285-297Abstract Full Text Full Text PDF PubMed Scopus (1355) Google Scholar, 2Pietrangelo A. N. Engl. J. Med. 2004; 350: 2383-2397Crossref PubMed Scopus (816) Google Scholar). Type 1 HH, the most common form, is caused by mutations of the HFE gene and is an late onset, autosomal recessive, low penetrance iron overload disorder (3Feder J.N. Gnirke A. Thomas W. Tsuchihashi Z. Ruddy D.A. Basava A. Dormishian F. Domingo R.J. Ellis M.C. Fullan A. Hinton L.M. Jones N.L. Kimmel B.E. Kronmal G.S. Lauer P. Lee V.K. Loeb D.B. Mapa F.A. McClelland E. Meyer N.C. Mintier G.A. Moeller N. Moore T. Morikang E. Prasss C.E. Quintana L. Starnes S.M. Schatzman R.C. Brunke K.J. Drayna D.T. Risch N.J. Bacon B.R. Wolff R.K. Nat. Genet. 1996; 13: 399-408Crossref PubMed Scopus (3314) Google Scholar). Type 2 HH or juvenile hemochromatosis (JH) is a rare autosomal recessive disease with high penetrance that affects young patients of both sexes and that leads to severe clinical complications typically in the first and second decades of life (4De Gobbi M. Roetto A. Piperno A. Mariani R. Alberti F. Papanikolaou G. Politou M. Lockitch G. Girelli D. Fargion S. Cox T.M. Gasparini P. Cazzola M. Camaschella C. Br. J. Haematol. 2002; 117: 973-979Crossref PubMed Scopus (148) Google Scholar, 5Camaschella C. Roetto A. De Gobbi M. Semin. Hematol. 2002; 39: 242-248Crossref PubMed Scopus (71) Google Scholar). JH results from mutations of either the HJV gene (Type 2A) or the hepcidin gene (Type 2B) in early iron loading with similar serum iron parameters and distribution of accumulated iron in organs (2Pietrangelo A. N. Engl. J. Med. 2004; 350: 2383-2397Crossref PubMed Scopus (816) Google Scholar, 6Papanikolaou G. Samuels M.E. Ludwig E.H. MacDonald M.L. Franchini P.L. Dube M.P. Andres L. MacFarlane J. Sakellaropoulos N. Politou M. Nemeth E. Thompson J. Risler J.K. Zaborowska C. Babakaiff R. Radomski C.C. Pape T.D. Davidas O. Christakis J. Brissot P. Lockitch G. Ganz T. Hayden M.R. Goldberg Y.P. Nat. Genet. 2004; 36: 77-82Crossref PubMed Scopus (821) Google Scholar). Both Type 1 HH and JH individuals have increased intestinal absorption and deposition of iron in vital organs, including the liver, heart, and pancreas. However, JH individuals have a higher rate of iron absorption and a more rapid and severe clinical course with a higher frequency of cardiomyopathy, diabetes, and hypogonadism (5Camaschella C. Roetto A. De Gobbi M. Semin. Hematol. 2002; 39: 242-248Crossref PubMed Scopus (71) Google Scholar). HJV is a newly cloned gene localized on chromosome 1q21 (6Papanikolaou G. Samuels M.E. Ludwig E.H. MacDonald M.L. Franchini P.L. Dube M.P. Andres L. MacFarlane J. Sakellaropoulos N. Politou M. Nemeth E. Thompson J. Risler J.K. Zaborowska C. Babakaiff R. Radomski C.C. Pape T.D. Davidas O. Christakis J. Brissot P. Lockitch G. Ganz T. Hayden M.R. Goldberg Y.P. Nat. Genet. 2004; 36: 77-82Crossref PubMed Scopus (821) Google Scholar). This region of chromosome 1 was previously linked to JH (7Roetto A. Totaro A. Cazzola M. Cicilano M. Bosio S. D'Ascola G. Carella M. Zelante L. Kelly A.L. Cox T.M. Gasparini P. Camaschella C. Am. J. Hum. Genet. 1999; 64: 1388-1393Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 8Papanikolaou G. Politou M. Roetto A. Bosio S. Sakelaropoulos N. Camaschella C. Loukopoulos D. Blood Cells Mol. Dis. 2001; 27: 744-749Crossref PubMed Scopus (30) Google Scholar, 9Papanikolaou G. Papaioannou M. Politou M. Vavatsi N. Kioumi A. Tsiatsiou P. Marinaki P. Loukopoulos D. Christakis J.I. Blood Cells Mol. Dis. 2002; 29: 168-173Crossref PubMed Scopus (15) Google Scholar, 10Rivard S.R. Lanzara C. Grimard D. Carella M. Simard H. Ficarella R. Simard R. D'Adamo A.P. Ferec C. Camaschella C. Mura C. Roetto A. De Braekeleer M. Bechner L. Gasparini P. Eur. J. Hum. Genet. 2003; 11: 585-589Crossref PubMed Scopus (26) Google Scholar). HJV has five predicted spliced transcripts encoding three different proteins of 426, 313, and 200 amino acids. Northern blot analysis has indicated high expression in adult and fetal liver, heart, and skeletal muscle with the full-length mRNA as the primary transcript. The encoded protein (HJV) possesses multiple protein domains, including an N-terminal signal peptide, an RGD motif, a partial von Willebrand factor D motif, and a C-terminal transmembrane domain (6Papanikolaou G. Samuels M.E. Ludwig E.H. MacDonald M.L. Franchini P.L. Dube M.P. Andres L. MacFarlane J. Sakellaropoulos N. Politou M. Nemeth E. Thompson J. Risler J.K. Zaborowska C. Babakaiff R. Radomski C.C. Pape T.D. Davidas O. Christakis J. Brissot P. Lockitch G. Ganz T. Hayden M.R. Goldberg Y.P. Nat. Genet. 2004; 36: 77-82Crossref PubMed Scopus (821) Google Scholar). Numerous mutations in HJV that cause Type 2A HH have been identified. These include missense, frameshift, and nonsense mutations in either homozygous or compound heterozygous individuals (6Papanikolaou G. Samuels M.E. Ludwig E.H. MacDonald M.L. Franchini P.L. Dube M.P. Andres L. MacFarlane J. Sakellaropoulos N. Politou M. Nemeth E. Thompson J. Risler J.K. Zaborowska C. Babakaiff R. Radomski C.C. Pape T.D. Davidas O. Christakis J. Brissot P. Lockitch G. Ganz T. Hayden M.R. Goldberg Y.P. Nat. Genet. 2004; 36: 77-82Crossref PubMed Scopus (821) Google Scholar, 11Huang F.W. Rubio-Aliaga I. Kushner J.P. Andrews N.C. Fleming M.D. Blood. 2004; 104: 2176-2177Crossref PubMed Scopus (49) Google Scholar, 12Lanzara C. Roetto A. Daraio F. Rivard S. Ficarella R. Simard H. Cox T.M. Cazzola M. Piperno A. Gimenez-Roqueplo A.P. Grammatico P. Volinia S. Gasparini P. Camaschella C. Blood. 2004; 103: 4317-4321Crossref PubMed Scopus (161) Google Scholar, 13Lee P.L. Beutler E. Rao S.V. Barton J.C. 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Genet. 2004; 36: 77-82Crossref PubMed Scopus (821) Google Scholar), the sporadic distribution of these mutations hints that Type 2A HH is due to the loss of HJV function. Interestingly, the hepcidin levels in these patients are found to be consistently depressed, suggesting that HJV may act as a modulator of hepcidin expression (6Papanikolaou G. Samuels M.E. Ludwig E.H. MacDonald M.L. Franchini P.L. Dube M.P. Andres L. MacFarlane J. Sakellaropoulos N. Politou M. Nemeth E. Thompson J. Risler J.K. Zaborowska C. Babakaiff R. Radomski C.C. Pape T.D. Davidas O. Christakis J. Brissot P. Lockitch G. Ganz T. Hayden M.R. Goldberg Y.P. Nat. Genet. 2004; 36: 77-82Crossref PubMed Scopus (821) Google Scholar). Hepcidin is a small peptide hormone synthesized predominantly in hepatocytes. It is critical for the maintenance of body iron homeostasis through down-regulation of the iron exporter FPN in intestinal endothelial cells and macrophages (17Krause A. Neitz S. Magert H.J. Schulz A. Forssmann W.G. Schulz-Knappe P. Adermann K. FEBS Lett. 2000; 480: 147-150Crossref PubMed Scopus (1047) Google Scholar, 18Park C.H. Valore E.V. Waring A.J. Ganz T. J. Biol. Chem. 2001; 276: 7806-7810Abstract Full Text Full Text PDF PubMed Scopus (1711) Google Scholar, 19Nemeth E. Tuttle M.S. Powelson J. Vaughn M.B. Donovan A. Ward D.M. Ganz T. Kaplan J. Science. 2004; 306: 2090-2093Crossref PubMed Scopus (3487) Google Scholar). Hepcidin expression is highly regulated by body iron status, hypoxia, and inflammation (20Nicolas G. Chauvet C. Viatte L. Danan J.L. Bigard X. Devaux I. Beaumont C. Kahn A. Vaulont S. J. Clin. Investig. 2002; 110: 1037-1044Crossref PubMed Scopus (1320) Google Scholar). In contrast, a more recent study found that HJV mRNA levels are increased by inflammation, but do not respond to iron status or erythropoietin in mice (21Krijt J. Vokurka M. Chang K.T. Necas E. Blood. 2004; 104: 4308-4310Crossref PubMed Scopus (58) Google Scholar). HJV shares considerable sequence similarity with the newly identified repulsive guidance molecules (RGMs) (6Papanikolaou G. Samuels M.E. Ludwig E.H. MacDonald M.L. Franchini P.L. Dube M.P. Andres L. MacFarlane J. Sakellaropoulos N. Politou M. Nemeth E. Thompson J. Risler J.K. Zaborowska C. Babakaiff R. Radomski C.C. Pape T.D. Davidas O. Christakis J. Brissot P. Lockitch G. Ganz T. Hayden M.R. Goldberg Y.P. Nat. Genet. 2004; 36: 77-82Crossref PubMed Scopus (821) Google Scholar). In the mouse (m) RGM family, there are at least three members, mRGMa, mRGMb, and mRGMc (22Schmidtmer J. Engelkamp D. Gene Expr. Patterns. 2004; 4: 105-110Crossref PubMed Scopus (81) Google Scholar, 23Oldekamp J. Kramer N. Alvarez-Bolado G. Skutella T. Gene Expr. Patterns. 2004; 4: 283-288Crossref PubMed Scopus (66) Google Scholar, 24Niederkofler V. Salie R. Sigrist M. Arber S. J. Neurosci. 2004; 24: 808-818Crossref PubMed Scopus (151) Google Scholar). Human HJV is the ortholog of mRGMc (Fig. 1). In situ hybridization analysis of the distribution of the various RGMs in mouse embryos has shown that mRGMa and mRGMb are expressed predominantly in distinct, mostly no-overlapping patterns in the developing and adult central nervous systems, whereas mRGMc is expressed mainly in skeletal and heart muscles (22Schmidtmer J. Engelkamp D. Gene Expr. Patterns. 2004; 4: 105-110Crossref PubMed Scopus (81) Google Scholar, 23Oldekamp J. Kramer N. Alvarez-Bolado G. Skutella T. Gene Expr. Patterns. 2004; 4: 283-288Crossref PubMed Scopus (66) Google Scholar, 24Niederkofler V. Salie R. Sigrist M. Arber S. J. Neurosci. 2004; 24: 808-818Crossref PubMed Scopus (151) Google Scholar). The in situ localization of mRGMc is consistent with the Northern blot analysis results of HJV mRNA in human tissues and the fact that no neural symptoms have been reported in Type 2A HH patients (6Papanikolaou G. Samuels M.E. Ludwig E.H. MacDonald M.L. Franchini P.L. Dube M.P. Andres L. MacFarlane J. Sakellaropoulos N. Politou M. Nemeth E. Thompson J. Risler J.K. Zaborowska C. Babakaiff R. Radomski C.C. Pape T.D. Davidas O. Christakis J. Brissot P. Lockitch G. Ganz T. Hayden M.R. Goldberg Y.P. Nat. Genet. 2004; 36: 77-82Crossref PubMed Scopus (821) Google Scholar). Functional studies of mRGMa in mouse embryos have revealed that it plays a critical role in the control of cephalic neural tube closure and formation of afferent connections in the dentate gyrus (24Niederkofler V. Salie R. Sigrist M. Arber S. J. Neurosci. 2004; 24: 808-818Crossref PubMed Scopus (151) Google Scholar, 25Brinks H. Conrad S. Vogt J. Oldekamp J. Sierra A. Deitinghoff L. Bechmann I. Alvarez-Bolado G. Heimrich B. Monnier P.P. Mueller B.K. Skutella T. J. Neurosci. 2004; 24: 3862-3869Crossref PubMed Scopus (80) Google Scholar). Chicken RGM, an ortholog of mRGMa, has also been reported to be essential for retinotectal map formation in the chick embryo (26Monnier P.P. Sierra A. Macchi P. Deitinghoff L. Andersen J.S. Mann M. Flad M. Hornberger M.R. Stahl B. Bonhoeffer F. Mueller B.K. Nature. 2002; 419: 392-395Crossref PubMed Scopus (250) Google Scholar). More recent studies have demonstrated that neogenin is the high affinity receptor for chicken and mouse RGMs and that their interaction is critical in the regulation of neuronal survival (27Rajagopalan S. Deitinghoff L. Davis D. Conrad S. Skutella T. Chedotal A. Mueller B.K. Strittmatter S.M. Nat. Cell Biol. 2004; 6: 756-762Crossref PubMed Scopus (218) Google Scholar, 28Matsunaga E. Tauszig-Delamasure S. Monnier P.P. Mueller B.K. Strittmatter S.M. Mehlen P. Chedotal A. Nat. Cell Biol. 2004; 6: 749-755Crossref PubMed Scopus (218) Google Scholar). Neogenin is a membrane protein. It is closely related to another receptor, DCC (deleted in Colon Cancer), with nearly 50% amino acid identity. Neogenin is widely expressed in different tissues, including muscles and liver (29Vielmetter J. Kayyem J.F. Roman J.M. Dreyer W.J. J. Cell Biol. 1994; 127: 2009-2020Crossref PubMed Scopus (141) Google Scholar, 30Meyerhardt J.A. Look A.T. Bigner S.H. Fearon E.R. Oncogene. 1997; 14: 1129-1136Crossref PubMed Scopus (76) Google Scholar, 31Keeling S.L. Gad J.M. Cooper H.M. Oncogene. 1997; 15: 691-700Crossref PubMed Scopus (77) Google Scholar). Functional studies in zebrafish embryos using morpholino oligonucleotides demonstrated that it is essential for neural tube formation and somitogenesis (32Mawdsley D.J. Cooper H.M. Hogan B.M. Cody S.H. Lieschke G.J. Heath J.K. Dev. Biol. 2004; 269: 302-315Crossref PubMed Scopus (53) Google Scholar), a function similar to mRGMa in mouse embryos (24Niederkofler V. Salie R. Sigrist M. Arber S. J. Neurosci. 2004; 24: 808-818Crossref PubMed Scopus (151) Google Scholar). This evidence supports the idea that the RGMa/neogenin interaction is indispensable for neural development. On the basis of the high sequence identity of RGMa to RGMc and human HJV, the lack of RGMa in the liver, and finally the presence of HJV and neogenin in the liver (6Papanikolaou G. Samuels M.E. Ludwig E.H. MacDonald M.L. Franchini P.L. Dube M.P. Andres L. MacFarlane J. Sakellaropoulos N. Politou M. Nemeth E. Thompson J. Risler J.K. Zaborowska C. Babakaiff R. Radomski C.C. Pape T.D. Davidas O. Christakis J. Brissot P. Lockitch G. Ganz T. Hayden M.R. Goldberg Y.P. Nat. Genet. 2004; 36: 77-82Crossref PubMed Scopus (821) Google Scholar, 22Schmidtmer J. Engelkamp D. Gene Expr. Patterns. 2004; 4: 105-110Crossref PubMed Scopus (81) Google Scholar, 23Oldekamp J. Kramer N. Alvarez-Bolado G. Skutella T. Gene Expr. Patterns. 2004; 4: 283-288Crossref PubMed Scopus (66) Google Scholar, 24Niederkofler V. Salie R. Sigrist M. Arber S. J. Neurosci. 2004; 24: 808-818Crossref PubMed Scopus (151) Google Scholar), we speculate that neogenin might also be a candidate receptor for HJV. In this study, we stably transfected HJV cDNA into human embryonic kidney 293 (HEK293) cells and characterized its effect on iron homeostasis. Our results indicate that HJV is processed similarly to other members of the RGM family of proteins. Immunoprecipitation analysis demonstrated that HJV interacts with neogenin, but not with DCC, and that the HJV G320V mutant implicated in Type 2A HH does not bind to neogenin. Immunoblot analysis of ferritin levels and transferrin (Tf)-bound 55Fe accumulation studies showed that the HJV-induced increases in intracellular iron levels in HEK293 cells are dependent on the presence of neogenin in the cells. Subcloning HJV into pcDNA3—The full-length HJV open reading frame was amplified from a human liver cDNA library by PCR using the Expand high fidelity PCR system (Roche Applied Science) with primers 5′-atgggggagccaggccagtcccctagtcccaggtcctccc-3′ (forward) and 5′-ttactgaatgcaaagccacagaacaaagagcccagaaagga-3′ (reverse). The amplicon was subsequently cloned into the pGEM-T vector (Promega). After the sequence was confirmed by sequencing, the HJV open reading frame was subcloned into the expression vector pcDNA3 (Invitrogen) to make the pcDNA3-HJV construct. To map the fragmentation pattern of HJV, we engineered a modified plasmid by addition of a Myc tag at the position 3 amino acid after the N-terminal signal peptide sequence. To do this, two portions of the HJV open reading frame (the first 111 bp at the 5′-end and the remaining 1170 bp at the 3′-end) were first amplified separately using the primers for the full-length HJV open reading frame cloning (described above) as well as the following primers: 5′-ctcgaggcattgagaatgagcatgtcc-3′ (reverse primer for the former) and 5′-ctcgaggagcagaaactcatctctgaagaggatctgaagatcctccgctgcaatgct-3′ (forward primer for the latter). An XhoI enzyme digestion site was also included in these primers to ligate the two HJV fragments. The PCR amplicons were first cloned into the pGEM-T vector, and their sequences and orientation in the vectors were verified. The 1170-bp 3′-portion of HJV was excised from pGEM-T with XhoI and SphI and subcloned into pGEM-T containing the 111-bp 5′-portion of HJV. The resulting MycHJV in pGEM-T (pGEM-T-Myc-HJV) was subcloned into the expression vector pcDNA3 as follows. Both the pGEM-T-Myc-HJV and pcDNA3 vectors were first linearized by SphI and ApaI digestion, respectively, followed by treatment with the Klenow fragment (New England Biolabs Inc.) to make blunt ends. Myc-HJV was then excised from linearized pGEM-T-Myc-HJV with NotI and ligated with linearized and NotI-digested pcDNA3 to form pcDNA3-Myc-HJV. The construct was verified by sequencing. The HJV G320V mutant was made using the QuikChange™ XL site-directed mutagenesis kit (Stratagene) following the manufacturer's instructions. The pcDNA3-HJV construct was used as a template. The primers used to introduce the mutation were 5′-gctctgtgttggggtgtgccctccaagtc-3′ (forward) and 5′-gacttggagggcacaccccaacacagagc-3′ (reverse). The G320V mutation in the resulting construct was confirmed by DNA sequencing. No other sequence changes were detected. The full-length human neogenin cDNA in pcDNA3 was kindly provided by Dr. Eric R. Fearon (University of Michigan Medical School, Ann Arbor, MI). The sequence has been published previously (30Meyerhardt J.A. Look A.T. Bigner S.H. Fearon E.R. Oncogene. 1997; 14: 1129-1136Crossref PubMed Scopus (76) Google Scholar). Transfection—HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1 mm pyruvate. All transfections were performed using Lipofectamine reagent (Invitrogen) according to the manufacturer's instruction. Transiently transfected cells were used in experiments ∼48 h post-transfection. For stable transfection, transfected cells were selected under 800 μg/ml G418. Positive clones were screened by Western blot analysis. HEK293 cells stably transfected with Myc-HJV, HJV G320V, and empty vector (pcDNA3) were designated Myc-HJV HEK293 cells, HJV G320V HEK293 cells, and control HEK293 cells, respectively. HT29 cells stably transfected either with DCC (HT29-DCC11) or with empty vector (HT29-neo) (33Velcich A. Corner G. Palumbo L. Augenlicht L. Oncogene. 1999; 18: 2599-2606Crossref PubMed Scopus (35) Google Scholar) were kindly provided by Dr. Anna Velcich (Montefiore-Einstein Cancer Center, New York). Generation of Soluble HJV (sHJV) and Anti-HJV Antibody—A construct encoding a soluble portion of HJV (residues 1–401 inclusive of the HJV signal sequence) with a C-terminal His tag was subcloned into the baculovirus transfer vector pVL1393 (Pharmingen). sHJV was purified from the supernatants of baculovirus-infected High5 cells and buffer-exchanged to 10 mm Tris (pH 8) and 200 mm NaCl, followed by nickel-nitrilotriacetic acid Superflow chromatography (Qiagen Inc.). Protein from an imidazole elution was further purified by gel filtration chromatography in 20 mm Tris (pH 8) and 300 mm NaCl at 4 °C (34Lebron J.A. Bennett M.J. Vaughn D.E. Chirino A.J. Snow P.M. Mintier G.A. Feder J.N. Bjorkman P.J. Cell. 1998; 93: 111-123Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar). sHJV was used as an antigen to generate polyclonal antiserum in rabbits (Pocono Rabbit Farm & Laboratory, Inc., Canadensis, PA). Fluorescence Microscopy Analysis—Two days prior to immunofluorescence analysis, Myc-HJV HEK293, HJV G320V HEK293 and control HEK293 cells were seeded onto poly-l-lysine-coated coverslips. After washing with phosphate-buffered saline, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline for 15 min at room temperature. The cells were either blocked directly in 10% fetal bovine serum in phosphate-buffered saline for 1 h at room temperature (non-permeabilized cells) or treated with 0.2% Triton X-100 in phosphate-buffered saline for 10 min, followed by blocking (permeabilized cells). The cells were then incubated either with affinity-purified anti-Myc monoclonal antibody (for Myc-HJV; a kind gift from Dr. Jan Christian, Oregon Health & Science University) at 1:20 dilution or with rabbit anti-HJV serum (for HJV G320V) at 1:400 dilution for 1 h at room temperature, followed by incubation with either Alexa 594-labeled donkey anti-mouse (1:500 dilution) or goat anti-rabbit (1:500 dilution) antibody (Molecular Probes, Inc., Eugene, OR) as the corresponding secondary antibody. Cells were mounted with ProLong antifade reagent (Molecular Probes, Inc.) and imaged using a Nikon fluorescence microscope (×60 oil immersion lens; Meridian Instrument Company, Inc., Kent, WA). Immunodetection on Western Blots—Cell lysates from HEK293 and HT29 cells were prepared as described previously (35Carlson H. Zhang A.S. Fleming W.H. Enns C.A. Blood. 2005; 105: 2564-2570Crossref PubMed Scopus (23) Google Scholar). Briefly, samples were subjected to 8 or 12% SDS-PAGE under reducing or nonreducing conditions as indicated below. After transferring onto nitrocellulose membrane and blocking with 5% nonfat milk in buffer containing 0.1 m Tris-HCl, 0.15 m NaCl (pH 7.4), and 0.05% Tween 20, immunoblot analysis was performed using mouse anti-Myc monoclonal antibody (1:500 dilution), rabbit anti-HJV peptide antibody (1:1000 dilution; a kind gift from Dr. Silvia Arber, University of Basel, Basel, Switzerland) (24Niederkofler V. Salie R. Sigrist M. Arber S. J. Neurosci. 2004; 24: 808-818Crossref PubMed Scopus (151) Google Scholar), rabbit anti-HJV serum (1:10,000 dilution), sheep anti-human ferritin antibody (1:1000 dilution; The Binding Site, Ltd., Birmingham, UK), rabbit anti-neogenin antibody H-175 (1:1000 dilution; Santa Cruz Biotechnology, Inc.), mouse anti-DCC monoclonal antibody (1:100 dilution; Oncogene Research Products), sheep anti-transferrin receptor (TfR) antibody (1:10,000), or mouse anti-β-actin monoclonal antibody (clone AC-15, 1:10,000 dilution; Sigma). Blots were visualized by incubation with the appropriate horseradish peroxidase-conjugated secondary antibody and chemiluminescence (SuperSignal, Pierce). Equal protein loading in each lane was verified by both Ponceau S staining and Western blotting for β-actin. Neutralization of pH in the HJV-processing Compartments—MycHJV HEK293 cells (clones 6 and 15) and control HEK293 cells were incubated overnight in the presence or absence of 25 mm NH4Cl. Cell lysates were prepared as described above and subjected to 12% SDS-PAGE. HJV was detected using anti-HJV peptide antibody at 1:1000 dilution. Phosphatidylinositol-specific Phospholipase C (PI-PLC) Cleavage of Cell-surface HJV—Approximately 5 × 105 intact HEK293 cells transiently transfected with HJV or empty vector were suspended in 0.2 ml of Dulbecco's modified Eagle's medium and incubated in the presence or absence of PI-PLC (ProZyme, San Leandro, CA) at a concentration of 1 unit/ml for 2 h at 37°C in 5% CO2 incubator. Cells were separated from the supernatant by centrifugation at 500 × g for 5 min. Both cell lysates and supernatants were subjected to 8% SDS-PAGE, followed by immunodetection of HJV using rabbit anti-HJV antibody at 1:10,000 dilution as described under "Immunodetection on Western Blots." Immunoprecipitation—Immunoprecipitation was performed as described previously (36Gross C.N. Irrinki A. Feder J.N. Enns C.A. J. Biol. Chem. 1998; 273: 22068-22074Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 37Zhang A.S. Davies P.S. Carlson H.L. Enns C.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9500-9505Crossref PubMed Scopus (36) Google Scholar) with some modifications. Briefly, 50 μlof Pansorbin (Calbiochem) was first coated with antibody by incubation with 2 μl of either rabbit anti-HJV serum or 2 μg of rabbit anti-neogenin antibody in 50 μl of NET/Triton buffer (150 mm NaCl, 5 mm EDTA, and 10 mm Tris (pH 7.4) with 1% Triton X-100) for 50 min at 4 °C. After removing the unbound antibody by washing with NET/Triton buffer, pre-absorbed cell lysate as indicated below was added and incubated at 4 °C for 50 min, followed by washing twice with NET/Triton buffer. Samples were eluted in 100 μl of 2× Laemmli buffer (125 mm Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol) (38Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar) and subjected to SDS-PAGE on 8% acrylamide gels under reducing conditions. Immunodetection was performed as described above, except that rabbit TrueBlot (catalog no. 18-8816, eBioscience) at 1:1000 dilution was used as the secondary antibody. This secondary antibody was used to immunodetect protein immunoprecipitated with rabbit antibodies. It does not recognize denatured IgG;

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