Hepatoma-derived Growth Factor

Abstract

Hepatoma-derived growth factor (HDGF) has proliferative, angiogenic, and neurotrophic activity. It plays a putative role in the development and progression of cancer. When expressed in cells, the mitogenic activity of HDGF depends on its nuclear localization, but it also stimulates proliferation when added to the cell culture medium. A cell surface receptor for HDGF has not been identified so far. We investigated the interaction of various purified recombinant HDGF fusion proteins with the cell surface of NIH 3T3 fibroblasts. We showed that binding of a HDGF-β-galactosidase fusion protein to the cell surface of NIH 3T3 fibroblasts was saturable, occurred with high affinity (KD = 14 nm), and had a proliferative effect. We identified a peptide comprising amino acid residues 81–100 within the amino-terminal part of HDGF that bound to the cell surface of NIH 3T3 cells with saturation and affinity values similar to those of HDGF. When added to primary human fibroblasts, this peptide stimulated proliferation. Substitution of a single amino acid (K96A) within this peptide was sufficient to abolish its binding to the cell surface and its proliferative activity. In contrast, when expressed transiently in NIH 3T3 cells, a HDGF-β-galactosidase fusion protein in which amino acid residues 81–100 were deleted still had proliferative activity, whereas a fusion protein containing only the 81–100 peptide did not. Our results suggest the existence of a plasma membrane-located HDGF receptor for which signaling depends on amino acid residues 81–100 of HDGF. This region differs from the one that has been recently identified to be essential for mitogenic activity depending on the nuclear localization of HDGF. Thus, HDGF exerts its proliferative activity via two different pathways. Hepatoma-derived growth factor (HDGF) has proliferative, angiogenic, and neurotrophic activity. It plays a putative role in the development and progression of cancer. When expressed in cells, the mitogenic activity of HDGF depends on its nuclear localization, but it also stimulates proliferation when added to the cell culture medium. A cell surface receptor for HDGF has not been identified so far. We investigated the interaction of various purified recombinant HDGF fusion proteins with the cell surface of NIH 3T3 fibroblasts. We showed that binding of a HDGF-β-galactosidase fusion protein to the cell surface of NIH 3T3 fibroblasts was saturable, occurred with high affinity (KD = 14 nm), and had a proliferative effect. We identified a peptide comprising amino acid residues 81–100 within the amino-terminal part of HDGF that bound to the cell surface of NIH 3T3 cells with saturation and affinity values similar to those of HDGF. When added to primary human fibroblasts, this peptide stimulated proliferation. Substitution of a single amino acid (K96A) within this peptide was sufficient to abolish its binding to the cell surface and its proliferative activity. In contrast, when expressed transiently in NIH 3T3 cells, a HDGF-β-galactosidase fusion protein in which amino acid residues 81–100 were deleted still had proliferative activity, whereas a fusion protein containing only the 81–100 peptide did not. Our results suggest the existence of a plasma membrane-located HDGF receptor for which signaling depends on amino acid residues 81–100 of HDGF. This region differs from the one that has been recently identified to be essential for mitogenic activity depending on the nuclear localization of HDGF. Thus, HDGF exerts its proliferative activity via two different pathways. Hepatoma-derived growth factor (HDGF) 1The abbreviations used are: HDGF, hepatoma-derived growth factor; HRP, HDGF-related protein; LEDGF, lens epithelium-derived growth factor; HATH, homologue to amino terminus of HDGF; FGF, fibroblast growth factor; HLGAG, heparin-like glycosaminoglycan; Erk, extracellular signal-regulated kinase; GFP, green fluorescent protein; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt.1The abbreviations used are: HDGF, hepatoma-derived growth factor; HRP, HDGF-related protein; LEDGF, lens epithelium-derived growth factor; HATH, homologue to amino terminus of HDGF; FGF, fibroblast growth factor; HLGAG, heparin-like glycosaminoglycan; Erk, extracellular signal-regulated kinase; GFP, green fluorescent protein; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt. was initially purified from the supernatant of human hepatoma cell lines (1Klagsbrun M. Sasse J. Sullivan R. Smith J.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2448-2452Crossref PubMed Scopus (199) Google Scholar, 2Nakamura H. Kambe H. Egawa T. Kimura Y. Ito H. Hayashi E. Yamamoto H. Sato J. Kishimoto S. Clin. Chim. Acta. 1989; 183: 273-284Crossref PubMed Scopus (106) Google Scholar, 3Nakamura H. Izumoto Y. Kambe H. Kuroda T. Mori T. Kawamura K. Yamamoto H. Kishimoto T. J. Biol. Chem. 1994; 269: 25143-25149Abstract Full Text PDF PubMed Google Scholar). It has been shown to have growth factor activity for hepatoma cells, fibroblasts, smooth muscle cells, and endothelial cells (1Klagsbrun M. Sasse J. Sullivan R. Smith J.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2448-2452Crossref PubMed Scopus (199) Google Scholar, 2Nakamura H. Kambe H. Egawa T. Kimura Y. Ito H. Hayashi E. Yamamoto H. Sato J. Kishimoto S. Clin. Chim. Acta. 1989; 183: 273-284Crossref PubMed Scopus (106) Google Scholar, 4Dietz F. Franken S. Yoshida K. Nakamura H. Kappler J. Gieselmann V. Biochem. J. 2002; 366: 491-500Crossref PubMed Scopus (76) Google Scholar, 5Everett A.D. Stoops T. McNamara C.A. J. Biol. Chem. 2001; 276: 37564-37568Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). In addition, it has recently been shown to be neurotrophic (6Zhou Z. Yamamoto Y. Sugai F. Yoshida K. Kishima Y. Sumi H. Nakamura H. Sakoda S. J. Biol. Chem. 2004; 279: 27320-27326Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In recent years, five homologous proteins have been identified by different approaches (4Dietz F. Franken S. Yoshida K. Nakamura H. Kappler J. Gieselmann V. Biochem. J. 2002; 366: 491-500Crossref PubMed Scopus (76) Google Scholar, 7Ge H. Si Y. Roeder R.G. EMBO J. 1998; 17: 6723-6729Crossref PubMed Scopus (254) Google Scholar, 8Ikegame K. Yamamoto M. Kishima Y. Enomoto H. Yoshida K. Suemura M. Kishimoto T. Nakamura H. Biochem. Biophys. Res. Commun. 1999; 266: 81-87Crossref PubMed Scopus (43) Google Scholar, 9Izumoto Y. Kuroda T. Harada H. Kishimoto T. Nakamura H. Biochem. Biophys. Res. Commun. 1997; 238: 26-32Crossref PubMed Scopus (118) Google Scholar). Four of these proteins have been termed HRP-1 to HRP-4, and the fifth protein has been named p52/75 or LEDGF. Because of the high similarity between the different family members, the amino-terminal region (amino acids residues 1–100) of these proteins has been termed the HATH region (9Izumoto Y. Kuroda T. Harada H. Kishimoto T. Nakamura H. Biochem. Biophys. Res. Commun. 1997; 238: 26-32Crossref PubMed Scopus (118) Google Scholar). In contrast, the length and amino acid sequences of carboxyl-terminal regions of HRPs vary considerably, suggesting a modular structure of these proteins, which is supported by NMR data analysis of human HDGF (10Sue S.C. Chen J.Y. Lee S.C. Wu W.G. Huang T.H. J. Mol. Biol. 2004; 343: 1365-1377Crossref PubMed Scopus (42) Google Scholar). Expression of HDGF mRNA as well as protein is developmentally regulated, and various results point to an important function of HDGF during tissue development, protection, and repair (6Zhou Z. Yamamoto Y. Sugai F. Yoshida K. Kishima Y. Sumi H. Nakamura H. Sakoda S. J. Biol. Chem. 2004; 279: 27320-27326Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 11Oliver J.A. Al-Awqati Q. J. Clin. Investig. 1998; 102: 1208-1219Crossref PubMed Scopus (117) Google Scholar, 12Everett A.D. Lobe D.R. Matsumura M.E. Nakamura H. McNamara C.A. J. Clin. Investig. 2000; 105: 567-575Crossref PubMed Scopus (117) Google Scholar, 13Enomoto H. Yoshida K. Kishima Y. Kinoshita T. Yamamoto M. Everett A.D. Miyajima A. Nakamura H. Hepatology. 2002; 36: 1519-1527Crossref PubMed Google Scholar, 14Cilley R.E. Zgleszewski S.E. Chinoy M.R. J. Pediatr. Surg. 2000; 35 (discussion, 119): 113-118Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 15Mori M. Morishita H. Nakamura H. Matsuoka H. Yoshida K. Kishima Y. Zhou Z. Kida H. Funakoshi T. Goya S. Yoshida M. Kumagai T. Tachibana I. Yamamoto Y. Kawase I. Hayashi S. Am. J. Respir. Cell Mol. Biol. 2004; 30: 459-469Crossref PubMed Scopus (44) Google Scholar, 16Nakamura H. Yoshida K. Ikegame K. Kishima Y. H. Enomoto H. J. 2002; PubMed Google Scholar, H. Yoshida K. Kishima Y. Y. Nakamura H. J. 2002; PubMed Google Scholar, F. Kappler J. Gieselmann V. Franken S. Biochem. J. 2004; PubMed Google Scholar). In addition, on different of suggest a role of this growth factor in development and K. E. K. Everett A.D. K. Res. Google Scholar, M. J.A. Everett A.D. M. Y. PubMed Scopus Google Scholar, T.H. Huang Lee PubMed Scopus (106) Google Scholar, A. H. K. Y. K. Mori M. Res. 2001; Google Scholar, Y. Nakamura H. Yoshida K. Enomoto H. H. T. M. Ito H. Everett A.D. T. Kawase I. Sci. PubMed Scopus Google Scholar, K. Nakamura H. Y. Enomoto H. Kishima Y. H. Ito H. T. S. Kawase I. J. PubMed Scopus (48) Google Scholar). with growth factor HDGF and HRPs is still localization of HDGF is in cells, HDGF be in the H. Izumoto Y. Kambe H. Kuroda T. Mori T. Kawamura K. Yamamoto H. Kishimoto T. J. Biol. Chem. 1994; 269: 25143-25149Abstract Full Text PDF PubMed Google Scholar, 11Oliver J.A. Al-Awqati Q. J. Clin. Investig. 1998; 102: 1208-1219Crossref PubMed Scopus (117) Google Scholar, 12Everett A.D. Lobe D.R. Matsumura M.E. Nakamura H. McNamara C.A. J. Clin. Investig. 2000; 105: 567-575Crossref PubMed Scopus (117) Google Scholar, A.D. J. PubMed Scopus Google Scholar). HDGF has two nuclear localization one in the HATH and the in the carboxyl-terminal for the different family nuclear localization has been shown to be a for the mitogenic activity of expressed HDGF A.D. Stoops T. McNamara C.A. J. Biol. Chem. 2001; 276: 37564-37568Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, Y. Yamamoto H. Izumoto Y. Yoshida K. Enomoto H. Yamamoto M. Kuroda T. Ito H. K. Nakamura H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). have shown that HDGF is mitogenic not only when expressed in cells but also when H. Kambe H. Egawa T. Kimura Y. Ito H. Hayashi E. Yamamoto H. Sato J. Kishimoto S. Clin. Chim. Acta. 1989; 183: 273-284Crossref PubMed Scopus (106) Google Scholar, 3Nakamura H. Izumoto Y. Kambe H. Kuroda T. Mori T. Kawamura K. Yamamoto H. Kishimoto T. J. Biol. Chem. 1994; 269: 25143-25149Abstract Full Text PDF PubMed Google Scholar). its from cell culture and its mitogenic as an HDGF a and is only by cell In this it and data for these family suggest that these growth be from cells via a of the E. J. PubMed Scopus Google Scholar, J. Biochem. PubMed Scopus Google Scholar, T. J. PubMed Scopus Google Scholar, J. Biochem. PubMed Scopus Google Scholar). HDGF and its LEDGF, it has also been shown that be and it has been that the mitogenic of HDGF depends on and nuclear Y. Yamamoto H. Izumoto Y. Yoshida K. Enomoto H. Yamamoto M. Kuroda T. Ito H. K. Nakamura H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Investig. Sci. 2000; Google Scholar). a cell surface and in this it is important to that HDGF heparin-like (4Dietz F. Franken S. Yoshida K. Nakamura H. Kappler J. Gieselmann V. Biochem. J. 2002; 366: 491-500Crossref PubMed Scopus (76) Google Scholar, S.C. Chen J.Y. Lee S.C. Wu W.G. Huang T.H. J. Mol. Biol. 2004; 343: 1365-1377Crossref PubMed Scopus (42) Google Scholar). the of has not been HDGF also to a plasma receptor to a proliferative In with of of HDGF to cells has recently been T. T. K. T. Biochem. Biophys. Res. Commun. 2000; PubMed Scopus Google Scholar). the of receptor for HDGF or the proteins has been identified so far. In this the of of HDGF on NIH 3T3 fibroblasts. various HDGF-β-galactosidase fusion proteins, for the HDGF region for binding to the cell We the of a peptide region in the amino terminus of HDGF that was sufficient to with the cell surface and proliferation of fibroblasts. of this peptide region that HDGF from the cell surface of fibroblasts, and to the of these were from of and of fusion proteins, the region of the containing residues the terminus was by the from which the for the fusion protein was via a and in the used for sequences of and of the recombinant protein and HDGF were by containing and for amino acids were not by but by of the and with and the were of the region in In the within the of the as the point of of and used to the fusion in and and and and in a fusion proteins were by the region of as for was with and and with the in the the sequences of the fusion proteins were from by and and with an containing an in of the HDGF was as (4Dietz F. Franken S. Yoshida K. Nakamura H. Kappler J. Gieselmann V. Biochem. J. 2002; 366: 491-500Crossref PubMed Scopus (76) Google Scholar). of recombinant were by on an to the by the and of recombinant proteins were and purified by of carboxyl-terminal as (4Dietz F. Franken S. Yoshida K. Nakamura H. Kappler J. Gieselmann V. Biochem. J. 2002; 366: 491-500Crossref PubMed Scopus (76) Google Scholar). and of the recombinant proteins were by of and the carboxyl-terminal were by with of on a and by the protein Cell and for cell culture were from and was from NIH 3T3 cells were in with and cells were by with for to human fibroblasts were in the as for NIH 3T3 cells and a HDGF were of a a of cells were in for and with protein binding were with of in for of the different fusion proteins was in binding and for with and and one with containing bound activity was by of in binding to development was in a cell fusion proteins were by with of in and for activity was by the of of cells I. I. A Scholar). human fibroblasts were a of and in with was by containing but containing different of the recombinant proteins or In addition, well activity, and cells were for an were and as I. I. A and of was in a the of cell the was to the by the of the was as H. PubMed Scopus Google Scholar). on cells by proteins as fusion proteins in cells and purified by of as cells were for binding of the fluorescent the In addition, for cells and were as by R. H. Biol. Chem. 2002; PubMed Scopus Google Scholar, F. Kappler J. H. 2004; PubMed Scopus Google Scholar). values shown as the analysis of was for of data between two or A of was to be and a of was to be were V. to NIH 3T3 and by a proliferation HDGF growth factor activity when added to the of various cell (1Klagsbrun M. Sasse J. Sullivan R. Smith J.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2448-2452Crossref PubMed Scopus (199) Google Scholar, 2Nakamura H. Kambe H. Egawa T. Kimura Y. Ito H. Hayashi E. Yamamoto H. Sato J. Kishimoto S. Clin. Chim. Acta. 1989; 183: 273-284Crossref PubMed Scopus (106) Google Scholar, 4Dietz F. Franken S. Yoshida K. Nakamura H. Kappler J. Gieselmann V. Biochem. J. 2002; 366: 491-500Crossref PubMed Scopus (76) Google Scholar, 5Everett A.D. Stoops T. McNamara C.A. J. Biol. Chem. 2001; 276: 37564-37568Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, H. Kishima Y. Kuroda T. Enomoto H. H. H. 2000; Google Scholar). A for this activity is the interaction of HDGF with the cell A HDGF has not been for a putative HDGF expressed and purified a HDGF-β-galactosidase fusion A and that the recombinant fusion protein for bound to NIH 3T3 cells, whereas used as a did not. Cell binding of the fusion protein that this interaction was binding occurred In addition, added to the cell culture stimulated the of primary human fibroblasts two and comprising amino acids 81–100 were of were added in to human fibroblasts, and proliferation was by the of a in mitogenic activity between and peptide be peptide and with were used as and as = values in the fusion proteins of and HDGF the or amino acids 81–100 were and purified as and of of the proteins was added in to human fibroblasts, and proliferation was by the of of was used as a In to HDGF, of the HDGF proteins were to proliferation with when added to the cell culture medium. as = not with as a of proliferation activity is in for fusion proteins of HDGF the or amino acids 81–100 and a fusion protein were as and and NIH 3T3 for and HDGF to were used as and cell were the whereas peptide 81–100 from the HATH region was not to proliferation when expressed as a fusion protein the as = between and the of in HDGF Cell the regions of the growth factor for cell surface various HDGF-β-galactosidase fusion proteins of the fusion proteins, and investigated the of the purified proteins to to the surface of and NIH 3T3 fibroblasts. of the HDGF family a amino-terminal acid HATH region and a carboxyl-terminal region (4Dietz F. Franken S. Yoshida K. Nakamura H. Kappler J. Gieselmann V. Biochem. J. 2002; 366: 491-500Crossref PubMed Scopus (76) Google Scholar, 9Izumoto Y. Kuroda T. Harada H. Kishimoto T. Nakamura H. Biochem. Biophys. Res. Commun. 1997; 238: 26-32Crossref PubMed Scopus (118) Google Scholar). which part of HDGF is for cell surface the HATH region and region to and cell binding as results shown in and that the amino-terminal HATH region the cell surface binding activity of HDGF, whereas the carboxyl-terminal region was to to the surface of NIH 3T3 fibroblasts. the region of HDGF for cell surface deleted various of the HATH region and the of the proteins to to the surface of NIH 3T3 fibroblasts of amino acids 81–100 cell surface binding Thus, amino acids 81–100 of the HATH region for cell surface this region is also sufficient for the fusion that the fusion protein was to to NIH 3T3 fibroblasts as as a fusion protein comprising the HATH In addition, cell surface binding with different of the fusion protein that binding of this protein to the cell surface was and occurred with the affinity as the HDGF-β-galactosidase fusion protein and the of this peptide region for surface a to in the and the fusion proteins, this amino acid the cell surface interaction of the protein and a in cell surface binding of the fusion protein containing HDGF Because HDGF has been shown to to the of the and fusion proteins to to In to the fusion the fusion protein did not to that binding to the cell surface not via not the that the data on the fusion protein and used which to of and on this expressed and purified proteins in which HDGF and amino acids 81–100 of the HATH region were to to of fluorescent in a for the different in in the of cells the of similar single for and in the of cells in of the cell and not showed in the of the proteins, which when fluorescent but bound to the cell these for the fusion proteins of and in to were that bound to the plasma for were for the of the binding of and to the identified peptide region with a cell surface binding for HDGF, cells were with HDGF of with 35 on the cells a of binding of when with cells not 81–100 of HDGF a peptide amino acids 81–100 of the HATH region for its mitogenic activity. This peptide stimulated the proliferation of primary human fibroblasts of and In contrast, a peptide had fusion proteins the or amino acids 81–100 were not to the proliferation of fibroblasts when added to the Thus, amino acid residues 81–100 of HDGF important for the mitogenic activity of this growth of the HATH 81–100 on Cell not only when added to cells but also when expressed within In the the mitogenic activity of HDGF depends on its nuclear In addition, it was shown that extracellular HDGF be This the that the fusion protein added to the not a cell surface receptor but be and HDGF this deleted amino acids 81–100 from fusion protein We expressed for and transiently in NIH 3T3 cells and the growth of cells by the and fusion stimulated proliferation whereas showed results that HDGF two different proliferative one the carboxyl-terminal region for activity Y. Yamamoto H. Izumoto Y. Yoshida K. Enomoto H. Yamamoto M. Kuroda T. Ito H. K. Nakamura H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, A.D. Stoops T. Nakamura H. A. Am. J. Cell Mol. 2004; PubMed Scopus Google and the in the region its mitogenic activity from the cell that extracellular HDGF its HATH region and to the investigated HDGF be on cells that a fusion protein added to the cell culture be and is to the within Y. Yamamoto H. Izumoto Y. Yoshida K. Enomoto H. Yamamoto M. Kuroda T. Ito H. K. Nakamura H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). We added a and HDGF-β-galactosidase fusion protein to the cell culture of primary human fibroblasts and NIH 3T3 cells for of to were investigated by or activity We were not to these fusion proteins in the or in the of these cells not the that these be to used the fusion proteins to This is to interaction and of single fluorescent in cells with high F. Kappler J. H. 2004; PubMed Scopus Google Scholar, Mol. Biol. 2004; Google Scholar, E. Scholar, 2004; PubMed Scopus Google Scholar, A. 2004; PubMed Scopus (43) Google Scholar, M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (76) Google Scholar). In to with a fusion and to the human which has been shown to of the fluorescent protein different of cells J. K. J. Mol. Google were used as and for the fusion proteins, of cells with the different were these the in which is is from the plasma to the the the plasma the medium. proteins that were be as fluorescent between the two of the plasma be in only for to the fluorescent activity of be the cell in the whereas or not be in of the cells Thus, in the used had that added HDGF fusion proteins be HDGF is a of a family of growth an amino-terminal HATH region (4Dietz F. Franken S. Yoshida K. Nakamura H. Kappler J. Gieselmann V. Biochem. J. 2002; 366: 491-500Crossref PubMed Scopus (76) Google Scholar, 9Izumoto Y. Kuroda T. Harada H. Kishimoto T. Nakamura H. Biochem. Biophys. Res. Commun. 1997; 238: 26-32Crossref PubMed Scopus (118) Google Scholar). of the of this it has been shown that growth factor activity to various cell (1Klagsbrun M. Sasse J. Sullivan R. Smith J.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2448-2452Crossref PubMed Scopus (199) Google Scholar, 2Nakamura H. Kambe H. Egawa T. Kimura Y. Ito H. Hayashi E. Yamamoto H. Sato J. Kishimoto S. Clin. Chim. Acta. 1989; 183: 273-284Crossref PubMed Scopus (106) Google Scholar, 3Nakamura H. Izumoto Y. Kambe H. Kuroda T. Mori T. Kawamura K. Yamamoto H. Kishimoto T. J. Biol. Chem. 1994; 269: 25143-25149Abstract Full Text PDF PubMed Google Scholar, 8Ikegame K. Yamamoto M. Kishima Y. Enomoto H. Yoshida K. Suemura M. Kishimoto T. Nakamura H. Biochem. Biophys. Res. Commun. 1999; 266: 81-87Crossref PubMed Scopus (43) Google Scholar, T. T. K. T. Biochem. Biophys. Res. Commun. 2000; PubMed Scopus Google Scholar). A putative HDGF has not been Our data high affinity binding of HDGF fusion proteins to the cell surface of NIH 3T3 cells with the existence of a HDGF were to amino acid residues 81–100 of the HATH as important for HDGF We have shown that amino acids 81–100 of the HATH region and sufficient to cell surface binding with similar affinity and saturation as the fusion protein the HDGF. In addition, a protein region for binding to the plasma be on the surface of a (10Sue S.C. Chen J.Y. Lee S.C. Wu W.G. Huang T.H. J. Mol. Biol. 2004; 343: 1365-1377Crossref PubMed Scopus (42) Google NMR data that of the HATH this the peptide to the surface of HDGF the peptide is well It one of two in the HATH region of HDGF It is in to the region for binding to but is not to it (10Sue S.C. Chen J.Y. Lee S.C. Wu W.G. Huang T.H. J. Mol. Biol. 2004; 343: 1365-1377Crossref PubMed Scopus (42) Google Scholar). This is in with data that the peptide not to it be that this region to binding on the cell of the peptide for the of HDGF is supported by its to a proliferative in the of amino acid residues to an essential Substitution of the by the cell surface binding activity of the fusion protein and the proliferative activity of the peptide mitogenic activity of expressed HDGF is in the carboxyl-terminal region and depends on its nuclear localization A.D. Stoops T. McNamara C.A. J. Biol. Chem. 2001; 276: 37564-37568Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, Y. Yamamoto H. Izumoto Y. Yoshida K. Enomoto H. Yamamoto M. Kuroda T. Ito H. K. Nakamura H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google but added HDGF has mitogenic activity H. Kambe H. Egawa T. Kimura Y. Ito H. Hayashi E. Yamamoto H. Sato J. Kishimoto S. Clin. Chim. Acta. 1989; 183: 273-284Crossref PubMed Scopus (106) Google Scholar, 3Nakamura H. Izumoto Y. Kambe H. Kuroda T. Mori T. Kawamura K. Yamamoto H. Kishimoto T. J. Biol. Chem. 1994; 269: 25143-25149Abstract Full Text PDF PubMed Google Scholar). results two for the mitogenic activity of added HDGF. the growth factor is and to the of the cell to its proliferative activity the as expressed HDGF. In this it is important to that and nuclear of HDGF and have been Y. Yamamoto H. Izumoto Y. Yoshida K. Enomoto H. Yamamoto M. Kuroda T. Ito H. K. Nakamura H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Investig. Sci. 2000; Google Scholar). HDGF of its nuclear localization a cell surface not In the used HDGF to its proliferative via of a cell surface receptor the of of HDGF when were Because on cells, in contrast, have of HDGF Y. Yamamoto H. Izumoto Y. Yoshida K. Enomoto H. Yamamoto M. Kuroda T. Ito H. K. Nakamura H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google it that the of cells to this growth factor depends on the cell In addition, the peptide is in the HATH whereas the proliferative activity of the growth factor expressed in the cell has been to the region A.D. J. PubMed Scopus Google Scholar, Y. Yamamoto H. Izumoto Y. Yoshida K. Enomoto H. Yamamoto M. Kuroda T. Ito H. K. Nakamura H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In with these of for HDGF fusion proteins proliferation when the region was In contrast, when a of a peptide fusion protein was proliferative was peptide mitogenic activity when but not when expressed within the In contrast, a HDGF this region is when but cell proliferation This the existence of two different which HDGF exerts its proliferative activity. depends on the region and localization of HDGF, whereas the depends on the HATH region and binding of the growth factor to an cell surface is supported by the that HDGF of protein to the cell culture of cells A.D. Stoops T. Nakamura H. A. Am. J. Cell Mol. 2004; PubMed Scopus Google Scholar). When to this in NIH 3T3 cells, the in of HDGF to the culture was and was not with or growth factor used as not Thus, to only a role in HDGF of fibroblasts. In the of of HDGF vary between different cell with to and nuclear as well as the of of pathways. the of this it be that of HDGF those of growth in Thus, also to and a cell surface but it also be 2000; PubMed Scopus Google Scholar, Biol. 2001; PubMed Scopus Google Scholar, J. J. Mol. 1997; PubMed Scopus Google Scholar, A. J. PubMed Google Scholar). its from the cell surface to the depends on HDGF, it it is E. J. PubMed Scopus Google Scholar, E. J. Biochem. PubMed Scopus Google Scholar). point to of the of by these growth We for