Fibroblast Growth Factor (FGF) Homologous Factors Share Structural but Not Functional Homology with FGFs

Fibroblast growth factors (FGFs) interact with heparan sulfate glycosaminoglycans and the extracellular domains of FGF cell surface receptors (FGFRs) to trigger receptor activation and biological responses. FGF homologous factors (FHF1–FHF4; also known as FGF11–FGF14) are related to FGFs by substantial sequence homology, yet their only documented interactions are with an intracellular kinase scaffold protein, islet brain-2 (IB2) and with voltage-gated sodium channels. In this report, we show that recombinant FHFs can bind heparin with high affinity like classical FGFs yet fail to activate any of the seven principal FGFRs. Instead, we demonstrate that FHFs bind IB2 directly, furthering the contention that FHFs and FGFs elicit their biological effects by binding to different protein partners. To understand the molecular basis for this differential target binding specificity, we elucidated the crystal structure of FHF1b to 1.7-Å resolution. The FHF1b core domain assumes a β-trefoil fold consisting of 12 antiparallel β strands (β1 through β12). The FHF1b β-trefoil core is remarkably similar to that of classical FGFs and exhibits an FGF-characteristic heparin-binding surface as attested to by the number of bound sulfate ions. Using molecular modeling and structure-based mutational analysis, we identified two surface residues, Arg52 in the β4–β5 loop and Val95 in the β9 strand of FHF1b that are required for the interaction of FHF1b with IB2. These two residues are unique to FHFs, and mutations of the corresponding residues of FGF1 to Arg and Val diminish the capacity of FGF1 to activate FGFRs, suggesting that these two FHF residues contribute to the inability of FHFs to activate FGFRs. Hence, FHFs and FGFs bear striking structural similarity but have diverged to direct related surfaces toward interaction with distinct protein targets. Fibroblast growth factors (FGFs) interact with heparan sulfate glycosaminoglycans and the extracellular domains of FGF cell surface receptors (FGFRs) to trigger receptor activation and biological responses. FGF homologous factors (FHF1–FHF4; also known as FGF11–FGF14) are related to FGFs by substantial sequence homology, yet their only documented interactions are with an intracellular kinase scaffold protein, islet brain-2 (IB2) and with voltage-gated sodium channels. In this report, we show that recombinant FHFs can bind heparin with high affinity like classical FGFs yet fail to activate any of the seven principal FGFRs. Instead, we demonstrate that FHFs bind IB2 directly, furthering the contention that FHFs and FGFs elicit their biological effects by binding to different protein partners. To understand the molecular basis for this differential target binding specificity, we elucidated the crystal structure of FHF1b to 1.7-Å resolution. The FHF1b core domain assumes a β-trefoil fold consisting of 12 antiparallel β strands (β1 through β12). The FHF1b β-trefoil core is remarkably similar to that of classical FGFs and exhibits an FGF-characteristic heparin-binding surface as attested to by the number of bound sulfate ions. Using molecular modeling and structure-based mutational analysis, we identified two surface residues, Arg52 in the β4–β5 loop and Val95 in the β9 strand of FHF1b that are required for the interaction of FHF1b with IB2. These two residues are unique to FHFs, and mutations of the corresponding residues of FGF1 to Arg and Val diminish the capacity of FGF1 to activate FGFRs, suggesting that these two FHF residues contribute to the inability of FHFs to activate FGFRs. Hence, FHFs and FGFs bear striking structural similarity but have diverged to direct related surfaces toward interaction with distinct protein targets. Fibroblast growth factors (FGF1–FGF23) 1The abbreviations used are: FGF, fibroblast growth factor; FGFR, FGF receptor; FHF, FGF homologous factor; HS, heparan sulfate glycosaminglycans; IB2, islet brain-2; mIB2, murine IB2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide1The abbreviations used are: FGF, fibroblast growth factor; FGFR, FGF receptor; FHF, FGF homologous factor; HS, heparan sulfate glycosaminglycans; IB2, islet brain-2; mIB2, murine IB2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide constitute one of the largest families of polypeptide growth factors, and fulfill vital functions in both the embryo and the adult (1Ornitz D.M. Itoh N. Genome Biol. 2001; 2: 1-12Crossref Google Scholar). During embryogenesis, FGFs govern the development of parenchymal organs, such as the lung and limb, which require directional epithelial-mesenchymal communication (2Martin G. Bioessays. 2001; 23: 865-868Crossref PubMed Scopus (58) Google Scholar, 3Ornitz D.M. Marie P.J. Genes Dev. 2002; 16: 1446-1465Crossref PubMed Scopus (713) Google Scholar, 4Ware L.B. Matthay M.A. Am. J. Physiol. 2002; 282: L924-L940Crossref PubMed Scopus (285) Google Scholar). In the adult, FGFs are important in wound healing, tissue repair, metabolism, and homeostasis (5Basilico C. Moscatelli D. Adv. Cancer Res. 1992; 59: 115-165Crossref PubMed Scopus (1049) Google Scholar, 6Powers C.J. McLeskey S.W. Wellstein A. Endocr. Relat. Cancer. 2000; 7: 165-197Crossref PubMed Scopus (1111) Google Scholar). FGFs exert their diverse actions by binding, dimerizing, and activating members of the FGF receptor family (FGFR1–FGFR4) of receptor tyrosine kinases. Both FGFs and FGFR must also interact with heparan sulfate glycosaminglycans (HS) for sustainable FGF-FGFR binding and dimerization to occur (7Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Crossref PubMed Scopus (558) Google Scholar, 8Yayon A. Klagsbrun M. Esko J.D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-848Abstract Full Text PDF PubMed Scopus (2073) Google Scholar, 9Rapraeger A.C. Krufka A. Olwin B.B. Science. 1991; 252: 1705-1708Crossref PubMed Scopus (1285) Google Scholar, 10Schlessinger J. Plotnikov A.N. Ibrahimi O.A. Eliseenkova A.V. Yeh B.K. Yayon A. Linhardt R.J. Mohammadi M. Mol. Cell. 2000; 6: 743-750Abstract Full Text Full Text PDF PubMed Scopus (948) Google Scholar).FGFs differ significantly in both size (17–29 kDa) and sequence, but all contain a core region of homology encompassing 120–130 residues. The FGF core homology region assumes a β-trefoil fold consisting of 12 β strands arranged in three sets of four-stranded β-sheets (11Zhu X. Komiya H. Chirino A. Faham S. Fox G.M. Arakawa T. Hsu B.T. Rees D.C. Science. 1991; 251: 90-93Crossref PubMed Scopus (329) Google Scholar, 12Eriksson A.E. Cousens L.S. Weaver L.H. Matthews B.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3441-3445Crossref PubMed Scopus (254) Google Scholar, 13Osslund T.D. Syed R. Singer E. Hsu E.W. Nybo R. Chen B.L. Harvey T. Arakawa T. Narhi L.O. Chirino A. Morris C.F. Protein Sci. 1998; 7: 1681-1690Crossref PubMed Scopus (30) Google Scholar, 14Plotnikov A.N. Eliseenkova A.V. Ibrahimi O.A. Shriver Z. Sasisekharan R. Lemmon M.A. Mohammadi M. J. Biol. Chem. 2001; 276: 4322-4329Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 15Bellosta P. Iwahori A. Plotnikov A.N. Eliseenkova A.V. Basilico C. Mohammadi M. Mol. Cell. Biol. 2001; 21: 5946-5957Crossref PubMed Scopus (60) Google Scholar, 16Yeh B.K. Igarashi M. Eliseenkova A.V. Plotnikov A.N. Sher I. Ron D. Aaronson S.A. Mohammadi M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2266-2271Crossref PubMed Scopus (146) Google Scholar). Based on sequence comparison, the 22 known mammalian FGFs (FGF1–FGF23) are grouped into eight subfamilies. FGF11–FGF14, initially termed FGF homologous factors 1–4 (FHF1–FHF4), constitute an FGF subfamily that was discovered by searching cDNA data bases for sequences with homology to the core region of FGFs (17Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (329) Google Scholar, 18Verdier A.S. Mattei M.G. Lovec H. Hartung H. Goldfarb M. Birnbaum D. Coulier F. Genomics. 1997; 40: 151-154Crossref PubMed Scopus (35) Google Scholar, 19Coulier F. Pontarotti P. Roubin R. Hartung H. Goldfarb M. Birnbaum D. J. Mol. Evol. 1997; 44: 43-56Crossref PubMed Scopus (184) Google Scholar, 20Hartung H. Feldman B. Lovec H. Coulier F. Birnbaum D. Goldfarb M. Mech. Dev. 1997; 64: 31-39Crossref PubMed Scopus (100) Google Scholar). Within the β-trefoil core, FHFs share the highest sequence identity (36–40%) with FGF9 subfamily members (FGF9, FGF16, and FGF20). Like FGF9 subfamily members, FHFs lack a recognizable secretory signal sequence. However, whereas FGF9 subfamily members are efficiently secreted as glycoproteins (21Miyamoto M. Naruo K. Seko C. Matsumoto S. Kondo T. Kurokawa T. Mol. Cell. Biol. 1993; 13: 4251-4259Crossref PubMed Scopus (392) Google Scholar, 22Miyake A. Konishi M. Martin F.H. Hernday N.A. Ozaki K. Yamamoto S. Mikami T. Arakawa T. Itoh N. Biochem. Biophys. Res. Commun. 1998; 243: 148-152Crossref PubMed Scopus (134) Google Scholar), FHFs remain intracellular when transfected into two different cultured cell lines (17Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (329) Google Scholar, 23Wang Q. McEwen D.G. Ornitz D.M. Mech. Dev. 2000; 90: 283-287Crossref PubMed Scopus (73) Google Scholar). It is note-worthy that the prototypical FGFs, FGF1 and FGF2, also lack a signal peptide and are only poorly secreted from transfected cell lines. Current data suggest that these FGFs may be released from cells via a mechanism independent of ER-Golgi such as cell injury (24Jackson A. Friedman S. Zhan X. Engleka K.A. Forough R. Maciag T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10691-10695Crossref PubMed Scopus (225) Google Scholar, 25Mignatti P. Morimoto T. Rifkin D.B. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11007-11011Crossref PubMed Scopus (191) Google Scholar, 26Florkiewicz R.Z. Majack R.A. Buechler R.D. Florkiewicz E. J. Cell. Physiol. 1995; 162: 388-399Crossref PubMed Scopus (191) Google Scholar, 27Florkiewicz R.Z. Anchin J. Baird A. J. Biol. Chem. 1998; 273: 544-551Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). These precedents have led to speculation that FHFs undergo localized nonclassical release from cells for interactions with HS and cell surface FGFRs (17Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (329) Google Scholar). However, no interactions of FHFs with HS and FGFRs have been reported.For each FHF, multiple isoforms differing only in the N-terminal region preceding the β-trefoil core are generated through alternative promoter usage and differential splicing of 5′-exons (20Hartung H. Feldman B. Lovec H. Coulier F. Birnbaum D. Goldfarb M. Mech. Dev. 1997; 64: 31-39Crossref PubMed Scopus (100) Google Scholar, 28Munoz-Sanjuan I. Smallwood P.M. Nathans J. J. Biol. Chem. 2000; 275: 2589-2597Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 29Yamamoto S. Mikami T. Konishi M. Itoh N. Biochim. Biophys. Acta. 2000; 1490: 121-124Crossref PubMed Scopus (10) Google Scholar). These FHF isoforms are evolutionarily well conserved and exhibit distinct tissue distribution and subcellular localization, implicating the N terminus as an intracellular trafficking signal (23Wang Q. McEwen D.G. Ornitz D.M. Mech. Dev. 2000; 90: 283-287Crossref PubMed Scopus (73) Google Scholar, 28Munoz-Sanjuan I. Smallwood P.M. Nathans J. J. Biol. Chem. 2000; 275: 2589-2597Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 30Munoz-Sanjuan I. Simandl B.K. Fallon J.F. Nathans J. Development. 1999; 126: 409-421PubMed Google Scholar).In situ hybridization studies in mice have revealed prominent expression of FHFs in the developing and adult nervous system (17Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9850-9857Crossref PubMed Scopus (329) Google Scholar, 20Hartung H. Feldman B. Lovec H. Coulier F. Birnbaum D. Goldfarb M. Mech. Dev. 1997; 64: 31-39Crossref PubMed Scopus (100) Google Scholar, 30Munoz-Sanjuan I. Simandl B.K. Fallon J.F. Nathans J. Development. 1999; 126: 409-421PubMed Google Scholar). In chickens, FHFs are also expressed in the developing limbs and face (31Munoz-Sanjuan I. Fallon J.F. Nathans J. Mech. Dev. 2000; 95: 101-112Crossref PubMed Scopus (13) Google Scholar, 32Munoz-Sanjuan I. Cooper M.K. Beachy P.A. Fallon J.F. Nathans J. Dev. Dyn. 2001; 220: 238-245Crossref PubMed Scopus (11) Google Scholar). Recent genetic findings have established the importance of FGF14 (FHF4) in neurological function. Targeted disruption of FGF14 (FHF4) in mice has been shown to cause ataxia and paroxysmal dyskinesia, and a loss of function mutation in FGF14 (FHF4) has been detected in patients with autosomal dominant cerebral ataxia (33Wang Q. Bardgett M.E. Wong M. Wozniak D.F. Lou J. McNeil B.D. Chen C. Nardi A. Reid D.C. Yamada K. Ornitz D.M. Neuron. 2002; 35: 25-38Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 34van Swieten J.C. Brusse E. de Graaf B.M. Krieger E. van de Graaf R. de Koning I. Maat-Kievit A. Leegwater P. Dooijes D. Oostra B.A. Heutink P. Am. J. Hum. Genet. 2003; 72: 191-199Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar).The apparent intracellular localization of FHF has also led to the contention that FHFs act intracellularly. Indeed, two cytoplasmic binding partners for FHFs have been identified. One such target is the mitogen-activated protein kinase scaffolding protein islet brain 2 (IB2), which was identified using FHF1b as bait in a yeast two-hybrid system and was then validated as a native binding partner through detection of FHF1·IB2 complexes in brain extracts (35Schoorlemmer J. Goldfarb M. Curr. Biol. 2001; 11: 793-797Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). This interaction is highly specific, since intracellular FGF1 fails to bind IB2, nor can FHF bind the related mitogen-activated protein kinase scaffold protein IB1 (JIP-1) (35Schoorlemmer J. Goldfarb M. Curr. Biol. 2001; 11: 793-797Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). It has been proposed that FHF-IB2 complex formation facilitates the recruitment of p38δ mitogen-activated protein kinase to IB2, allowing p38δ to be phosphorylated and activated by IB2-associated upstream kinases (36Schoorlemmer J. Goldfarb M. J. Biol. Chem. 2002; 277: 49111-49119Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Other potential intracellular targets for FHFs are voltage-gated sodium channels. The cytoplasmic tails of two such channels, Nav1.5 and Nav1.9, have been shown to bind FHF1b in a yeast two-hybrid screen and GST fusion protein pull-down assays (37Liu C. Dib-Hajj S.D. Waxman S.G. J. Biol. Chem. 2001; 276: 18925-18933Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 38Liu C.J. Dib-Hajj S.D. Renganathan M. Cummins T.R. Waxman S.G. J. Biol. Chem. 2003; 278: 1029-1036Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). More recent data suggest that FHF binding may modulate the electrophysiological properties of Nav1.5 (38Liu C.J. Dib-Hajj S.D. Renganathan M. Cummins T.R. Waxman S.G. J. Biol. Chem. 2003; 278: 1029-1036Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar).In this report, we prepared purified recombinant FHF proteins to assay them for the ability to interact with FGFRs and IB2. Our data show that FHFs do not activate any of seven principal cell surface FGFRs but do bind directly to the intracellular protein IB2. Using the newly determined FHF1b crystal structure and structure-based mutagenesis, we identified two residues unique to FHFs that contribute to the differential target specificity of FHFs. The data provide details of how, despite striking structural homology, FHFs and FGFs have diverged to direct related surfaces toward interaction with distinct protein targets.EXPERIMENTAL PROCEDURESProtein Purification—The DNA fragment encoding the “b” isoform of human FHF1 (FHF1b; residues 1–181) (see Fig. 4B for residue numbering) (20Hartung H. Feldman B. Lovec H. Coulier F. Birnbaum D. Goldfarb M. Mech. Dev. 1997; 64: 31-39Crossref PubMed Scopus (100) Google Scholar) was amplified by PCR and subcloned into the pET-3 bacterial expression vector. In order to prevent disulfide-mediated dimer formation, Cys144 of FHF1b was mutated to alanine (the corresponding residue in all other FHFs). Monomeric FHF1b was expressed in E. coli strain BL21 (DE3) pLysS cells and was purified by heparin affinity, ion exchange, and size exclusion chromatography. The C-terminally truncated form of FHF1b (FHF1b1–142) was generated by PCR with mutagenic primers. The FHF1bV95N, FHF1bR52G, and FHF1bV95N/R52G mutants were generated by multicycle DNA synthesis with Pfu DNA polymerase and complementary mutagenic primers (QuikChange mutagenesis; Stratagene). FHF1b1–142, FHF1bV95N, and FHF1bR52G were expressed and purified using the same protocol as for FHF1b.To express FHF4b, the DNA fragment encoding residues 64–252 of the “b” isoform of human FHF4 (FHF4b) (23Wang Q. McEwen D.G. Ornitz D.M. Mech. Dev. 2000; 90: 283-287Crossref PubMed Scopus (73) Google Scholar) was amplified and subcloned into the pET-28a bacterial expression vector. FHF4b was expressed in E. coli strain BL21 (DE3) cells and was purified using the same protocol as for FHF1b. Consistent with having an alanine in the position homologous to Cys144 of FHF1b, FHF4b did not form disulfide-linked dimers.Bacterially expressed human FGF1 (residues 22–155) was purified by heparin affinity as previously described (39Wang J.K. Gao G. Goldfarb M. Mol. Cell. Biol. 1994; 14: 181-188Crossref PubMed Scopus (205) Google Scholar). FGF1N110V and FGF1G67R mutants were generated by multicycle DNA synthesis with Pfu DNA polymerase and complementary mutagenic primers (QuikChange™ mutagenesis; Stratagene). The FGF1N110V and FGF1G67R mutants were expressed and purified using the same protocol as for FGF1.Crystallization, Structure Determination, and Refinement—Repeated attempts to crystallize freshly purified FHF1b failed; however, after a few weeks of storage at 4 °C, the FHF1b sample crystallized. Analysis of FHF1b crystals by mass spectrometry yielded a molecular mass of 16.3 kDa corresponding to FHF1b residues 1–144. Crystals of the truncated FHF1b fragment were grown by vapor diffusion at 20 °C using the conventional hanging drop method. 2 μl of FGF1b protein (45 mg/ml in 25 mm Hepes, pH 7.5, 300 mm NaCl) were mixed with an equal volume of crystallization buffer (20–25% PEG 400, 200 mm ammonium sulfate). The FHF1b crystals belong to the orthorhombic space group P212121 with unit cell dimensions α = 30.62 Å, β = 58.85 Å, γ = 65.42 Å. The asymmetric unit contains a single FHF1b molecule with a solvent content of 38.67%. Crystals were flash-frozen in a dry nitrogen stream using mother liquor with 10% glycerol as cryoprotectant. Diffraction data were collected on a charge-coupled device detector at beamline X4A at the National Synchrotron Light Source, Brookhaven National Laboratory. The data were processed with DENZO and SCALEPACK (40Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38368) Google Scholar).The program AMORE (41Navaza J. Acta Crystallogr. A. 1994; 50: 157-163Crossref Scopus (5027) Google Scholar) was used to find a molecular replacement solution using the structure of FGF9 (Protein Data Bank identification code 1IHK) as the search model (14Plotnikov A.N. Eliseenkova A.V. Ibrahimi O.A. Shriver Z. Sasisekharan R. Lemmon M.A. Mohammadi M. J. Biol. Chem. 2001; 276: 4322-4329Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Rigid body, positional, and B-factor refinement and simulated annealing were performed using CNS (42Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16930) Google Scholar). The program O was used for model building into the 2Fo -Fc and Fo -Fc maps (43Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard G. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar). The final refined model consists of FHF1b residues 6–143, 36 water molecules, and four sulfate ions. The N-terminal residues 1–5 are disordered. The average B factors are 21.93 Å2 for all atoms, 21.50 Å2 for FHF1b, 25.46 Å2 for the water molecules, and 39.86 Å2 for the sulfate ions.BaF3/FGFR Viability Assay—BaF3 cell lines transfected to express each of the seven principal FGFR isoforms (FGFR1-IIIb, FGFR1-IIIc, FGFR2-IIIb, FGFR2-IIIc, FGFR3-IIIb, FGFR3-IIIc, and FGFR4) have been described previously (44Ornitz D.M. Xu J. Colvin J.S. McEwen D.G. MacArthur C.A. Coulier F. Gao G. Goldfarb M. J. Biol. Chem. 1996; 271: 15292-15297Abstract Full Text Full Text PDF PubMed Scopus (1414) Google Scholar). For agonist viability assays, cells were plated in 96-cluster miniwells at 104 cells/well in RPMI 1640 medium supplemented with 10% fetal bovine serum, 5 μg/ml heparin, and increasing concentrations of wild-type or mutant FGF1 or FHFs. Viable cells were quantified by MTT assay after 4 days of culture. To test the ability of FHFs to antagonize FGF1 signaling, cells were plated in media as above containing a submaximal concentration of FGF1 (150 pm) plus increasing concentrations of FHF. Growth/viability was quantified as above. All experiments were performed in duplicate.Assays for FHF·IB2 Complex Formation—The DNA fragment encoding residues 226–421 of murine IB2 (numbered according to GenBank™ AF220195) was subcloned into the pET3d bacterial expression vector with an in-frame C-terminal 6× histidine tag. The resulting construct (mIB2226–421) was expressed in Escherichia coli strain BL21 (DE3) pLysS cells. Cell pellets were lysed in the presence of 6 m sodium isothiocyanate and loaded onto a nickel-agarose column. The column was washed in denaturing and then nondenaturing buffers, and mIB2226–421 was eluted with 200 mm imidizole. Purified FHF and mIB2226–421 were incubated together at 4 °C for 15 min and then subjected to gel filtration fast protein liquid chromatography on an analytical Superdex 75-HR10/30 (Amersham Biosciences). Column fractions were analyzed by SDS-PAGE and Coomassie Blue or silver staining.RESULTSA characteristic biochemical feature of all FGFs studied to date is the ability to interact with heparin. Indeed, this feature has been routinely used for FGF affinity purification (45Burgess W.H. Maciag T. Annu. Rev. Biochem. 1989; 58: 575-606Crossref PubMed Google Scholar). A primary sequence analysis of FHFs indicated that several basic residues in FGFs that are shown to mediate the FGF-heparin interaction are also present in FHFs. Therefore, it was pertinent to check whether FHFs bind heparin and, if so, to utilize this interaction as a tool for affinity purification. As shown in Fig. 1, both FHF1b and FHF4b bound to heparin-Sepharose and were eluted using 0.75 m sodium chloride. The ionic strength required to elute FGF from heparin-Sepharose has been routinely used as a measure of FGF-heparin affinity (46Thompson L.D. Pantoliano M.W. Springer B.A. Biochemistry. 1994; 33: 3831-3840Crossref PubMed Scopus (277) Google Scholar). Thus, the affinity of FHFs for heparin is comparable with that of FGF7 and FGF10 (0.75 m) but is significantly lower than that of the FGF1- and FGF2-heparin interaction (1.5 and 1.8 m, respectively). Both FHFs were further purified to homogeneity using ion exchange and size exclusion chromatography.Fig. 1FHFs bind heparin. Bacterially expressed soluble FHF1b and FHF4b samples were analyzed by heparin affinity chromatography. The FHFs were eluted using increasing concentrations of sodium chloride in a step gradient (dashed line). FHF-containing peak fractions were analyzed by SDS-PAGE followed by Coomassie Blue staining. LD, load material; FT, flow-through.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FHFs Fail to Activate All Seven Known FGFRs—In addition to bearing homology with FGFs at the heparin binding site, all FHFs also share homology with FGFs at the surface regions that were shown to interact with FGFR in FGF-FGFR crystal structures (16Yeh B.K. Igarashi M. Eliseenkova A.V. Plotnikov A.N. Sher I. Ron D. Aaronson S.A. Mohammadi M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2266-2271Crossref PubMed Scopus (146) Google Scholar, 47Plotnikov A.N. Schlessinger J. Hubbard S.R. Mohammadi M. Cell. 1999; 98: 641-650Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar, 48Plotnikov A.N. Hubbard S.R. Schlessinger J. Mohammadi M. Cell. 2000; 101: 413-424Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 49Stauber D.J. DiGabriele A.D. Hendrickson W.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 49-54Crossref PubMed Scopus (211) Google Scholar). Therefore, it was pertinent to check whether FHFs bind and activate FGFR in a heparin-dependent manner. To test this possibility, we made use of previously characterized BaF3 lymphoid cell lines, each expressing one of the seven principal FGFR isoforms. These cell lines survive and proliferate in response to FGF/heparin-induced receptor activation (7Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Crossref PubMed Scopus (558) Google Scholar, 44Ornitz D.M. Xu J. Colvin J.S. McEwen D.G. MacArthur C.A. Coulier F. Gao G. Goldfarb M. J. Biol. Chem. 1996; 271: 15292-15297Abstract Full Text Full Text PDF PubMed Scopus (1414) Google Scholar). Each cell line was tested for viability in response to FHF concentrations ranging up to 12.5 nm using an MTT assay (Fig. 2, A–G). Whereas FGF1 had significant activity toward all receptors at concentrations as low as 300 pm, FHFs had no activity at all tested concentrations.Fig. 2FHFs do not interact with FGFRs. A–G, assays for FHF activation of FGFRs. BaF3 cells expressing each of the seven FGFRs were plated in medium containing 5 μg/ml heparin and increasing concentrations of FHFs or FGF1 as a control. Viable cells were quantitated 4 days later by MTT assay, and data are expressed as percentage of maximum MTT values achieved with highest concentration of FGF1. H, FHFs do not antagonize FGF-FGFR interaction. BaF3 cells expressing FGFR1-IIIb were cultured with FGF1 (150 pm) and increasing concentrations of FHF1b or FHF4b. Data are expressed as percentage of MTT value attained with 3 nm FGF1 in the absence of FHFs. MTT values for treatment with different FGF1 concentrations alone (expressed as log10m) are indicated on the left. ♦, FGF1; ▴, FHF1b; ▵, FHF4b. Experiments for A–H were performed in duplicate and include error bars.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The failure of FHFs to activate any FGFR despite significant homology with FGFs at the receptor binding residues left open the possibility that FHFs may have evolved to antagonize FGF signaling. To assay for receptor antagonism, FHFs at concentrations ranging up to 12.5 nm were tested for their ability to antagonize viability/proliferation mediated by a threshold concentration of FGF1 (150 pm). Fig. 2H shows data for the BaF-FGFR1-IIIb cell line, which is representative of all cell lines tested, and demonstrates that FHFs fail to antagonize FGF1 activity. Taken together, these data substantiate the premise that FHFs are functionally unrelated to FGFs.FHFs Interact Directly with the Cytoplasmic Protein IB2— Multiple isoforms of FHF1, FHF2, and FHF4 (FGF14) have been shown to form intracellular complexes with IB2 (35Schoorlemmer J. Goldfarb M. Curr. Biol. 2001; 11: 793-797Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). 2D. M. Ornitz and M. Goldfarb, unpublished data. The FHF-IB2 interaction requires the presence of both FGF core homology region and the C-terminal tail of FHF and a 260-residue long segment (residues 212–471) of murine IB2 not shared by the related scaffold IB1 (JIP-1b) (35Schoorlemmer J. Goldfarb M. Curr. Biol. 2001; 11: 793-797Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). In more recent studies, we have shown that a shorter fragment of this region consisting of residues 226–421 is sufficient for FHF binding. 3S. K. Olsen, M. Garbi, N. Zampieri, A. V. Eliseenkova, D. M. Ornitz, M. Goldfarb, and M. Mohammadi, unpublished results.To determine whether the FHF-IB2 interaction is direct, the IB2 fragment consisting of residues 226–421 was expressed as a His6-tagged fusion protein (mIB2226–421) in E. coli and purified using Ni+ chelating chromatography. FHF1b or FHF4b (10 μm) were preincubated with or without an excess of mIB2226–421 (28 μm) and chromatographed through a gel filtration column, and fraction aliquots were analyzed by SDS-PAGE. Fig. 3, A–D, shows that each FHF alone has a long column retention time, whereas in the presence of added mIB2226–421, each FHF more rapidly co-elutes with the added IB2. To confirm the requirement for the C-terminal tail of FHF1b for IB2 binding, we also expressed and purified a C-terminally truncated version of FHF1b and tested the capacity of this construct (FH