©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ligand-specific Structural Domains in the Fibroblast Growth Factor Receptor (*)

Fen Wang , Mikio Kan , Jianming Xu , Guochen Yan (§) , Wallace L. McKeehan (¶)

From the (1) Albert B. Alkek Institute of Biosciences and Technology, Department of Biochemistry and Biophysics, Texas A & M University, Houston, Texas 77030-3303

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Two tandem immunoglobulin-like disulfide loops (Loops II and III) linked by a short connecting sequence in the ectodomain of the fibroblast growth factor receptor kinase compose the binding sites for glycosaminoglycan and fibroblast growth factor (FGF) ligands. Alternate splicing of exons IIIb and IIIc coding for the COOH-terminal half of Loop III confers high affinity for FGF-7 or FGF-2, respectively, on the fibroblast growth factor receptor ectodomain without effect on the binding of FGF-1. Here we show that a 139-amino acid fragment composed of Loop II, the inter-Loop II/III sequence, and a short segment of the NHterminus of Loop III is sufficient and near the minimal requirement for binding of FGF-1, FGF-2, and FGF-7. Extension of the fragment by five additional highly conserved residues (SD(P/A)QP) within a distinct constitutive structural domain (fl1) in Loop III restricts the binding of FGF-7 without effect on FGF-1 and FGF-2. Since the presence of exon IIIc in the full-length ectodomain does not change this ligand binding profile, we suggest that alternately spliced exon IIIc plays no active role in binding of the three ligands. In contrast, exon IIIb actively abrogates the restriction on the binding of FGF-7 and concurrently lowers the affinity for FGF-2.


INTRODUCTION

The heparin-binding fibroblast growth factor (FGF)() family consists of nine genetically distinct polypeptides that elicit receptor-mediated effects on growth, function, and differentiation (1, 2) . The FGF receptor (FGFR) is a trimolecular complex of the glycosaminoglycan portion of a heparan sulfate proteoglycan, an FGF ligand, and the ectodomain of a tyrosine kinase transmembrane glycoprotein (3, 4) , each component of which exhibits an interactive domain for the two other components of the complex (5, 6) . Although the functional role of the direct interaction of heparan sulfate with the receptor kinase ectodomain is unclear, we show in the accompanying paper (7) that the affinity of the interaction of heparin with it varies with the structure of the ectodomain and correlates with the affinity of the glycosaminoglycan-ectodomain complex for ligand.

The tyrosine kinase component of the complex consists of the products of four genes, FGFR1, FGFR2, FGFR3, and FGFR4 (2) . At least 16 single-site splice variations in the FGFR1 and FGFR2 genes form combinatorial variants in the extracellular and intracellular juxtamembrane and intracellular kinase and COOH-terminal domains (2, 8, 9, 10, 11, 12) . The heparan sulfate- and ligand-binding domain of the FGFR transmembrane tyrosine kinase is composed of Ig-like disulfide loop units (13, 14) and interloop sequences encoded by one or two exons (9) . Tandem Loops II and III form the heparan sulfate- and ligand-binding sites (5) . A highly conserved 18-amino acid sequence in the NHterminus of Loop II of all FGFR kinase isoforms composes a heparan sulfate-binding domain that is obligatory for ligand binding (5) . Although coding sequences for Loop II, the inter-Loop II/III sequence, and the NH-terminal half of Loop III are not alternately spliced, the COOH terminus of Loop III in the FGFR1 and FGFR2 genes varies as a result of alternate splicing (2) . Alternate splicing of exons IIIb and IIIc in the FGFR2 gene dramatically alters specificity for FGF ligands (15, 16, 17) . While both FGFR2(IIIb) and FGFR2(IIIc) bind FGF-1 with equal affinity, FGFR2(IIIb) binds FGF-7 and exhibits a lower affinity for FGF-2. Both FGFR1(IIIc) and FGFR2(IIIc) isoforms bind FGF-2 with high affinity, but they reject FGF-7 at all concentrations.

Although FGF-1, FGF-2, and FGF-7 are expressed in many tissues, FGF-7 is a specific product of stromal fibroblasts in tissues with distinct epithelial and stromal cell compartments (11, 18, 19) . Epithelial cells display the FGFR2(IIIb) isoform, while stromal fibroblasts and other non-parenchymal cells display the FGFR1(IIIc) isoform (11, 16, 18) . The stromal cell-specific expression of FGF-7 and epithelial cell-specific expression of FGFR2(IIIb) appear to constitute a directionally specific paracrine communication system from stromal to epithelial cells. Subversion of the directional specificity occurs in a variety of tumors by a switch from expression of the FGFR2(IIIb) to the FGFR2(IIIc) isoform and subsequent activation of the FGF-2 gene, other embryonic isoforms of the FGF ligand family, and the normally mesenchymal cell-associated FGFR1(IIIc) isoform of the receptor in epithelial cells (11, 20, 21) . Since the differential specificity of the FGFR(IIIb) and FGFR(IIIc) isoforms for FGF ligands may underlie the autonomous growth and independence of tumor epithelial cells on stroma, we undertook the current study to define the ligand-specific structural domains within the extracellular domain of the FGFR tyrosine kinase component of the FGFR complex.


EXPERIMENTAL PROCEDURES

Construction of FGFR cDNAs

In the oligonucleotide sequences below, residues in linker and restriction sites are underscored, mutant residues are in boldface, and residues not in the FGFR cDNAs are in lowercase. Where possible, restriction sites (silent mutations) were introduced into oligonucleotide primers to aid in identification of cloned cDNAs. DNAs were purified by agarose gel electrophoresis, excised, and electroeluted. Orientation of cloned constructions was determined by restriction enzyme analysis. Wild-type cDNAs coding for the extracellular domain of human FGFR1 extending through the transmembrane and 18 residues into the intracellular juxtamembrane sequence were cloned and prepared as described (8) . The cDNAs for the extracellular domain of the rat FGFR2(IIIb) (GenBanknumber L19104) and FGFR2(IIIb) (GenBanknumber L19107) isoforms extending from the translational initiation site through the transmembrane domain and 20 residues into the intracellular juxtamembrane domain were generated in the polymerase chain reaction (PCR) with human sense primer pB1 (gcctgaattcCACATGGAGATATGGAAGAGGAC) and antisense primer pB2 (gcctgaattcAGCTTGTGCACAGCCGGCTGGCT) with rat prostate cDNA template (11, 12) . The cDNA coding for the intracellular kinase domain of FGFR2 (12) (GenBanknumber L19106) was ligated to the FGFR2(IIIb) to generate the cDNA coding for the full-length FGFR2(IIIb) type 1 kinase (12) . The FGFR2 portion of the chimeric human FGFR1 and rat FGFR2 cDNAs consisted of FGFR2 residues corresponding to FGFR1(IIIc) residues Glu-Leu. The FGFR2 segment was generated in the PCR with sense primer pB3 (TGGATCAAACATGTCGAAAAGAAC) containing a TaqI restriction site and antisense primer pB2, which contained an EcoRI site using the FGFR2(IIIb) cDNA template. After digestion with TaqI and EcoRI, the 367-base pair (bp) fragment from the PCR was ligated at the TaqI site of both wild-type and mutant (described below) human FGFR1 cDNA fragments (621 bp) prepared by digestion with PstI and TaqI. The FGFR1 portion began 13 bp upstream of the translation initiation site and extended through the coding sequence for residue Ileof FGFR1.

Mutant cDNAs were generated by overlap extension with pairs of complementary oligonucleotide primers in the PCR as described (5, 8, 23) . Mutant DNA fragments (621 bp) for the FGFR1(P194L,Q195Q,P196L) and FGFR1(S192I,D193L) constructions were generated in the same PCR from the FGFR1 cDNA template with sense primer pR1a (AAAGAAACAGATAACACCAAA) and antisense primer pR1b (GTTACCCGCCAAGCACGTATA) and a pair of asymmetric mutant primers (sense: mpFL1a GTGTACAGTGACCTGCAGCTGCACATC; antisense: mpFL1b GATGTGCGGCTGCGGGACAATGTACAC). A PstI site was incorporated into the mpFL1a primer, which was used to discriminate clones of the FGFR1(S192I,D193L) from the FGFR1(P194L,Q195Q,P196L) cDNA. The 275-bp core of the mutant DNA fragments from the PCR was prepared by digestion with BstXI and TaqI and ligated at the BstXI site with wild-type FGFR1 cDNA (401 bp) on the 5` side and at the TaqI site with wild-type FGFR1(IIIc) or wild-type and mutant FGFR2(IIIb) cDNAs on the 3` side. Mutant cDNA fragments in which Hisor Hiswas substituted with phenylalanine were also generated in the same PCR from FGFR1 cDNA template with sense primer pR1a and antisense primer pR1b and a pair of asymmetric mutant primers (sense (mpHa), CAGCCGTTCATCCAGTGGCTAAAGCACATCGAG; antisense (mpHb), CTCGATGAACTTTAGCCACTGGATGTGCGGCTG). After digestion with BstXI and TaqI, the resultant core 275-bp mutant fragments were inserted between flanking wild-type FGFR1 cDNAs at the BstXI and TaqI sites.

Mutants in the fl2 domain of exon IIIb in the FGFR2 portion of the FGFR1/R2(IIIb) chimeric construction (described above) were generated with 5` sense primer mpFL1a complementary to the FGFR1 sequence and 3` antisense primer pB2 complementary to the FGFR2(IIIb) sequence as flanking primers in the presence of specific mutant primers. The FGFR1/R2(IIIb)(H313T,S314A,+KE) mutant cDNA was generated with sense mutant primer mpFL2a (GGGATTAATAGTTCCAATAAGGAAGCAGAA) coding for the Lys-Glu insert and antisense mutant primer mpFL2b (ATTGGAACTATTAATCCCCGCGGTCTT) coding for the H313T,S314A substitution were used in the PCR with the FGFR1/R2(IIIb) cDNA template. To generate the individual mutant FGFR1/R2(IIIb)(H313T,S314A) and FGFR1/R2(IIIb)(+KE) cDNAs, sense mutant primer mpFL2a and an antisense mutant primer mpFL2c (TGCATTGGAACTATTAATCCC) coding for the Lys-Glu deletion in exon IIIb were employed in the PCR with the mutant FGFR1/R2(IIIb) (H313T,S314A,+KE) cDNA template. After digestion with TaqI and EcoRI, the mutant DNA fragments were ligated with the TaqI site of the FGFR1 cDNA.

cDNAs coding for wild-type and mutant secreted truncates of the FGFR1 extracellular domain (SFR1) were prepared and cloned into transfer vector pVL1393 for preparation of recombinant baculovirus (21, 22) . Partial cDNAs coding for the wild-type and mutant FGFR1 extracellular domains through Ilewere prepared by treatment of the FGFR1 cDNA with TaqI followed by an end-fill with Klenow enzyme and a digestion with XbaI. They were then cloned into the pBluescript SK vector at XbaI and SmaI sites. After partial digestion with EcoRI, the 679-bp cDNA was cloned into the EcoRI sites of the pVL1393 vector. The ligation resulted in an expression product ending with Ileof the FGFR1 sequence with a tail of 14 unrelated amino acid residues due to an in-frame ligation with the transfer vector. SFR1 cDNA constructions that coded for secreted extracellular domains truncated at Pro(SFR1P), Tyr(SFR1Y), Lys(SFR1K), and Lys(SFR1K) were constructed by generation of DNA fragments containing a stop codon in the antisense mutation primers, which resulted in the desired truncation. Mutation antisense primers pSFP(TAGGCACTGCAGGTGCTACGGCTG), pSFY(GTGCGGCTGCAGGTCTCAGTACAC), pSFK(GGGCTGCAGGTCACTGTACTACTT), and pSFK(ACCCACCTGCAGTCATTTGTTGGC) containing the stop codons and a PstI restriction site were used with sense primer pR1a and FGFR1 cDNA template. After digestion with PstI and BstXI, the cDNA fragments were ligated with the XbaI- BstXI FGFR1 cDNA fragment described above at the BstXI site and cloned into the pVL1393 vector at XbaI and PstI sites.

PCR was performed for 40 cycles at 94 °C for 1 min, 55 °C for 2 min, and 72 °C for 3 min. Reaction mixtures (100 µl) contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl, 0.2 mM each of deoxynucleotide triphosphates, 1 µg of template, and 15 pM of each primer. cDNAs were cloned into pBluescript SK vector for restriction enzyme and sequence analysis. The complete sequence of mutant cDNA fragments generated by the PCR and the sequence across ligation sites with wild-type and mutant FGFR cDNAs were determined. cDNAs coding for transmembrane forms of FGFR extracellular domains were partially digested with EcoRI and cloned into mammalian expression vector P91023B for transient expression in mammalian cell hosts (5, 7, 8, 21) . cDNAs coding for SFR1 were cloned into baculoviral transfer vector pVL1393 (InVitrogen, San Diego, CA) for preparation of recombinant baculovirus and expression in Spodoptera frugiperda (Sf9) insect cells (21, 22) .

Expression of Recombinant FGFR Variants

Wild-type and mutant FGFR cDNAs in mammalian expression vector P91023B were transiently transfected into monkey kidney cells (COS-7) in replicate monolayer cultures (10cells in 60-mm tissue culture dishes) by the DEAE-dextran method as described (8, 21, 23) . After exposure to individual plasmids bearing cDNA constructions indicated under ``Results'' for 5 h, transfected cells were harvested by trypsin from replicate 60-mm dishes, pooled, and then divided equally among 60-mm dishes and cultured for 2 days prior to analysis of binding and covalent cross-linking of I-labeled FGF-1 (I-FGF-1), I-FGF-2, and I-FGF-7. For concurrent analysis of expression of surface antigen and ligand binding without covalent cross-linking, transfected cells harvested from a 60-mm dish were distributed into 24-well plates and cultured for 2 days prior to the analysis. In a typical experiment, experimental groups consisted of cells transfected with three or four different cDNA constructions.

Recombinant baculovirus bearing cDNA coding wild-type or mutant FGFR constructions was prepared by co-infection of Sf9 cells with the pVL1393 vector bearing FGFR cDNAs and BaculoGold viral DNA (PharMingen, San Diego, CA) by the Lipofectin method (21, 22, 24) . The recombinant expression products were identified and purified by immunochemical methods as described (21) . Each recombinant viral stock was standardized by analysis of the level of expression of antigen determined by immunoblot (10) .

Binding and Cross-linking of I-FGF Ligands

FGF-1, FGF-2, and FGF-7 were iodinated, activated by reduction, and purified to a specific activity of 2-5 10cpm/ng, and analysis of binding and covalent affinity cross-linking of I-FGF were performed as described (5, 10, 21, 23, 24, 25) . Binding mixtures contained 2 ng/ml labeled ligand and 2 µg/ml heparin. Prior to analysis of ligand-labeled receptors by SDS-polyacrylamide gel electrophoresis and autoradiography (5, 7, 8) , radioactivity of extracts was counted by -counter. Specific binding was determined, and specificity of ligand-labeled bands was confirmed by addition of a 100-fold excess of unlabeled ligand. Since FGF-1 bound to products of all constructions that exhibited specific ligand binding, it served as a qualitative standard for comparison of FGF-2 and FGF-7 binding. Levels of receptor antigen present in binding assays were standardized by the immunoassays described below.

For analysis of secreted recombinant FGFR antigens, the medium of baculovirus-infected Sf9 cell cultures (5 10cells in 25-cmculture flasks) was changed to 3 ml of Grace's medium containing 1 mg/ml bovine serum albumin and 2 µg/ml heparin 12 h after infection, and then culture was continued for 24 h. Portions (0.60 ml) of neutralized medium (pH 7.2) were immobilized on anti-FGFR1 antiserum A50 (21) bound to 60 µl of protein A-agarose beads for 1 h at room temperature or 4 h at 4 °C; the immunocomplexes were washed once with 0.50 ml of PBS, washed once with PBS containing 1 M NaCl, washed again with PBS, and then suspended in 0.6 ml of binding assay buffer. Three equal portions of the same preparation were used for comparison of the binding of I-FGF-1, I-FGF-2, and I-FGF-7 (5) . Harvested soluble receptor fragments were used immediately in binding assays. Products stored in culture medium or as purified immunoprecipitates exhibited a time-dependent decrease in ligand affinity indicated by non-linear Scatchard plots and an increase in heparin-independent and nonspecific binding followed by loss of total ligand binding activity.

Scatchard and Displacement Analysis of I-FGF-1 Binding

For Scatchard analysis and competition assays, 1.5 ml of medium from infected cells was immobilized on protein A-agarose beads as above, and the immunocomplexes were divided equally for binding assays. The binding was carried out in 0.4 ml of binding buffer. For Scatchard analysis, the binding buffer contained various concentrations of I-FGF-1 and 2 µg/ml heparin. For competition assays, the binding buffer contained 2 ng/ml I-FGF-1, 2 µg/ml heparin, and various concentrations of unlabeled FGF-1, FGF-2, and FGF-7. After incubation at room temperature for 2 h, the beads were washed with PBS twice and heparin-PBS once, and the bound radioactivity was quantitated by -counter.

Analysis of Recombinant FGFR Antigens

FGFR antigens displayed on the surface of transfected COS-7 cells were analyzed by double antibody radioimmunoassay as described (5, 21) . Triplicate wells from the same 24-well replicates used for ligand binding were analyzed.

Receptor antigen levels from constructions expressed in baculovirus-infected Sf9 cells were standardized by immunoblot analysis (5, 21) . For analysis of secreted antigens in the medium, antigens were enriched from a 0.20-ml portion of neutralized medium used for binding assays by treatment with 20 µl of heparin-agarose beads. The beads were extracted, and immunoblot analysis of the extract was performed (21) . In the tryptic fragmentation experiments (see Fig. 3 ), portions of receptor antigens immobilized on heparin-agarose before and after exposure to trypsin were similarly analyzed.


Figure 3: Binding of FGF ligands to fragments of the FGFR ectodomain. A, FGF binding to tryptic fragments. The lysate (0.40 ml in 0.5% Triton X-100 and PBS) of 5 10Sf9 cells infected with recombinant baculovirus bearing the cDNA coding for full-length FGFR1(IIIc) or FGFR2(IIIb) was diluted to 2 ml with PBS containing 1 mg/ml bovine serum albumin and was immobilized on 120 µl of heparin-agarose beads by incubation at 4 °C for 2 h. After a wash with PBS, the beads were divided into two portions. Aliquots of one portion ( part a) were incubated in PBS for 1 h at 4 °C prior to performance of ligand binding assays. Aliquots of the second portion ( part b) were incubated with 0.2 ml of 10 µg/ml trypsin in PBS for 1 h at 4 °C as described (5). The beads were then washed with 0.2 mM phenylmethylsulfonyl fluoride in PBS twice and then used in ligand binding assays. FGF binding was determined as described under ``Experimental Procedures.'' Bands between the 66- and 97-kDa markers in part a are the truncated ectodomain resulting from an endogenous protease (5, 21). B, FGF binding to recombinant fragments truncated at specific residues. Secreted recombinant fragments ending at the indicated residues were expressed in baculovirus-infected Sf9 cells and immobilized to antibody-agarose beads, and covalent affinity cross-linking of I-FGF-1, I-FGF-2, and I-FGF-7 to each construction was determined. The SF9 lane indicates an immunocomplex prepared from the medium of uninfected Sf9 cells. Sf9 cells infected with wild-type baculovirus yielded the same result. The three upper panels are autoradiographs, and the lower panel is an immunoblot analysis of a portion of each of the same samples used in the binding analysis. C, FGF binding to secreted SFR1Ifragments with the indicated mutations in the fl1 sequence. PQP LQL, P194L,Q195Q,P196L; SD IL, S192I,D193L.




RESULTS

Hypothetical Model of the FGFR Extracellular Domain

To visualize the FGFR ectodomain in a three-dimensional context and to guide structure-function analysis, structural domains were assigned to the sequence of tandem Ig Loops II and III of FGFR using a template of the well defined structural domains exhibited by the constant loop in immunoglobulins for which the three-dimensional structure is well characterized (13, 14, 21) (Fig. 1 A). The constant Ig homology unit is composed of two -sheets composed of four (A, B, D, and E) and three (C, F, and G) antiparallel -strands connected by a disulfide. -Strands are connected by front (fl) and back (bl) interstrand loops designated according to whether they are on the front or back ends of the cylindrical Ig unit. Consistent with the idea that interaction of the back of Loop II and the front of Loop III may form the binding site for the glycosaminoglycan-FGF complex, the interstrand loops and flanking strand sequences on the back face of Ig Loop II and the front face of Loop III as well as the inter-Loop II/III sequence are highly conserved across isoforms of the four FGFR genes in all species (21) . The A-bl1-B domain in Loop II is the major heparan sulfate-binding domain (5) . The variant COOH-terminal domain of Loop III encoded by alternately spliced exons begins with the fl2 domain at the COOH terminus of the D strand. Important residues within the B-fl1-C and the fl2-E domains of Loop III that are discussed in the text are indicated in Fig. 1 A and in boldface in Fig. 1B.


Figure 1: Model of the FGFR complex. A, sequence domains of Ig Loops II and III and the interloop sequence of FGFR1 were assigned by combined Chou-Fasman, Garnier, and EMBL PHD methods using a template constructed from the x-ray coordinates for a constant Ig loop unit from the light chain of IgG1 (21). The putative disulfides between strands B and F that connect the two -sheets within each unit are indicated in black. The Ig units, which are actually cylinders whose back and front faces are composed of interactive structural domains, are flattened out and separated for clarity. The heparan sulfate( HS)-binding domain (A-bl1-B) in Loop II and the fl1 domain of Loop III are indicated in black. Key residues discussed in the text are indicated. Numbering of residues is from the translational initiation site of FGFR1 (8). The open triangle and hatched area within strand E indicate location of the variant TA/HS and KE residues in exons IIIb and IIIc, respectively. The -carbon backbone of FGF-2 is from Eriksson et al. (29). SO indicates the location of a bound sulfate ion in crystals of FGF-2, which is thought to be the heparan sulfate-binding pocket (29). The interstrand 9/10 domain of FGF-2 is shaded. B, sequence of B-fl1-C ( top) and fl2-E-bl3 ( bottom) domains of FGFR. Conserved residues in all variants and species are indicated with closed circles (21). Serine/threonine and valine/isoleucine/leucine residues were scored as conserved. Key residues discussed in the text are in reverse print.



Alternately Spliced Exons IIIb and IIIc Modify the Affinity for FGF-2 and FGF-7

cDNAs encoding extracellular domains of FGFR1 and FGFR2 beginning from the translational initiation site extending through the transmembrane domain and 18-20 residues into the intracellular juxtamembrane domain, respectively, were constructed, cloned into mammalian expression vector P91023B, and transiently transfected into monkey kidney COS cells (5, 7, 8, 21, 22, 23) . Binding and covalent cross-linking of I-FGF-1, I-FGF-2, or I-FGF-7 to the membrane-anchored recombinant extracellular domains on the cell surface was distinguished from endogenous full-length FGFR isoforms by size on SDS-polyacrylamide gel electrophoresis after autoradiography. The results confirmed that FGF-1 and FGF-2 but not FGF-7 bound to FGFR1(IIIc) (Fig. 2). In contrast, both FGF-1 and FGF-7 bound to FGFR2(IIIb) while the binding of FGF-2 was significantly reduced. A chimeric construction FGFR1/R2(IIIb) in which the exon IIIc sequence and 19 residues upstream of it in FGFR1 were substituted with exon IIIb and homologous upstream residues from FGFR2 exhibited a similar ligand binding profile to the FGFR2(IIIb) variant (Fig. 2). This indicated that the determinants for differences in binding of FGF-2 and FGF-7 were encoded in the portion of the FGFR2 gene containing exon IIIb independent of whether the NH-terminal fragment was from FGFR1 or FGFR2. Throughout this study, we detected no significant difference in the FGFR2(IIIb) and chimeric FGFR1/R2(IIIb) product in respect to affinity for the three FGF ligands.


Figure 2: Impact of alternately spliced exons IIIb and IIIc on binding of FGF-1, FGF-2, and FGF-7. COS-7 monkey kidney cells were transfected with cDNAs coding for the membrane-anchored ectodomains of the indicated isoforms followed by analysis of covalent affinity cross-linking of the I-labeled ligands to the products expressed on the cell surface. The antisense lane indicates a sample from cells transfected with an FGFR1(IIIc) cDNA cloned in the antisense orientation. R1(IIIc) and R1/R2(IIIb) refer to the 2-loop isoforms, while R2(IIIb) was the 3-loop variant.



The Conserved fl1 Domain of Ig Loop III Restricts FGF-7 Binding in the Absence of Alternately Spliced Exons IIIb and IIIc

Heparin interacts with and protects 30- and 33-kilodalton (kDa) fragments of the extracellular domain of recombinant baculovirus-infected insect cell-derived FGFR1(IIIc) from degradation by trypsin (8) . Immunochemical and biochemical analysis of the fragments indicated that they began at residues Gluand Pro, respectively, in the NHterminus of FGFR1 and spanned Loop II, the inter-Loop II/III sequence, and the NHterminus of Loop III through tryptic cut sites at Lys, Lys, or Lys(Fig. 1). A complex of the 30-33-kDa fragment and heparin still bound FGF-1 and FGF-2, similar to the parent FGFR1(IIIc) molecule (8) (Fig. 3 A). Surprisingly, the fragment acquired the ability to bind FGF-7, which was restricted in the intact FGFR1(IIIc) isoform. A similar treatment of the FGFR2(IIIb) isoform, which binds FGF-7 but has low affinity for FGF-2, yielded a fragment that appeared to bind FGF-1, FGF-2, and FGF-7 to nearly equal extents (Fig. 3 A). Competition experiments indicated that FGF-2 and FGF-7 competed with I-FGF-1 binding to the tryptic fragments from either isoform to an extent equal to or better than unlabeled FGF-1. A 3-fold excess of unlabeled FGF-1, FGF-2, and FGF-7 reduced labeled FGF-1 binding by 60, 90, and 85%, respectively, with standard errors of 20-30% among triplicates (not shown). The results suggested that (i) FGF-1, FGF-2, FGF-7, and heparin share a binding site that resides in the 30-33-kDa fragment and (ii) the tryptic fragments independent of parent isoform have lost specificity for FGF ligands. Therefore, structural domains downstream of the tryptic cleavage site confer the specificity for FGF ligands. In the exon IIIc isoform, the COOH-terminal domains restrict the binding of FGF-7 while downstream domains in the IIIb isoform lower the affinity for FGF-2.

To determine whether sequence domains between the COOH terminus of the 30-33-kDa fragment and the beginning of alternately spliced exons IIIb and IIIc were determinants of specific ligand binding, we constructed recombinant baculoviruses bearing cDNAs coding for SFR1 ending at Ile, Pro, Tyr, Lys, and Lys(Fig. 1). SFR1Iextended through residue Ilein strand C of Loop III. Ileis 15 residues downstream of Lys, a candidate for the COOH terminus of the 30-33-kDa tryptic fragments (5) , and is 19 residues upstream of the beginning of the alternate exons IIIb. Immobilization of the SFR1Ifragment from the medium of baculovirus-infected Sf9 cells on antibody-protein A-agarose beads followed by analysis of I-FGF binding revealed that a complex of heparin and SFR1Ibound FGF-1 and FGF-2 but failed to bind FGF-7 (Fig. 3 B). The secreted product SFR1Pending at proline 196 also bound FGF-1 and FGF-2 but not FGF-7 (Fig. 3 B). However, truncation of the secreted product (SFR1Y) to tyrosine 191 restored FGF-7 binding. This suggested that in the absence of downstream sequence domains, five residues or fewer within the constitutive fl1 domain restrict FGF-7 binding with little or no effect on the binding of FGF-1 and FGF-2.

To determine the minimal requirement for ligand binding, we then constructed and expressed cDNAs coding for secreted fragments, SFR1Kand SFR1K, which ended at lysine 189 and lysine 176, respectively. Both lysine residues were potential COOH termini of the 30-33-kDa fragment that resulted from trypsin treatment of full-length FGFR1(IIIc) in the presence of heparin (5) (Fig. 3 A). Similar to the 30-33-kDa tryptic fragment, the SFR1Kproduct bound FGF-1, FGF-2, and FGF-7 (Fig. 3 B). Reduction of the ectodomain to Lyseliminated the binding of all three FGF ligands (Fig. 3 B).

Scatchard analyses confirmed that FGF-1 bound to the four immobilized active FGFR1 fragments with a similar affinity ( K= 400-1000 pM) (see Fig. 5 A), which is of the same order exhibited by the full-length membrane-anchored receptor in intact cells (2, 8, 10, 20) . In agreement with the results of the covalent affinity cross-linking analysis, quantitative displacement analysis of I-FGF-1 demonstrated that unlabeled FGF-1, FGF-2, and FGF-7 competed with I-FGF-1 binding to SFR1Kwith similar efficiencies. In contrast, unlabeled FGF-7 competed with the binding of I-FGF-1 to SFR1Pand SFR1Imuch less effectively than FGF-1 and FGF-2, which is the characteristic pattern exhibited by the intact FGFR1(IIIc) variant (Fig. 4 B).


Figure 5: FGF binding to membrane-anchored FGFR with mutations in the fl1 domain. A and B, the indicated variants of the full-length FGFR ectodomain extending through the transmembrane domain were expressed on the surface of transfected COS-7 cells, and FGF binding was then analyzed by covalent affinity cross-linking. The mutants in A were in the FGFR1(IIIc) isoform. Binding to the FGFR2(IIIb) isoform was included as a positive control for FGF-7 binding assays. Mutants in B were in the fl1 domain of the FGFR1 portion of the FGFR1/R2(IIIb) chimera. Antisense indicates cells transfected with a vector bearing the FGFR1(IIIc) A or FGFR2 (IIIb) B cDNAs cloned into the vector in the antisense direction. C, both FGF binding and expression of cell surface receptor antigen were measured on COS-7 cells that were transfected with the indicated FGFR1 cDNAs. Solid bars, FGF-1; hatched bars, FGF-2. Surface antigen was determined with anti-FGFR1 antiserum A50 and a secondary I-labeled donkey anti-rabbit IgG (5). Data are the mean of duplicates. D, FGF binding to the indicated constructions of FGFR1(IIIc) containing histidine-to-phenylalanine mutations was determined as in A. Mutants were in the FGFR1(IIIc) isoform. Binding to COS cells transfected with the FGFR1/R2(IIIb) chimera was included as a positive control for FGF-7 binding ( lower panel). PQP LQL, P194L,Q195Q,P196L; SD IL, S192I,D193L.




Figure 4: Quantitative analysis of ligand binding to recombinant fragments of the FGFR1 ectodomain. Secreted recombinant fragments ending at the indicated residues were expressed in baculovirus-infected Sf9 cells and immobilized to antibody-agarose beads. The immunocomplexes were divided into equal portions and used for the following analyses. A, Scatchard analysis of FGF-1 binding. B, displacement analysis of FGF-1 binding by FGF-1, FGF-2, and FGF-7. Unlabeled FGF-1 ( diamonds), FGF-2 ( squares), and FGF-7 ( circles) were added to assays containing the indicated fragments of the ectodomain to result in the indicated ratios of unlabeled FGF to I-FGF-1 (2 ng/ml). 100% binding was about 40,000 cpm. Data are the mean of duplicates.



These combined results suggested that (i) the 30-33-kDa fragment that is protected against tryptic degradation by heparin and binds FGF-1, FGF-2, and FGF-7 most likely results from tryptic cleavage at lysine 189; (ii) a complex of glycosaminoglycan and the 30-33-kDa fragment is the minimal structure required to bind FGF ligands in general; (iii) the putative fl1 domain, which is highly conserved and constitutive in all FGFR isoforms, restricts the binding of FGF-7 to the base ligand-binding site in the FGFR fragment; and (iv) structural domains encoded in variant exon IIIb abrogate the restriction placed on FGF-7 binding and lower the affinity for FGF-2.

To determine whether the binding of FGF-7 might be restored by alteration of specific residues within the fl1 domain of Loop III, we constructed two mutant cDNAs coding for the secreted fragment SFR1Iin which the two proline residues and the serine-aspartate (SD) residues within the SDPQPHI sequence were substituted with bulky hydrophobic leucine or isoleucine residues. Similar to the wild-type SFR1Iconstruction, both mutant expression products bound FGF-1 and FGF-2 but not FGF-7 (Fig. 3 C). This indicated that the restrictive role of the fl1 domain on FGF-7 binding in the absence of alternate exons IIIb and IIIc is not dependent on specific sequences. In contrast, the same substitutions in the fl1 sequence significantly affected ligand binding in the presence of the complete COOH-terminal domain of Loop III when the full-length extracellular domain was expressed as a transmembrane product. The S192I,D193L substitution impaired binding of all three FGF ligands in both exon IIIc and IIIb isoforms (Fig. 5, A and B). The P194L,Q195Q,P196L substitution abrogated the binding of FGF-2 to the FGFR1(IIIc) isoform and the binding of FGF-7 to the FGFR1/R2(IIIb) isoform without effect on FGF-1 binding (Fig. 5, A and B). Analysis of expression of cell surface antigen on the same transfected cells indicated that the lack of FGF binding was not due to inadequate expression of the mutant constructions (Fig. 5 C). In contrast, mutants in which histidine 197 within the fl1 domain and histidine 203 within strand C of FGFR1(IIIc) (Fig. 1) were substituted with phenylalanine bound both FGF-1 and FGF-2 with equal efficiency with no change in the rejection of FGF-7 (Fig. 5 D). Results not shown here indicate that the impact of several of the mutations in the full-length ectodomain described here is not dependent on whether ligand binding was performed on membrane-anchored or detergent-solubilized expression products. These results suggest that, although alterations in specific residues within the fl1 domain of Loop III do not alter the affinity of FGF-2 and FGF-7 for the shared active site core, specific residues within the sequence may be important in the mechanism of how exons IIIb and IIIc impact ligand specificity in the context of the full-length ectodomain. In ascending order, FGF-1, FGF-2, and FGF-7 are sensitive to changes in structural domains downstream of the common base binding site.

Dipeptide Residues in the fl2 and E Domain of Alternate Exon IIIb Indirectly Modify FGF-2 and FGF-7 Binding

The exon IIIb sequence in both the FGFR1 and FGFR2 genes is distinguished from exon IIIc and homologous sequences in the FGFR3 and FGFR4 genes by histidine-serine (HS) residues at the NHterminus of the fl2 domain combined with the absence of lysine-glutamate (KE) residues within the E strand of Loop III (Fig. 1). To determine whether the HS and lack of the KE dipeptide sequences were determinants of the permissive effects of exon IIIb on FGF-7 binding, we substituted the HS residues with TA residues and inserted the KE sequence into the chimeric FGFR1/FGFR2(IIIb) construction, which permits FGF-7 binding (Fig. 3). Both substitutions significantly reduced the binding of FGF-7 without effect on FGF-1 (Fig. 6). However, an apparent increase in FGF-2 binding to the FGFR1/R2IIIb(H313T,S314A) construction indicated that the HS residues at the NHterminus of exon IIIb may have greater impact on the reduction of the affinity for FGF-2 than the absence of the KE residues.


Figure 6: Impact of dipeptide residues in the fl2-E domain of exon IIIb on the binding of FGF-7. FGF binding to the indicated mutants in the sequence of exon IIIb in the FGFR2 portion of the chimeric FGFR1/R2(IIIb) is shown. cDNA constructions coding for the full-length membrane-anchored ectodomain were expressed on the surface of transfected COS-7 cells. HS TA, H313T,S314A.




DISCUSSION

The extracellular domain of the transmembrane tyrosine kinase component of the heparan sulfate-FGF receptor complex is a member of the immunoglobulin supergene family of cell surface recognition proteins (14) . The glycosaminoglycan-FGF-binding site is within two tandem Ig units separated by an interloop sequence, the individual units of which conform to a template based on the well characterized three-dimensional structures of Ig units (8, 21) (Fig. 1). We have previously proposed that clusters of highly conserved sequences that compose the back of Loop II (A-bl1-B, bl2-D, and E-bl3-F domains), the inter-Loop II/III domain, and the B-fl1-C domain on the front of Loop III interact to form the FGF ligand-binding site (21) (Fig. 1). Here we show that the minimal ligand-binding site within the two tandem Ig loops is shared by FGF-1, FGF-2, and FGF-7 and is composed of NH-terminal Loop II, the sequence that connects Loops II and III, and a short segment of the NHterminus of Loop III that ends between lysine residues 176 and 189. The active core of the receptor is precisely the fragment of the ectodomain that is resistant to degradation by trypsin when complexed with heparin or heparan sulfate. This is consistent with the integral and obligatory role of the glycosaminoglycan in formation of the active FGFR complex (5) . The conserved A-bl1-B domain within Loop II has been identified as an obligatory binding site for heparan sulfate glycosaminoglycan (5) .

Although the active core extending through lysine 189 binds FGF-1, FGF-2, and FGF-7, addition of the sequence SDPQP, which composes most of the fl1 domain (SD(P/A)PQPHI) specifically restricted the binding of FGF-7 with little impact on the binding of FGF-1 and FGF-2. Residues SD(P/A)PQP are invariant throughout the four genes and all isoforms of FGFR. Since this is the same ligand binding profile exhibited by exon IIIc variants of FGFR, we conclude that exon IIIc sequence domains are passive and do not affect the ligand binding profile set by subdomains extending through the fl1 sequence. In contrast, exon IIIb actively abrogates the restriction on the binding of FGF-7 concurrent with a reduction in affinity for FGF-2 by an indirect interaction with the fl1 and potentially other sequence domains upstream of it. These conclusions differ significantly from those that suggest that sequence domains encoded in alternately spliced exons coding for the COOH-terminal half of Loop III interact directly with different FGF ligands (26, 27) . In our model of FGFR (Fig. 1), the fl2 domain, which is the NHterminus of alternate exons IIIb and IIIc, is in sufficient proximity to interact with the invariant fl1 domain of Loop III. The fl2 domain of exon IIIb is distinguished from that of exon IIIc by the presence of HS residues instead of (T/A)A residues and the absence of KE residues within the E strand (Fig. 1). Substitution of the HS residues of exon IIIb with the TA residues of exon IIIc and insertion of the KE sequence of exon IIIc indicated that both dipeptide sequence domains in exon IIIb are necessary to permit high affinity FGF-7 binding, while the HS residues appear to have the most impact on the lowered affinity for FGF-2.

In sum, our results suggest that the affinity or specificity of an FGF ligand bound to the active site core (the back face of Loop II, the inter-Loop II/III sequence, the NHterminus of Loop III, and glycosaminoglycan) is determined by how the other side of the ligand interacts with the highly conserved invariant fl1 domain on the front face of Loop III. Variant domains encoded in the COOH-terminal half of Loop III either play no role in ligand binding ( e.g. exon IIIc) or in the case of exon IIIb indirectly interact with constitutive upstream domains to alter affinity for both FGF-2 and FGF-7. The requirement for a precise relationship between sequence domains in the COOH-terminal half of Loop III, the fl1 domain of Loop III, and the core ligand-binding site is consistent with the fact that several mutations of residues within or downstream of the fl1 domain in the membrane-anchored full-length ectodomain interfere with the binding of different FGF ligands to different extents. We reported previously that substitution of the COOH-terminal cysteine 252 with serine in Loop III impaired both FGF-1 and FGF-2 binding in both the FGFR1(IIIc) and FGFR1(IIIc) isoforms (23) . The S192I,D193L mutation in the fl1 domain shown in the current study had a similar effect, while the P194L,Q195Q,P196L substitution interfered with both FGF-2 and FGF-7 binding without effect on FGF-1. X-ray crystallographic analysis of FGF-1 and FGF-2 revealed that FGF exhibits dual structural domains separated by 25 Å on opposite faces of the ligand, which consist of the glycosaminoglycan-binding domain formed by the NH- and COOH-terminal amino acid sequences on one side (Fig. 1 A) and a distinct solvent-accessible loop between -strands 9 and 10 within the core sequence domain on the other side. The latter domain varies among the nine genetically distinct FGF ligands (28, 29, 30) . This is compatible with our model of the FGFR ectodomain, which includes a common 139-residue base FGF-binding site (Loop II, inter-Loop II/III, A-bl1-B domain of Loop III) whose central feature is the Loop II glycosaminoglycan-binding domain A-bl1-B, which anchors the FGF-glycosaminoglycan complex to the receptor (Fig. 1 A). Interaction of the spatially distinct FGF type-specific interstrand 9/10 domain with domains on the front face of Loop III is potentially a distinct second determinant of specificity and affinity of the FGF-FGFR interaction (Fig. 1). Two recent reports that show evidence for two interactive domains on FGF-2 for the heparin-FGFR1 complex (31) and show that the interstrand 9/10 domain appears to be the basis of the low affinity of FGF-2 for the FGFR2(IIIb) ectodomain (32) support this model.

Mounting evidence suggests that the mutually exclusive splicing of exon IIIb in the FGFR2 gene in epithelial cells underlies the normal paracrine control of epithelial cell growth and differentiation by stromal cells, which is mediated by stromal cell-derived FGF-7 (11, 18, 19) . A switch from exclusive expression of exon IIIb to exclusive expression of exon IIIc concurrent with overexpression of FGF-2 and embryonic and oncogenic FGF ligands in epithelial cells correlates with the stromal independence, the lack of differentiation, and the malignancy of tumor-derived epithelial cells (11) . The observations presented here should be of utility in design of antagonists to block interaction of specific FGF ligands with specific FGFR complexes.


FOOTNOTES

*
This work was supported by Public Health Service Grants DK35310 and DK38639 from NIDDKD and Grant CA59971 from NCI, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Howard Hughes Medical Inst., Program in Molecular Medicine, University of Massachusetts Medical Center, 373 Plantation St., Worcester, MA 01605.

To whom correspondence should be addressed: Albert B. Alkek Inst. of Biosciences and Technology, Texas A & M University, 2121 W. Holcombe Blvd., Houston, TX 77030-3303. Tel.: 713-677-7522; Fax: 713-677-7512; E-mail: wmckeeha@ibt.tamu.edu.

The abbreviations used are: FGF, fibroblast growth factor; FGFR, FGF receptor; PCR, polymerase chain reaction; bp, base pair(s); Sf9, S. frugiperda cells; PBS, phosphate-buffered saline (pH 7.0); SFR1, secreted fragment(s) of the FGFR1 extracellular domain.


ACKNOWLEDGEMENTS

We thank Maki Kan, George McBride, and Kerstin McKeehan for excellent technical assistance.


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