©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Alternately Spliced NH-terminal Immunoglobulin-like Loop I in the Ectodomain of the Fibroblast Growth Factor (FGF) Receptor 1 Lowers Affinity for both Heparin and FGF-1(*)

Fen Wang , Mikio Kan , Guochen Yan (§) , Jianming Xu , 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

Alternate splicing of a single exon encoding an NH-terminal immunoglobulin (Ig) disulfide loop in the ectodomain of the fibroblast growth factor receptor (FGFR) types 1 and 2 results in and isoforms that exhibit 3- and 2-Ig loops, respectively. Previously we demonstrated that alternately spliced Loop I has no independent ligand binding activity but is sufficiently interactive with the ligand- and heparin-binding site formed by Loops II and III to lower affinity for the same fibroblast growth factor (FGF) ligand. Here we show that a lower affinity of FGFR1 for heparin parallels the lower affinity for FGF-1. A mutant of FGFR1 in which the sequence between Loops I and II was deleted exhibits high affinity for both FGF-1 and heparin and other properties of the FGFR1 isoform, which include resistance to degradation by trypsin and display of specific antibody epitopes. This suggests that the interloop sequence facilitates the interaction of Loop I with Loops II and III. Lack of expression of both exons coding for Loop I and the sequence between Loops I and II in the FGFR2 gene characterizes rat prostate tumor cells, which exhibit a loss of the low affinity class of FGF receptors. Although the exon coding for the sequence between Loops I and II is alternately spliced in the FGFR2 isoform, coordinate expression with the exon coding for Loop I results in the functional differences between the FGFR and FGFR variants.


INTRODUCTION

Two tandem Ig-like disulfide loops (Loops II and III) connected by an interloop sequence are sufficient for the binding of glycosaminoglycan and ligand to the fibroblast growth factor receptor (FGFR)() kinase ectodomain (1) . Loop II, the interloop sequence, and the NHterminus of Loop III appear to comprise the minimal structure required for the binding of members of the FGF ligand family in general, while both the invariant core and COOH-terminal domains encoded in alternately spliced exons of Loop III determine specificity for particular FGF ligands (1) . The NHterminus of Loop II contributes a conserved heparan sulfate binding domain that is obligatory for ligand binding (2) . Alternate splicing of a single exon in the FGFR1 and FGFR2 genes results in an isoform, FGFR, which exhibits an additional Ig-like loop (Loop I) NH-terminal to Loops II and III of the FGFR isoform (3) . Only the 3-loop products of the FGFR3 and FGFR4 genes have been reported (3) . The NH-terminal Loop I is the least conserved domain among the FGFR isoforms (21-38% relative to the 63-82% conservation between Loops II and III) (3) . In the FGFR isoform, Loops I and II are separated by an interloop sequence encoded by a single exon that exhibits a characteristic cluster of acidic residues called the ``acidic box'' (3) . The sequence composes the NHterminus of the FGFR isoform, which can be alternately spliced (3) . Both the natural variants of FGFR2 and mutants of FGFR1 with a deletion of all or a portion of the exon coding for the acidic box sequence had no qualitative effects on ligand binding (3, 4, 5) . However, the presence of Loop I causes recombinant FGFR1 to exhibit an affinity for FGF-1 that is 12.5% of that of the FGFR1 isoform in the presence of heparin and complete loss of binding to FGF-1 in the absence of the glycosaminoglycan (6) . The fact that distal antibody epitopes within Loop I, the inter-Loop II/III sequence, and the COOH terminus of Loop III are masked in specifically native FGFR1 relative to denatured FGFR1 or FGFR1 suggested that Loop I and Loops II and III are sufficiently interactive to affect both the interaction of glycosaminoglycan and ligand with the 2-loop structure (7) .

Here we report that FGFR1 and FGFR1 exhibit differences in affinity for heparin that parallel the differences in affinity for FGF-1. Deletion of the interloop sequence between Loops I and II caused reversion of multiple properties (affinity for heparin and FGF, lability to trypsin, display of antibody epitopes) of the FGFR1 isoform to those of the FGFR1 isoform despite the presence of the Loop I structure. We show that lack of expression of both exons coding for Loop I and the sequence between Loops I and II in the FGFR2 gene in prostate tumor cells correlates with the loss of low affinity binding sites. Although the interloop sequence is alternately spliced in the FGFR2 isoform, the two exons are expressed coordinately in the FGFR variant, which results in unique properties of the isoform.


EXPERIMENTAL PROCEDURES

Construction of Mutant FGFR1 cDNAs

The following oligonucleotide primer sequences (5` to 3`) were synthesized on an ABI PCRMATE DNA synthesizer (Applied Biosystems, Inc., Foster City, CA): P1a, atatgaattcCGAGCTCACTGTGGAGTATCCATG; ADP1, TTTGGTGTTATCTGTTTCTTTCGAGGAGGGGAGAGCATC; ADP2, AAAGAAACAGATAACACCAAA; P1b, GACCTTGTAGCCTCCAATTCTGTGG; ADP3, TCCGTCAATGTTTCACCCGTAGCTCCATAT; and ADP4, ATATGGAGCTACGGGTGAAACATTGACGGA. Residues not in human FGFR1 cDNA are indicated in lowercase, and restriction sites are indicated in boldface. Mutant cDNA fragments for construction of FGFR1(A) (see Fig. 1) were generated by previously described methods (1, 2, 8) between primers P1a and ADP1 and between primers ADP2 and P1b in the polymerase chain reaction (PCR) using FGFR1 cDNA as a template. PCR reactions were performed as described (1) . Mutant fragments for construction of FGFR1(AE) were generated between P1a and ADP3 and between ADP4 and P1b. After digestion with BglI and BstXI, the mutant cDNAs were ligated with flanking FGFR1 cDNA sequences at BglI and BstXI sites and cloned into pBlueScript SK vector (Stratagene, La Jolla, CA) at EcoRI and SalI sites. The resulting 1222- and 1273-base pair (bp) cDNAs were recovered by partial digestion with EcoRI and cloned into mammalian expression vectors p91023B and the baculovirus transfer vector pVL1393 at EcoRI sites. Recombinant baculoviruses bearing mutant FGFR1 cDNAs were prepared as described (1, 7) . The nucleotide sequences of all cDNA fragments generated by the PCR and the sequence across ligation sites in all cDNA constructions were determined.


Figure 1: Schematic diagram of the FGFR extracellular domain. The three disulfide Ig loops (I, II, and III), primer positions, and restriction enzyme sites described under ``Experimental Procedures'' are indicated. Dotted lines indicate sequence deletions, and zigzag lines indicate vector sequence. The translational initiation site and secretory signal sequence ( S), the acidic box ( A), the putative heparin binding domain ( HB), and the transmembrane domain ( TM) are indicated by solid rectangles. The Loop I and interloop exons are indicated by arrows. Inset, covalent cross-linking of labeled FGF-1 to wild-type ( , ) and mutant constructions of the FGFR1 ectodomain expressed in COS-7 cells. Each analysis is from an assay that contained 5 10cells.



Binding of Heparin and FGF-1 to Recombinant FGFR

I-Labeled FGF-1 binding and covalent affinity cross-linking were performed in transfected COS-7 cells and baculovirus-infected Sf9 cells as described (1) . Scatchard analysis was carried out by addition of various concentrations of I-labeled FGF-1 and 2 µg/ml heparin. Specific binding was calculated by subtraction of the amount of labeled FGF-1 bound in the presence of a 50-fold excess of unlabeled FGF-1. For heparin binding, Sf9 insect cells were infected with recombinant baculovirus for 2 days and then harvested and distributed into 1.7-ml Eppendorf tubes at 5 10cells/tube by modification of procedures described for FGF-1 binding (6) . Concentrations (0.7-8 µg/ml) of [H]heparin ( M= 6000-20,000; 0.7 Ci/mg, DuPont) were added to each sample in 0.5 ml of binding buffer. After incubation for 4 h at 4 °C, the cells were collected in 96-well filtration plates (Millipore, Bedford, MA) and washed three times with phosphate-buffered saline, pH 7 (PBS). The membrane from each well was excised, and radioactivity was determined by liquid scintillation. Specific binding was determined by the difference between total binding and nonspecific binding remaining in the presence of a 100-fold excess of unlabeled heparin or the amount of [H]heparin bound to an equivalent amount of cells infected with an unrelated recombinant virus at the same titer.

Tryptic Fragmentation of Recombinant FGFR1

Complexes of recombinant FGFR1 constructions and heparin were fragmented with trypsin by modification of a previously described procedure (1, 2) . Sf9 cells infected with baculoviruses bearing recombinant FGFR1 cDNAs were lysed with 0.2% Triton X-100 in PBS, and receptor products were then immobilized by incubation of the cell lysates with heparin-agarose beads (1 ml of packed beads/10cells) for 4 h at 4 °C. After washing with PBS, half of the complex was retained as an untreated control, and the other half was treated with 100 µg/ml trypsin in PBS at 4 °C for 1 h. After a wash with PBS containing 2 mM p-methylsulfonyl fluoride, material retained on the beads was eluted with 1 M NaCl in PBS, concentrated, and subjected to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot (1, 7) .

Analysis of FGFR2 Isoforms in Rat Prostate Tissues by PCR

Variants of the ectodomain of FGFR2 were analyzed by paired 5` and 3` primers PB1 (5`-gccgaatTCAAGTGTGCAGATGGGATTAACG) and CB (5`-AGGATGGGCCGGTGCGGTGATCGCTC). Primer PB1 began 67 bp upstream of the translational initiation site, and CB was complementary to an invariant sequence within Loop II (see Fig. 1). A 5` primer PB9 (5`-AGCCACCAACCAAATACCAA) complementary to a coding sequence within Loop I of FGFR2 was paired with the CB primer to detect specifically the FGFR isoform. Template cDNAs were prepared, and the PCR was performed as described previously (9, 10) .


RESULTS

Impact of Loop I and the Inter-Loop I/II Domains on FGF-1 Binding

Previous studies indicated that deletion of the 39-bp coding sequence for the 13 core residues (EDDDDDDDSSSEE) that compose the acidic box (denoted as the A domain in Fig. 1) in the NHterminus of FGFR1 (5) or splice variants of the FGFR2 gene that do not exhibit the entire exon (acidic exon designated AE in Fig. 1) containing the acidic residues (3, 4) had no effect on the binding of FGF-1 to recombinant products expressed on the surface of transfected monkey kidney COS cells. To test whether the two deletions affected ligand binding to the 3-loop FGFR1 isoform, we constructed mutants FGFR1(A) and FGFR1(AE) with deletions of the 13 residues (Glu-Glu) of the acidic box and the whole exon coding for the interloop sequence (Asp-Met), respectively. Neither deletion had a notable qualitative effect on the amount of FGF-1 covalently cross-linked to recombinant product in assays containing heparin and I-FGF-1 at 120 pM (Fig. 1, inset). We previously reported that although both isoforms bind FGF-1 when expressed on the surface of baculovirus-infected Sf9 cells, the affinity of FGFR1 for FGF-1 was 12.5% that of FGFR1 in the presence of heparin (6) . Fig. 2 demonstrates that mammalian COS cells transfected with the FGFR1 ectodomain also exhibit a lower affinity for FGF-1 than those transfected with FGFR1. The FGFR1(A) construction, which exhibits a deletion of only the 13-residue acidic box between Loops I and II, retained the lower affinity exhibited by wild-type FGFR1; however, deletion of the entire interloop sequence resulted in a higher affinity similar to two-loop FGFR1 despite the presence of Loop I directly at the NHterminus of Loop II (Fig. 2).


Figure 2: Scatchard analysis of I-FGF-1 binding to variant FGFR1 ectodomains. Specific binding of different concentrations of I-FGF-1 to COS-7 cells transfected with the indicated cDNA constructions was performed as described under ``Experimental Procedures.'' The values are means of duplicate samples. B, bound ligand; F, free ligand.



Impact of Loop I and the Interloop Domains on Interaction with Heparin

When expressed on the surface of baculovirus-infected Sf9 cells, FGFR1 failed to bind FGF-1 at all in the absence of heparin relative to the FGFR1 isoform, which continues to bind FGF-1 with a reduced affinity (6) . Both deletions in the FGFR1(A) and FGFR1(AE) constructions relieved the absolute requirement for exogenous heparin for the binding of FGF-1 to wild-type FGFR1 on the surface of Sf9 cells (Fig. 3). Since heparan sulfate glycosaminoglycans bind directly to the FGFR ectodomain in the absence of FGF (2) , we designed a radioreceptor assay to quantitate the binding of [H]heparin to recombinant FGFR isoforms expressed on the surface of infected Sf9 cells. Binding was saturable and in the presence of unlabeled heparin reversible to a plateau level exhibited by cells infected with virus bearing an unrelated cDNA. Parallel to the differences in affinities for FGF-1, FGFR1 exhibited a lower affinity for the glycosaminoglycan than FGFR1. Absence of the 13-residue acidic box in FGFR1(A) did not lower affinity for heparin, while deletion of the entire interloop sequence increased the affinity to that of FGFR1 (Fig. 4). Solubilized ectodomains immobilized either on antibody-agarose beads or constructions bearing a fusion partner (glutathione S-transferase) at the COOH terminus immobilized on GSH-agarose exhibited similar differences (not shown).


Figure 3: Requirement for heparin for the binding of FGF-1 to variant FGFR1 ectodomains. Covalent affinity cross-linking of I-FGF-1 to Sf9 cells (10) infected with recombinant baculovirus bearing cDNA coding for the indicated variant ectodomains of FGFR1 from binding assays performed in the absence () or presence (+) of 2 µg/ml heparin was analyzed by SDS-polyacrylamide gel electrophoresis as described under ``Experimental Procedures.''




Figure 4: Scatchard analysis of [H]heparin binding to variant ectodomains of FGFR1. Specific binding of various concentrations of [H]heparin in the presence or absence of a 100-fold excess of unlabeled heparin to Sf9 cells expressing the indicated cDNA constructions was determined. For example, in a typical binding assay, 10cells infected with FGFR1 bound 271,540 dpm of labeled heparin, and the same number of cells infected with virus bearing an unrelated cDNA bound 64,859 dpm. Addition of excess unlabeled heparin to FGFR1-infected cells reduced the binding to near that exhibited by uninfected or mock-infected cells. The values are the mean of duplicate samples. B, bound ligand; F, free ligand.



The interaction of the Sf9 cell-derived recombinant FGFR ectodomain with heparin protects an active 30-kDa core fragment against proteolysis by trypsin (2) (Fig. 5). In contrast, heparin failed to protect the FGFR1 isoform from proteolysis. The FGFR1(A) product retained the properties of intact FGFR1, while the FGFR1(AE) product exhibited properties of FGFR1 despite the presence of Loop I.


Figure 5: Differential protection of variant ectodomains of FGFR1 against tryptic degradation in the presence of heparin. The indicated variants of the FGFR1 ectodomain were absorbed on heparin-agarose beads from lysates of baculovirus-infected 10Sf9 cells. Equal portions of the beads were then incubated in the absence ( top) and presence ( bottom) of trypsin as described under ``Experimental Procedures.'' Eluates of the beads were then analyzed by SDS-polyacrylamide gel electrophoresis and immunoblot.



Impact of Loop I and the Interloop Domains on Reaction of FGFR1 with Domain-specific Antibodies

Specific antibody epitopes within defined sequence domains in Loop I and the two loops of FGFR1 are masked by the native conformation of the FGFR1 isoform (7) . To determine whether the inter-Loop I/II sequence affected the display of epitopes within native FGFR1, we tested the reaction of the mutant construction products with monoclonal M5G10, which reacts with an epitope within the sequence spanning the COOH terminus of Loop II, the inter-Loop II/III sequence, and the NHterminus of Loop III (7) . As reported previously (7) , M5G10 fails to react with native FGFR1 but reacts with denatured FGFR1 (Fig. 2) and FGFR1 (Fig. 6). Deletion of the 13 residues of the acidic box and the entire interloop exon sequence in the mutant FGFR1(A) and the FGFR1(AE) constructions, respectively, restores reactivity with antibody M5G10 (Fig. 6).


Figure 6: Differential reactivity of variant native ectodomains with monoclonal antibody M5G10. The indicated native recombinant ectodomains from lysates of baculovirus-infected Sf9 cells were reacted with anti-FGFR1 mouse monoclonal antibody M5G10 and rabbit polyclonal antiserum (Ab) A50 immobilized to protein A beads as described (7). Extracts of immunocomplexes of both antibodies were subjected to SDS-polyacrylamide gel electrophoresis (denaturing conditions) and analyzed by immunoblot using antibody M5G10 followed by reaction with rabbit anti-mouse IgG conjugated to alkaline phosphatase.



Lack of Expression of FGFR2 Isoforms in Rat Prostate Tumors

A distinctive difference between isolated epithelial cells from normal rat prostate and transplantable rat prostate tumors is the loss of the low affinity class of FGF receptor sites in the tumor cells (11) . To determine whether the difference reflected loss of expression of the isoform of the FGFR2 gene, which is selectively expressed in the epithelial cells relative to the FGFR1 gene (9) , mRNAs coding for isoforms of the FGFR2 ectodomain were analyzed by PCR in normal prostate; a slow growing, well differentiated, androgen-responsive tumor (Dunning R3327PAP tumor); and a malignant variant that arises from the latter after castration of male hosts (Dunning R3327AT3 tumor) (9) . Normal prostate tissue exhibits three mRNA species characteristic of FGFR2 at 849 bp, FGFR2 at 585 bp, and FGFR2(AE) at 504 bp, while both types of tumors exhibited predominantly the FGFR2(AE) isoform (Fig. 7 A). The FGFR2 isoform exhibiting the NH-terminal AE sequence is detectable in Dunning R3327PAP tumors but undetectable in the Dunning R3327AT3 tumors. Analysis of the cDNA template from normal prostate tissue with a primer complementary to specific coding sequences for Loop I of FGFR2 revealed a single 664-bp band characteristic of the FGFR2 isoform containing the interloop exon coding sequence AE (Fig. 7 B). No band at 583 bp indicative of the FGFR2(AE) variant could be detected.


Figure 7: Lack of expression of FGFR2 in rat prostate tumors. A, paired primers for invariant coding sequences of the FGFR2 ectodomain that span the exons coding for Loop I and the inter-Loop I/II sequence (Fig. 1) were employed in the PCR with cDNA templates from the indicated rat prostate tissues. AT3, fast growing, undifferentiated malignant Dunning R3327AT3 tumor; DT, slow growing, nonmalignant, differentiated Dunning R3327PAP tumor; NP, normal rat prostate tissue. B, a Loop I-specific 5` primer was employed together with the same 3` primer (CB) used in panel A. NP, normal prostate; , FGFR2 cDNA; -, no cDNA template. Lanes at the right of panels A and B are standards.




DISCUSSION

In the accompanying paper (1) , we demonstrated that the base ligand-binding site of the FGFR kinase ectodomain that is shared by FGF-1, FGF-2, and FGF-7 consists of one Ig-like disulfide loop (Loop II), which contains a heparan sulfate binding domain, a short interloop sequence that connects Loop II and Loop III, and a part of the NHterminus of Loop III extending through lysine 189. A constitutive structural domain just downstream of lysine 189 within Loop III and mutually exclusive alternately spliced exons coding for the COOH-terminal half of Loop III interact to determine the affinity for FGF-2 and whether FGF-7 binds at all. In addition to tandem Loops II and III, which compose the FGFR isoform, alternate splicing of a single exon in the FGFR1 and FGFR2 genes results in an isoform (FGFR) containing an NH-terminal Ig-like loop (Loop I) that is connected to Loop II by an interloop sequence that is also encoded by a single exon. In previous reports, we have proposed that although Loop I does not directly participate in formation of a ligand-binding site, it is sufficiently interactive with the base-binding site formed by structural domains within Loops II and III to alter affinity for the same FGF ligand and to influence the nature of the interaction of the base-binding site with heparin (6, 7) . In this report, we show by Scatchard analysis that FGFR1 exhibits a lower affinity for heparin that parallels its lower affinity for FGF. This and the fact that heparin fails to protect the active binding site from degradation by trypsin in the FGFR1 isoform confirmed that the nature of the interaction of the glycosaminoglycan with FGFR1 is quite different from that of FGFR1 and may underlie the differences in affinity between the two splice variants for the same ligand.

Molecular models suggest that the interloop exon between Loops I and II may serve as a hinge that facilitates the interaction of Loop I with Loops II and III in the FGFR1 isoform (7) . Here we show that a mutant of FGFR1 (FGFR1(AE)) in which the inter-Loop I/II sequence was deleted exhibits properties of FGFR1 despite the presence of Loop I immediately adjacent to the NHterminus of Loop II. This included high affinity binding to both FGF and heparin, loss of the absolute requirement for heparin for FGF-1 binding, resistance to tryptic degradation in the presence of heparin, and display of antibody epitopes. Deletion of only the 13 residues that compose what is commonly referred to as the acidic box within the interloop sequence was insufficient to abrogate the low affinity of the FGFR1 isoform for FGF or heparin or the resistance of the active core of the ligand-binding site to trypsin in the presence of heparin. This suggests that the remaining 17 residues of the 30-residue interloop sequence are sufficient to facilitate the functional impact of Loop I with the active ligand- and heparin-binding site. It is noteworthy that deletion of the 13-residue sequence in FGFR1 was sufficient to expose a monoclonal antibody epitope within Loops II and III that is normally hidden and relieved the qualitative requirement for heparin for FGF-1 binding to the FGFR1 variant despite the fact that the deletion had no impact on the Kfor heparin and FGF-1.

The model presented here in which the major glycosaminoglycan binding domain of FGFR enjoys greater exposure to nearby co-factors explains the lack of requirement of FGFR relative to FGFR for exogenous heparin when expressed on the surface of baculovirus-infected Sf9 insect cells, which express unidentified mimetic co-factors for the FGFR ectodomain (6) but do not express the type and amounts of long chain heparan sulfate glycosaminoglycans comparable with mammalian cells.() The impact of the differences between the FGFR and FGFR ectodomains in requirement for heparan sulfates in a physiological context remains to be established. Although differences in the binding of FGF-1 and FGF-2 to FGFR1 and FGFR1 isoforms are quantitative when heparin is used as the glycosaminoglycan in the FGFR ternary complex, the presence of Loop I may restrict the range of natural heparan sulfate co-factors that will support the ternary FGFR complex with a particular FGF ligand. Regulation of the ratio of expression of FGFR and FGFR at the level of alternate splicing has been reported. In well differentiated human hepatoma cells that are growth-inhibited by high concentrations of FGF, expression of the isoform of the FGFR1 gene exceeds that of the variant, and only the ectodomain is combined with a kinase-defective intracellular domain, which also arises by an alternate splice event (6) . A selective reduction in expression of the FGFR1 variant correlates with malignancy of human pancreatic (12) and brain (13) tumors. Here we showed that lack of expression of the splice variant of the FGFR2 variant characterizes transplantable rat prostate tumors relative to normal prostate tissue. This correlates with the loss of low affinity receptors on cells derived from the tumors relative to normal epithelial cells (11) . The FGFR2 gene is specifically expressed in the epithelial cells from normal prostate and well differentiated prostate tumors that exhibit both epithelial and stromal compartments (9) . Alternate splicing of exons coding for the COOH terminus of Loop III of FGFR2 determine whether the epithelial cells respond to stromal cell-derived FGF-7 or to other FGF ligands that are abnormally activated in malignant tumors (9) . Last, we showed that, in normal prostate tissue that expresses the FGFR2 isoform, the single exons coding for Loop I and the inter-Loop I/II sequence are coordinately expressed, although the interloop exon is alternately spliced in the FGFR2 variant (3, 4) . This is consistent with the failure to demonstrate the AE variant of FGFR1 and FGFR2 in a variety of tissues (3, 13) () and the results of this study that indicate that the splice variant would be functionally redundant in respect to the FGFR isoform.


FOOTNOTES

*
This work was supported by NIDDKD Public Health Service Grants DK35310 and DK38639 and NCI Grant CA59971, 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: FGFR, fibroblast growth factor receptor; FGF, fibroblast growth factor; PCR, polymerase chain reaction; bp, base pair(s); PBS, phosphate-buffered saline (pH 7.0).

M. Kan and R. Owens, unpublished results.

M. Kan and W. L. McKeehan, unpublished results.


ACKNOWLEDGEMENTS

We thank Maki Kan, Kerstin McKeehan, and Suzanne Tran for excellent technical assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.