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. 1998 Jul;18(7):3762–3770. doi: 10.1128/mcb.18.7.3762

Identification of the Cytoplasmic Regions of Fibroblast Growth Factor (FGF) Receptor 1 Which Play Important Roles in Induction of Neurite Outgrowth in PC12 Cells by FGF-1

Hsien-Yi Lin 1,†, Jingsong Xu 2, Irene Ischenko 3, David M Ornitz 2, Simon Halegoua 4, Michael J Hayman 3,*
PMCID: PMC108959  PMID: 9632759

Abstract

Fibroblast growth factor 1 (FGF-1) induces neurite outgrowth in PC12 cells. Recently, we have shown that the FGF receptor 1 (FGFR-1) is much more potent than FGFR-3 in induction of neurite outgrowth. To identify the cytoplasmic regions of FGFR-1 that are responsible for the induction of neurite outgrowth in PC12 cells, we took advantage of this difference and prepared receptor chimeras containing different regions of the FGFR-1 introduced into the FGFR-3 protein. The chimeric receptors were introduced into FGF-nonresponsive variant PC12 cells (fnr-PC12 cells), and their ability to mediate FGF-stimulated neurite outgrowth of the cells was assessed. The juxtamembrane (JM) and carboxy-terminal (COOH) regions of FGFR-1 were identified as conferring robust and moderate abilities, respectively, for induction of neurite outgrowth to FGFR-3. Analysis of FGF-stimulated activation of signal transduction revealed that the JM region of FGFR-1 conferred strong and sustained tyrosine phosphorylation of several cellular proteins and activation of MAP kinase. The SNT/FRS2 protein was demonstrated to be one of the cellular substrates preferentially phosphorylated by chimeras containing the JM domain of FGFR-1. SNT/FRS2 links FGF signaling to the MAP kinase pathway. Thus, the ability of FGFR-1 JM domain chimeras to induce strong sustained phosphorylation of this protein would explain the ability of these chimeras to activate MAP kinase and hence neurite outgrowth. The role of the COOH region of FGFR-1 in induction of neurite outgrowth involved the tyrosine residue at amino acid position 764, a site required for phospholipase C gamma binding and activation, whereas the JM region functioned primarily through a non-phosphotyrosine-dependent mechanism. In contrast, assessment of the chimeras in the pre-B lymphoid cell line BaF3 for FGF-1-induced mitogenesis revealed that the JM region did not play a role in this cell type. These data indicate that FGFR signaling can be regulated at the level of intracellular interactions and that signaling pathways for neurite outgrowth and mitogenesis use different regions of the FGFR.

The fibroblast growth factor (FGF) family currently comprises 14 members, FGF-1 through -14 (6). These growth factors play roles in development, angiogenesis, wound healing, and tumorigenesis (for reviews, see references 1 and 20). FGF actions are mediated by the binding and activation of FGF receptor (FGFR) tyrosine kinases. FGFRs are a gene family of four members, termed FGFR-1 to -4. These receptors are widely expressed in many tissues and different cell types, and the temporal expression of the receptors and ligands is regulated during development (reviewed in reference 17).

Signals by FGFRs appear to control differentiation as well as proliferation. Mutations in these receptors have indicated that they may control the differentiation of specific cell types during development. Point mutations of the genes encoding human FGFR-1, -2, or -3 cause different syndromes that involve bone development (reviewed in references 24 and 47), and some of these syndromes (Apert syndrome and thanatophoric dysplasia) may also manifest effects in the central nervous system. In particular, point mutations that activate FGFR-3 (25, 46) cause dwarfism such as achondroplasia (34, 36), hypochondroplasia (2, 32), or thanatophoric dysplasia (39, 40). Studies analyzing the consequences of null mutations in FGFRs in mice also implicated these receptors as playing a role in development. The knockouts of FGFR-1 or FGFR-2 (9, 48) in mice result in embryonic lethality, whereas the knockout of FGFR-3 in mice was nonlethal. The FGFR-3-deficient mice developed an overgrowth of the long bones and an abnormal curvature of the spine and tail (5, 8) and were deaf (5).

The FGFRs are very similar in structure. In particular, their tyrosine kinase domains are highly conserved and they can be activated by overlapping subsets of ligands (17, 29). Presently, it is not clear how the specificity of signal transduction is achieved. Regulation can take place at two different levels. For example, the temporal control of the expression of both ligands and receptors undoubtedly is an important mechanism for regulating signal transduction during development. However, in addition, the receptors seem to have differing signaling capabilities. Studies have indicated that FGFR-1 is much better at producing mitogenic signals than FGFR-3 and FGFR-4 when assayed in BaF3 cells (3, 29, 44). In addition, we have demonstrated previously that there is a difference between FGFR-1 and -3 in their abilities to induce neurite outgrowth in PC12 cells when activated by FGF-1 (19). FGFR-3 can barely induce neurite outgrowth, whereas activation of FGFR-1 induces rapid and robust neurite outgrowth. These studies were made possible by the use of a PC12 variant, the fnr-PC12 cell line, that is unable to respond to FGF-1 because it does not express functional FGFR-1. When this cell line is transfected with FGFR-1, the ability of FGF-1 to induce neurite outgrowth is restored. By using chimeras of these two receptors, we were able to map the difference in intracellular signaling to the transmembrane and cytoplasmic region.

In this report, we have extended these observations to identify more specifically the regions of FGFR-1 that are responsible for the induction of neurites. By utilizing chimeras containing various regions of the FGFR-1 cytoplasmic domain exchanged with the FGFR-3 cytoplasmic domain, two regions were identified as playing a role. The cytoplasmic carboxy-terminal region was able to induce a moderate level of neurite outgrowth. However, introduction of amino acids 399 to 508 of FGFR-1, which contain the juxtamembrane (JM) region, was shown to be sufficient to confer robust neurite outgrowth to FGFR-3. Analysis of intracellular signaling by the different chimeras indicated that the carboxy-terminal tyrosine at amino acid 764 was responsible for inducing phospholipase Cγ (PLCγ) activation, whereas the JM region of FGFR-1 was capable of converting the normal transient nature of FGFR-3 signaling into the more sustained signaling associated with FGFR-1 and this potentially was mediated via SNT/FRS2 phosphorylation. However, analysis of the JM-containing chimera in BaF3 cells demonstrated that this region was not important for the induction of mitogenesis in this cell type. This indicates that there are cell type-specific intracellular interactions that can play a role in controlling FGF signaling.

MATERIALS AND METHODS

Construction of chimeric receptors.

The chimeric molecules shown in Fig. 1 were assembled with restriction fragments, PCR-generated fragments, and standard molecular cloning techniques. The FGFR31 clone was described previously (25). The chimeric receptors FGFR31-Q through -Z (Fig. 2) were constructed as follows. The SalI site at the carboxyl end of the transmembrane domain (position A in Fig. 1 and 2) was introduced into the FGFR-3 coding sequence with silent point mutations as described previously (3, 25). Oligonucleotide primers overlapping this SalI site were used to amplify downstream sequences for either FGFR-1 or FGFR-3. Primer DO130 amplifies sequences from FGFR-3, and primer JX21 amplifies sequences from FGFR-1 and changes three amino acid residues (see below). FGFR31-N and -M were derived from FGFR31-Q and FGFR-3, respectively, by exchanging carboxy-terminal restriction fragments at the common BglII site. Chimeric junctions B, C, and D were made by using PCR primers that overlap the junction. In some cases, short PCR fragments themselves were used as primers when the junction between FGFR-1 and FGFR-3 contained identical sequences.

FIG. 1.

FIG. 1

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Amino acid sequence alignment between the intracellular domains of FGFR-1 and -3. Subdomains of the FGFR intracellular region are labeled and demarcated. TM, transmembrane domain; JM, juxtamembrane domain; TK, tyrosine kinase domain; KI, kinase insert domain; CT, carboxy-terminal domain. Sites where chimeric molecules were fused together are indicated with letters A to E. A single asterisk indicates the tyrosine residue (Y461) in the FGFR-1 JM domain that was mutated to a phenylalanine residue (construct FGFR31b-RF). Double asterisks identify the tyrosine residue (Y764; PLCγ binding site [residue 764 in mice is equivalent to residue 766 in humans]) in the FGFR-1 carboxy-terminal domain that was changed to a phenylalanine residue (construct FGFR31b-MF). Residues crl, indicated above the FGFR-1 sequence at position A, replaced the corresponding residues in FGFR-1 when the SalI restriction site was introduced into the FGFR-1 coding sequence.

FIG. 2.

FIG. 2

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Schematic diagram of the structural organization of the FGFR-3/FGFR-1 chimeric receptors. ECD, extracellular domain; TM, transmembrane domain; JM, juxtamembrane domain; TK, tyrosine kinase domain; KI, kinase insert domain; CT, carboxy-terminal domain. Symbols: open bars, sequences derived from FGFR-3; hatched bars, sequences derived from FGFR-1. Sites A to E indicate locations of chimeric junctions and correspond to the same sites indicated in Fig. 1. Arrows indicate the locations of oligonucleotide primers used to synthesize chimeric receptor fragments.

In general, for each chimeric construct, FGFR-1 and -3 sequences were generated by PCR separately and then, using these PCR products as either templates or primers, a second-step PCR was performed to piece together individual fragments with a SalI site at the 5′ end and a BglII site at the 3′ end. All chimeric molecules were verified by sequencing. PCR-generated chimeric fragments were then cloned between the SalI site (position A) and the BglII site (position E), common to both receptors 1 and 3 (Fig. 2). The primers used to generate junctions A through E, shown in Fig. 2, are as follows (uppercase letters are nucleotides derived from FGFR-3, lowercase letters are nucleotides derived from FGFR-1, letters in italic type represent nucleotides that have been added or changed, and letters in boldface type represent nucleotides held in common by FGFR-1 and FGFR-3; SalI and BglII sites are underlined): for junction A, primers DO130 (5′-GTGATACTCTGTCGACTGC-3′ [SalI site underlined]) and JX21 (5′-catctgtcgactgaagagcggcaccaa-3′); for junction B, primers JX24 (5′-TCAGcatcttcacggccacttt-3′) and JX30 (5′-CCgtggccgtgaagatgttgaagtcc-3′); for junction C, primer JX23 (5′-ggcGcggCggcctcctgggctgga-3′); for junction D, primer JX22 (5′-CACAGGACACTAGATCCTTggaagaca-3′); for junction E, primer DO48 (5′-CCCAGAGTAAAGATCTCCCA-3′ [BglII site is underlined]).

Mitogenic assays.

Suspension cultures of BaF3 cells (21, 30) were maintained in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% neonatal bovine serum (NBS), 10% conditioned medium from WEHI-3 cells, l-glutamine, penicillin-streptomycin, and β-mercaptoethanol. To express chimeric FGFRs in BaF3 cells, 106 cells were electroporated with 20 μg of ClaI-linearized MIRB-FGFR as previously described (12, 27, 28). Cells were selected in medium containing 600 μg of G418 (Gibco BRL Inc.) per ml and 10% WEHI-3 conditioned medium for 10 to 12 days. Pools of transfected cells were assayed for mitogenic responsiveness to FGF-1 and then subcloned by limiting dilution.

For mitogenic assays, pools of BaF3 cells expressing specific FGFRs were washed and resuspended in RPMI 1640 medium–10% NBS–l-glutamine. A total of 22,500 cells were plated per well in a 96-well assay plate in medium containing 2 μg of heparin per ml. FGF-1, diluted in medium containing 2 μg of heparin per ml, was added to each well for a total volume of 200 μl per well. The cells were then incubated at 37°C for 36 to 48 h. To each well, 1 μCi of [3H]thymidine was added to 50 μl of medium. Cells were harvested after 4 to 5 h by filtration through glass fiber paper. Incorporated [3H]thymidine was counted on a Wallac β plate scintillation counter. Cell surface receptor cross-linking studies were done essentially as described previously (44, 49).

To measure fos activation in response to FGF stimulation, cells were starved in RPMI 1640 medium containing 1% fetal calf serum for 10 h and then stimulated with 100 ng of FGF-1 per ml plus 25 μg of heparin per ml for 0, 15, 30, or 60 min. Total RNA was prepared by lysing cells in guanidine HCl followed by centrifugation through CsCl2 (4). A Northern blot using 10 μg of RNA was probed with a 2.2-kb c-fos cDNA probe (43).

Cross-linking of 125I-FGF-1 to FGFR was performed as previously described (49).

Cell culture, transfection, and assay of neurite outgrowth.

fnr-PC12 cells have been described previously (19). fnr-PC12-derived transfectant lines were grown on tissue culture dishes in Dulbecco modified Eagle medium (DMEM; Life Technologies) supplemented with 10% donor horse serum (JRH Biosciences, Lenexa, Kans.), 5% FBS (JRH Biosciences), and 1% penicillin-streptomycin (Life Technologies) in an atmosphere of 10% CO2 at 37°C.

Transfection of the expression vectors to fnr-PC12 cells was performed as described previously (7). The transfection was carried out by electroporation with a Gene Pulser (Bio-Rad, Richmond, Calif.). Approximately 4 × 106 cells were electroporated in 0.4 ml of DMEM containing 20 μg of the expression vectors encoding the FGFRs described in this report, with settings of 250 V and 500 μF. Two days after transfection, the cells were incubated in 800 μg of Geneticin (Life Technologies). After ∼3 weeks, Geneticin-resistant clones were isolated and then screened for FGF-1-induced neurite outgrowth and the level of expression of the transfected receptors. Recombinant human FGF-1 (16) was kindly provided by M. Jaye (Rhône-Poulenc Rorer Central Research). β-Nerve growth factor (NGF) was purified from mouse submaxillary glands (22).

To assay for neurite outgrowth, cells were grown on tissue-culture plastic dishes coated with a solution of polylysine (25 μg/ml; Sigma, St. Louis, Mo.) and laminin (10 μg/ml; Collaborative Research). The cells were plated at ∼104 cells/35-mm-diameter dish and treated with 50 ng of FGF-1 per ml (plus 5 U of heparin [Upjohn, Kalamazoo, Mich.] per ml). Cells were scored positive for neurites if growth cone-containing neurites with a length of at least two cell body diameters were observed. Photographs of the cells were taken with a Nikon phase-contrast ELWD 0.3 microscope with Kodak technical pan film.

Immunoprecipitation and Western blotting.

For immunoprecipitation, cells were lysed in lysis buffer (20 mM Tris [pH 7.6], 150 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 5 mM benzamidine, 1 mM EDTA, 10 μg of leupeptin per ml, 10 μg of aprotinin per ml, 1 mM phenylmethylsulfonyl fluoride, 1% Nonidet P-40). The lysates were clarified by the addition of protein A-Sepharose 4B (Zymed, South San Francisco, Calif.) and centrifugation. The protein concentrations in the supernatant were determined by the bicinchoninic acid method (Pierce, Rockford, Ill.). Aliquots of the supernatant (1 mg) of each sample were incubated with polyclonal antisera for 1 h on ice. Protein A-Sepharose 4B was added for an additional 0.5 h. Pellets were collected by centrifugation, washed, boiled in sample buffer, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blotting was performed as described below.

For Western blotting, (i) to detect FGF-1-stimulated tyrosine phosphorylation of cellular proteins, cells were treated with 50 ng of FGF-1 per ml at 37°C for the time indicated in the text. Aliquots of the lysates (75 μg) of each sample were fractionated on an SDS–7.5% polyacrylamide gel. Western blotting was performed, and membranes were probed with a solution consisting of the antiphosphotyrosine monoclonal antibody 4G10 at a concentration of 1 μg/ml in blocking buffer. (ii) To detect FGF-1-stimulated phosphorylation of MAP kinase, cells were treated with 50 ng of FGF-1 per ml at 37°C for the time indicated in the text. Aliquots of the lysates (75 μg) of each sample were fractionated on an SDS–12.5% polyacrylamide gel. Western blotting was performed, and membranes were probed with a solution consisting of an anti-ERK2 monoclonal antibody in blocking buffer. Phosphorylation of the proteins was determined by mobility shift (19). Phosphorylation of SNT/FRS2 was monitored by using gst-suc1 agarose (UBI, Lake Placid, N.Y.) as described in the manufacturer’s instructions followed by Western blotting with antiphosphotyrosine antibodies.

RESULTS

Identification of the JM and carboxy-terminal regions of FGFR-1 as playing a role in the induction of neurite outgrowth in PC12 cells.

Our previous comparison of signaling by FGFR-1 and -3 had indicated that the initial kinase activations of these two receptors by FGF-1 were identical (19). However, the tyrosine phosphorylation signal induced by FGFR-1 was sustained whereas the FGFR-3 signal was relatively transient in nature. This difference mapped to the cytoplasmic domains of these two receptors. To extend our analysis of the different signaling capabilities of the cytoplasmic domains of FGFR-1 and -3, we prepared various chimeras in which different regions of the FGFR-1 transmembrane and cytoplasmic domains were swapped into the FGFR-3 protein as described in Materials and Methods. The structures of the initial six chimeras tested (FGFR31b-M, -N, -Q, -R, -X, -Y, and -Z) and the specific amino acids involved are shown in Fig. 1 and 2. These chimeric receptors were then introduced into the FGF-nonresponsive PC12 cells, fnr-PC12 cells, and stable cell lines that expressed similar levels of receptors as judged by Western blot analysis were selected (Fig. 3). The different cell lines were then assessed for their ability to induce neurite outgrowth in response to FGF-1. Based on our previous analysis, the difference between FGFR-1 and FGFR-3 is best demonstrated 1 to 2 days after the addition of FGF-1, at which time FGFR-1 typically induces neurites in greater than 40% of the cells whereas FGFR-3 induces neurites in only 5 to 10% of the cells (19). Figure 4 shows a quantitation of the analysis of a typical experiment comparing the different chimeras. Two days after treatment with FGF-1, the cells expressing FGFR-3 or the chimeras FGFR31b-Y and FGFR31b-Z had less than 7% neurite-bearing cells. The cells expressing the chimera FGFR31b-M had a moderate level of neurite outgrowth, which varied from 15 to 25% in three separate experiments. Cells expressing all the other chimeric receptors had levels of neurite-bearing cells equivalent to FGFR-1 in that they were greater than 40% by day 2. The results described above were confirmed by analyzing two or three additional clones of all of the chimeras. To demonstrate that all the cell lines isolated had retained the ability to produce neurites, they were stimulated with NGF to induce neurite outgrowth through a different receptor, namely, the NGF receptor. In all cases, the cell lines produced similar high levels of neurite outgrowth (50 to 80%) when stimulated by NGF (Table 1). This indicates that the difference in neurite induction by FGF-1 is due to the expression of the different chimeras and not to some general signaling defect. In summary, the chimeras FGFR31b, FGFR31b-Q, FGFR31b-N, FGFR31b-X, and FGFR31b-R conferred robust levels of neurite outgrowth, whereas FGFR-3b, FGFR31b-Y, and FGFR31b-Z conferred low levels of neurite outgrowth and FGFR31b-M induced a more moderate level of neurite outgrowth. An examination of Fig. 1 and 2 reveals that FGFR31b-M includes the carboxy-terminal (COOH) region of FGFR-1, whereas all receptors which conferred high levels of neurite outgrowth contained the region encompassing amino acids 399 to 508. These amino acids encompass the JM domain of FGFR-1 together with a few amino acids from the kinase domain. For the sake of simplicity, this region will be referred to as the JM region throughout the rest of the paper.

FIG. 3.

FIG. 3

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Expression level of the different chimeras in fnr-PC12 cells. fnr-PC12 cells were transfected with the receptors shown in Fig. 2. The cell lines stably expressing the receptors were lysed, and equivalent amounts of protein were analyzed for receptor expression by Western blotting.

FIG. 4.

FIG. 4

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FGF-stimulated neurite outgrowth of fnr-PC12 cells transfected with FGFR-3/FGFR-1 chimeric receptors. The cell lines stably expressing the receptors were incubated in cultural medium plus 50 ng of FGF-1 per ml. The percentage of neurite-bearing cells was scored 1 and 2 days after incubation with FGF-1. The results shown are the average from triplicate samples ± standard deviation.

TABLE 1.

Neurite outgrowth of cell lines stimulated by NGF

Clone Cells with NGF-induced neurites (%)a
Fnr 80
FGFR3b 50
FGFR31b-Q 50
FGFR31b-M 80
FGFR31b-G 50
FGFR31b-N 80
FGFR31b-R 80
FGFR31b-X 80
FGFR31b-Y 80
FGFR31b-Z 60
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a

fnr-PC12 cells expressing the different chimeras were treated with NGF (100 ng/ml), and the percentage of cells extending neurites after 7 days was assessed.  

From the results described above, we know that the JM and the COOH regions of FGFR-1 conferred robust and moderate abilities for neurite outgrowth, respectively, to FGFR-3. To test whether the JM region of FGFR-1 is essential for conferring robust neurite outgrowth, the chimeric receptor FGFR31b-G was made by swapping the JM region of FGFR-3 into the FGFR31b-Q protein (Fig. 2). This chimeric receptor was then introduced into fnr-PC12 cells, and its ability to induce neurite outgrowth in response to FGF-1 in comparison with that of FGFR31b-Q was assessed. Figure 5 shows that the ability of FGFR31b-G to confer robust neurite outgrowth was greatly reduced in comparison with the ability of FGFR31b-Q and was comparable to that of FGFR31b-M. This is to be predicted since it contains the COOH terminal region of FGFR-1. These experiments demonstrate that the JM region of FGFR-1 is essential for conferring robust neurite outgrowth to the receptor.

FIG. 5.

FIG. 5

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The JM region of FGFR-1 is essential for robust induction of neurite outgrowth. Induction of neurite outgrowth by FGFR31b-Q and FGFR31b-G is shown. Two fnr-PC12 lines which stably express equivalent levels of FGFR31b-Q and FGFR31b-G were treated with 50 ng of FGF-1 per ml. The percentage of neurite-bearing cells was determined 1 and 2 days after FGF treatment.

The JM region of FGFR-1 confers sustained signaling upon FGFR-3 in fnr-PC12 cells.

In earlier studies, we demonstrated that the autophosphorylation levels of FGFR-1 and FGFR-3 were not markedly different; however, we wanted to demonstrate that this was also the case for the chimeras, so we tested some of the chimeras for autophosphorylation. Figure 6 shows that there is no correlation between autophosphorylation and the ability of the chimeras to induce neurite outgrowth. For example, mutants FGFR31b-R, FGFR31b-Q, and FGFR31b-N are good inducers of neurite outgrowth whereas FGFR31b-G is a poor one, yet the levels of autophosphorylation are similar. We have previously shown that the different abilities of FGFR-1 and -3 to induce neurite outgrowth correlated with the ability of FGFR-1 to induce a sustained level of tyrosine phosphorylation of cellular proteins. This, in turn, gives rise to sustained activation of the enzyme MAP kinase. It has been demonstrated that sustained activation of this enzyme can lead to neurite outgrowth in PC12 cells. Thus, we examined whether the JM region conferred this ability of sustained signaling upon FGFR-3. Figure 7 shows a comparison of phosphorylation of cellular proteins and activation of MAP kinase mediated by FGFR3b and the mutant FGFR31b-R that contains the JM region of FGFR-1. Cells expressing these receptors were treated with FGF-1 for different time periods. After stimulation with FGF-1 for 2 to 60 min, FGFR3b and FGFR31b-R cells showed increases in tyrosine phosphorylation of a similar array of proteins. The level and duration of increase in phosphorylation of p90, p56, p44, and p42 mediated by FGFR31b-R were stronger and more sustained than those mediated by FGFR-3 (Fig. 7A). These proteins were identified as being the signaling proteins SNT/FRS2, SHC, and MAP kinases (see below and data not shown). Analysis of MAP kinase activation using a mobility shift assay demonstrated that the activation of this enzyme was also more sustained in cells expressing FGFR31b-R than in cells expressing FGFR-3 (Fig. 7B). The position of the activated form of this enzyme is indicated in Fig. 7B. These results suggest that the JM region of FGFR-1 conferred sustained signaling upon FGFR-3.

FIG. 6.

FIG. 6

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Receptor autophosphorylation. Cell lines were untreated (−) or treated (+) with FGF-1 for 5 min, the cells were lysed, and the lysates were subjected to immunoprecipitation with antiphosphotyrosine antibodies. The immune complexes were then subjected to SDS-PAGE and analyzed by Western blotting with FGFR-3-specific antibody.

FIG. 7.

FIG. 7

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Effect of the JM region of FGFR-1 on activation of signal transduction. (A) FGF-stimulated tyrosine phosphorylation of cellular proteins in fnr-PC12 cells expressing FGFR3b and FGFR31b-R cell lines were untreated (−) or treated with FGF-1 for 2, 5, 10, 30, or 60 min. The cells were lysed, and the lysates were subjected to SDS-PAGE followed by a Western blot analysis with an antiphosphotyrosine antibody, 4G10. The phosphorylated proteins of special interest, including p90, p52, p44, and p42, are indicated by arrows. (B) FGF-stimulated activation of MAP kinase in fnr-PC12 cells expressing FGFR3b and FGFR31b-R. The treatment was the same as that described for panel A. The Western blotting was done by using an anti-ERK2 antibody. The activation of MAP kinase was determined by mobility shift in the SDS-PAGE. MAPK, inactive form of MAP kinase; MAPK*, activated form of MAP kinase.

Recently, it was shown that the SNT/FRS2 protein can serve as a scaffolding protein to link FGFR signaling to MAP kinase (18). All the chimeras containing the JM domain from FGFR-1 were able to induce phosphorylation of proteins with similar molecular weights to SNT/FRS2. This raised the possibility that it is the ability to phosphorylate SNT/FRS2 that is the important property of these JM-containing chimeras. To assess this, we used gst-suc1 beads to isolate SNT and then assayed for tyrosine phosphorylation by Western blotting. Figure 8 shows that the FGFR31b-Q chimera induces strong phosphorylation of SNT whereas the phosphorylation of SNT by FGFR3b is very weak. This difference could potentially explain the ability of the JM-containing chimeras to activate MAP kinase in a strong sustained fashion.

FIG. 8.

FIG. 8

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SNT/FRS2 phosphorylation. fnr-PC12 cells expressing either FGFR31b or FGFR31b-Q were treated with FGF for the times shown, and lysates were prepared. Proteins that bind to gst-suc1 beads were isolated and analyzed by Western blotting with antiphosphotyrosine antibodies for their phosphorylation state.

Tyrosine 764 of FGFR-1 plays a role in induction of neurite outgrowth in fnr-PC12 cells.

A comparison of the cytoplasmic regions of FGFR-1 and FGFR-3 is shown in Fig. 1. As indicated in Fig. 1, the COOH region used in generating the FGFR31b-M chimera contains a tyrosine residue at amino acid position 764 of mouse FGFR-1. This tyrosine residue has been shown to be a major site of autophosphorylation in FGFR-1. To determine if this tyrosine residue was responsible for the modest increase in neurite outgrowth associated with the FGFR31b-M chimera, we mutated this tyrosine to phenylalanine (Fig. 9A, FGFR31b-MF) and analyzed FGF-stimulated neurite outgrowth mediated by FGFR31b-M and FGFR31b-MF. As can be seen in Fig. 9B, mutation of tyrosine 764 to phenylalanine abolished the ability of the FGFR31b-M chimera to induce neurite outgrowth. Similarly, mutation of this tyrosine to phenylalanine in the context of the FGFR31b-G chimera also abolished the ability of this receptor to induce a moderate level of neurite outgrowth (data not shown).

FIG. 9.

FIG. 9

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Effect of tyrosine 461 and tyrosine 764 residues of FGFR-1 in induction of neurite outgrowth and activation of signal transduction. (A) Schematic representation of FGFR31b-M, FGFR31b-R, and their tyrosine-to-phenylalanine mutants. The Y764 and the Y461 residues on FGFR31b-M and FGFR31b-R were respectively mutated to F764 and F461. FGFR31b-MF and FGFR31b-RF are the mutated forms of the receptors. The black areas indicate regions from FGFR-1. (B) FGF-stimulated neurite outgrowth of FGFR31b-M, FGFR31b-R, and their Y-to-F mutants. These four cell lines were treated with 50 ng of FGF-1 per ml. The percentage of neurite-bearing cells was determined 1 and 2 days after FGF treatment.

The result described above suggests that tyrosine 764 of FGFR-1 plays a role in FGF-stimulated neurite outgrowth. This tyrosine residue has been shown previously to be involved in the FGFR-1-mediated tyrosine phosphorylation of PLCγ. Furthermore, comparison of the abilities of FGFR-1 and FGFR-3 to phosphorylate PLCγ revealed that FGFR-1 was more efficient (Fig. 10A and B). To determine if this was the case for FGFR31b-M, we analyzed tyrosine phosphorylation of PLCγ in cells expressing FGFR31b-M. The phosphorylation induced by FGFR31b-M is strong and sustained like that of FGFR-1 and unlike that induced by FGFR-3b, (Fig. 10A to C). A comparison of tyrosine phosphorylation of cellular proteins in cells expressing FGFR31b-M and FGFR31b-MF indicated that the only difference was in the tyrosine phosphorylation of a 150-kDa protein (data not shown). To confirm that this protein was PLCγ, the lysates were immunoprecipitated with anti-PLCγ serum and analyzed by SDS-PAGE. The result demonstrates that FGFR31b-M, but not FGFR31b-MF, mediates tyrosine phosphorylation of PLCγ (Fig. 10D). These results suggest that the efficient tyrosine phosphorylation of PLCγ by FGFR31b-M correlated with the presence of the tyrosine 764 residue of FGFR-1 and that activation of PLCγ played a role in induction of neurite outgrowth.

FIG. 10.

FIG. 10

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FGF-stimulated tyrosine phosphorylation of PLCγ. fnr-PC12, FGFR31b, FGFR3b, FGFR31b-M, and FGFR31b-MF cells were untreated (−) or treated with 50 ng of FGF-1 per ml for various times as indicated. Lysates from the untreated and treated cells were immunoprecipitated with an antiserum against PLCγ, followed by SDS-PAGE and a Western blot analysis with the antiphosphotyrosine antibody 4G10. (A to C) Comparison of tyrosine phosphorylation of PLCγ in FGFR31b, FGFR3b, and FGFR31b-M, respectively; (D) comparison of tyrosine phosphorylation of PLCγ at the 5-min time point in fnr-PC12, FGFR31b-M, and FGFR31b-MF cells.

Examination of the JM region of FGFR-1 that was used to create the FGFR31b-R chimera revealed that it contains a tyrosine residue, tyrosine 461 in FGFR-1, that is absent from the similar region in FGFR-3 (Fig. 1). Furthermore, this tyrosine has been shown to be an autophosphorylation site in FGFR-1 (23). To determine if this was an important residue for induction of neurite outgrowth, we mutated it to phenylalanine (Fig. 9A, FGFR31b-RF). However, in this case, the replacement of tyrosine 461 with a phenylalanine residue had only a relatively minor effect on the ability of this chimera to induce neurite outgrowth (Fig. 9B), with neurite outgrowth induction by FGFR31b-RF being greater than 40% after 2 days. This indicates that the mechanism by which the JM region influences neurite outgrowth does not primarily involve tyrosine phosphorylation.

The JM region does not play a role in the induction of mitogenesis in BaF3 cells by FGFR-1.

In addition to having differing abilities to induce neurite outgrowth in PC12 cells, FGFR-1 and -3 have also been demonstrated to differ in their abilities to induce mitogenesis in BaF3 cells. In this pre-B cell line, FGFR-1 has been demonstrated to induce a strong mitogenesis response, whereas FGFR-3 cannot. To determine if this difference in signaling capability also resided in the JM region, we assessed the ability of the FGFR31b-R chimera to induce mitogenesis. BaF3 cells were transfected with the FGFR31b and FGFR31b-R chimeras (Fig. 2), and cells expressing equivalent amounts of receptors on their surfaces were identified by 125I-FGF binding (Fig. 11B). These cells were then stimulated with FGF-1, and the induction of the expression of c-Fos and mitogenesis was measured as described in Materials and Methods. Figure 11A and C demonstrate that the FGFR31b chimera induced c-Fos expression and mitogenesis, whereas the FGFR31b-R chimera was incapable of inducing either of these two events. This indicates that the regulation of FGFR signaling that involves the JM region is cell type specific.

FIG. 11.

FIG. 11

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Activity of chimeric FGFR expression constructs in BaF3 cells. (A) (Top panel) Comparison of single BaF3 clones expressing FGFR31b or FGFR31b-R to induce c-fos gene expression following stimulation with FGF-1 for the indicated period of time. (Bottom panel) Ethidium bromide stain of the gel shown in the top panel. (B) The same clones shown in panel A analyzed by cross-linking to 125I-FGF-1 followed by polyacrylamide gel electrophoresis and autoradiography. (C) Mitogenic activity of BaF3 cells expressing FGFR31b or FGFR31b-R in response to FGF-1.

DISCUSSION

The cytoplasmic signaling domains of FGF receptors are highly conserved, yet data is accumulating that they do not all signal equivalently. A comparison of the abilities of FGFR-1, -3, and -4 to induce proliferation of the lymphoid cell line BaF3 indicated that only FGFR-1 was able to induce cell division (3, 29, 45). Similarly, we had noted previously that FGFR-3 was very poor in its ability to induce neurite outgrowth in PC12 cells, whereas FGFR-1 could induce very efficient neurite outgrowth (19). In this report, we have addressed the structural determinants within the cytoplasmic domains of these two receptors that are responsible for their differing signaling capabilities and induction levels of neurite outgrowth.

The carboxy terminus of these two receptors seems to play differential roles in signal transduction. Introduction of the carboxy terminus of FGFR-1 into the cytoplasmic domain of FGFR-3 moderately increased the ability of this chimera to induce neurites. Our analysis of the chimeric receptor FGFR31b-M and its mutant receptor FGFR31b-MF demonstrated that the tyrosine residue at amino acid 764 of FGFR-1 allowed the tyrosine phosphorylation of PLCγ and gave rise to this modest increase in neurite outgrowth. Although both FGFR-1 and FGFR-3 domains contain a tyrosine residue in a similar context, the phosphorylation of PLCγ was more robust and sustained when this tyrosine was present in the context of the FGFR-1 carboxy terminus. The reason for this is presently unknown, but this difference in PLCγ phosphorylation may explain the difference in neurite outgrowth, since PLCγ activation by FGFR-1 has been reported to give rise to MAP kinase activation, which could then lead to neurite outgrowth (15).

Our data indicates a potential role for PLCγ and the subsequent signaling events in neurite outgrowth. Previous studies utilizing mutant FGFR-1 had indicated that the activation of this pathway was not essential for neurite outgrowth (37). However, in this previous study, the mutant FGFR-1 proteins analyzed all had the FGFR-1 JM region, which on the basis of our results would be predicted to generate signals that would mask the more modest signals emanating from tyrosine 764. In addition, these other studies utilized PC12 cells that expressed endogenous wild-type FGFR-1 as well as the mutant FGFR-1. Thus, the ancillary role of this pathway detected in our analysis could have potentially been masked by signals emanating either from the endogenous FGFR-1 or from the signals that require the FGFR-1 JM region. In TrkA signaling, a role for PLCγ signaling in neurite outgrowth has been indicated by mutational analysis (26, 38). This observation is consistent with our studies with the FGFRs.

We identified the JM region as playing an important role in neurite outgrowth in PC12 cells. When the JM region from FGFR-1 was swapped into the cytoplasmic domain of FGFR-3, robust neurite outgrowth ensued. In contrast, when the JM region of FGFR-3 was introduced into the FGFR-1 cytoplasmic domain, then neurite outgrowth was reduced. Analysis of the induction of tyrosine phosphorylation of cellular proteins by these chimeric receptors revealed that this region restored sustained tyrosine phosphorylation of cellular proteins. Analysis of activation of MAP kinase also indicated that the activation of this enzymatic activity was also sustained. Sustained activation of MAP kinase has been correlated previously with neurite outgrowth (11, 13, 14, 33, 42). The SNT/FRS2 protein has been shown to link FGFR signaling to MAP kinase activation (18), and SNT also plays a role in NGF signaling (31). We were able to demonstrate that SNT/FRS2 phosphorylation was robust in chimeras that contain the JM domain of FGFR-1. This would potentially be sufficient to induce robust MAP kinase activation and, hence, neurite outgrowth. Our data indicates that interactions between the JM domain and SNT/FRS2 may play important roles in the differing capabilities of FGFR-1 and -3 to activate MAP kinase.

The JM region used to make the chimera includes the JM domain and several amino acids from the kinase domain. At the present time, it is unclear which amino acids within this region are important for these effects. As we described previously, receptor autophosphorylation and the initial signaling effects are the same when FGFR-1 and -3 are compared (19). It is the ability to sustain the tyrosine phosphorylation signals that differs between these two receptors, and this is the property that maps to this region. The JM region of FGFR-1 contains a single tyrosine residue, Tyr 461, that can be phosphorylated upon activation of this receptor. However, previous studies have demonstrated that phosphorylation of this residue does not seem to play a major role in signaling by FGFR-1 in PC12 cells (23). Similarly, when we mutated this tyrosine to phenylalanine within the context of the FGFR31b-R chimeric receptor, we found that this mutation had only minor effects on the ability of this chimera to induce efficient neurite outgrowth. Recently, analysis of neurite outgrowth induced by activation of TrkA has identified the triplet of amino acids KFG within the JM region of this receptor as playing an important role in neurite outgrowth (31). However, this motif is not apparent within the JM region of FGFR-1 used in these studies. Similarly, non-phosphotyrosine-containing regions within the JM domains of the epidermal growth factor receptor and its family member HER-2 have been found to control the differing mitogenic responses elicited in 3T3 and 32D cells (10, 35). The studies reported here with FGFR signaling in PC12 cells provide another example in which non-tyrosine-containing regions can play important roles in signal transduction by tyrosine kinase receptors.

Although the JM region was a major determinant controlling neurite outgrowth in PC12 cells, analysis of the chimeric receptors in the BaF3 cell line revealed that the presence of this region did not correlate with the differing abilities of these two FGFRs to elicit mitogenesis and activate c-fos expression in this cell line. Similar comparative studies on FGFR-1 and FGFR-4 signaling in BaF3 cells had indicated that the JM domains did not play a role; rather, it appeared as if the kinase insert, together with parts of the kinase domain, was the important determinant (45). Activation of c-fos expression in BaF3 cells has been shown to involve activation of Ras and MAP kinase (41), and neurite outgrowth in PC12 cells is also dependent on activation of this pathway. This would indicate that the intracellular interactions involved in the activation of the same signaling pathway by the FGFRs are cell type specific. These interactions would be most easily explained by the cell type-specific expression of proteins that interact with this region, with SNT/FRS2 proteins being prime candidates. This would represent a novel level of regulation of signaling by the FGFR family.

ACKNOWLEDGMENTS

We thank the various members of our laboratories for helpful comments on the manuscript. Recombinant human FGF-1 was kindly provided by M. Jaye (Rhône-Poulenc Rorer Central Research).

This work was supported by Public Health Service grants CA28146 and CA42573 (to M.J.H.), CA60673 (to D.M.O.), and NS18218 (to S.H.).

H.-Y. Lin and J. Xu contributed equally to this work.

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