(Received for publication, March 21, 1995; and in revised form, August 14, 1995)
From the
We have raised specific antibodies to the second immunoglobulin-like domain of fibroblast growth factor receptors (FGFRs) and used these to investigate the expression and subcellular localization of FGFR-1, -2, -3, and -4 in breast epithelial cells. All four receptors classes could be detected in breast cell lines; however, FGFR-4 and FGFR-2 appeared to be expressed at a higher level in breast cancer cell lines than in normal epithelial cells. Surprisingly, FGFR-3 localized in the cell nucleus by immunofluorescence. A second antibody to a separate epitope confirmed this finding and showed that the form of FGFR-3 present must contain an intact kinase domain as well as the growth factor binding domain. Western analysis of fractionated cells revealed the presence of two forms of FGFR-3 of 135 and 110 kDa. The 110-kDa form was predominantly found in the nucleus, whereas the 135 kDa form was sometimes found in the nucleus. RT-PCR analysis of FGFR-3 mRNA showed the presence of a splice variant in which exons 7 and 8 are deleted. This results in the translation of FGFR-3 missing the transmembrane domain but with an intact kinase domain, which could be a soluble, intracellular receptor. Transfection experiments showed that FGFR-3 containing this deletion and no signal peptide gave an identical nuclear staining pattern to that seen in breast epithelial cells. We conclude that two forms of FGFR-3 are present in breast epithelial cells; a full-length 135-kDa receptor, which has a conventional membrane localization, and a novel soluble form of 110 kDa.
The fibroblast growth factor (FGF) ()family
constitutes a family of nine structurally related polypeptides sharing
30-55% homology (Burgess and Maciag 1989). Acidic FGF (FGF-1) and
basic FGF (FGF-2) are the best characterized members of the family,
other members including products of the oncogenes int-2 (FGF-3) and hst/K-FGF (FGF-4), keratinocyte growth factor
(FGF-7), and the recently discovered androgen-induced growth factor
(FGF-8) and glial-activating factor (FGF-9) (Tanaka et al.,
1992; Miyamoto et al., 1993). FGFs are involved in the control
of a variety of biological functions including mitogenesis, chemotaxis,
neuronal survival and neurite extension, mesoderm induction,
angiogenesis, and wound healing (Burgess and Maciag, 1989).
Biological responses to FGFs are mediated through specific high affinity receptors. Four structurally related genes encoding FGFRs have been identified (Jaye et al., 1992; Johnson et al., 1991; Keegan et al., 1991; Partanen et al., 1991). Each receptor contains an intracellular split tyrosine kinase domain and an extracellular domain containing up to three immunoglobulin-like domains. In addition, low affinity receptors for FGF are present on the surface of cells. These have been identified as heparin sulfate proteoglycans (Moscatelli, 1987). A ternary complex of FGF heparin sulfate proteoglycans and high affinity receptor appears to be required for biological activity (Yayon et al., 1991; Rapraeger et al., 1991; Roghani et al., 1994). Structural variants of the high affinity receptors can be generated by alternative splicing of their RNA transcripts (Hou et al., 1991). Ligand binding properties of the receptor are changed by the use of alternative third exons for receptors FGFR-1, FGFR-2, and FGFR-3 (Werner et al., 1992; Chellaiah et al., 1994), and the affinity of FGF binding may be affected by the loss of the first immunoglobulin-like domain (Johnson et al., 1990; Shi et al., 1993). Putative intracellular forms of FGF receptors may also be generated by alternative RNA splicing mechanisms by the substitution of a 267-base pair sequence encompassing the first Ig domain by an unrelated 144-base pair sequence (Hou et al., 1991). The 144-base pair insert contains stop codons leading to premature termination, but reinitiation of translation would lead to a receptor without signal peptide or acidic box having an intracellular localization. A rat analogue of FGFR-4 with this conformation has been reported (Horlick et al., 1992).
FGF-1 and -2 have both been detected in the cell
nucleus (Presta et al., 1993; Gualandris et al.,
1993; Cao and Pettersson, 1993; Cao et al., 1993, Amalric et al. 1994). When FGF2 is synthesized in a cell, only the
higher molecular mass forms (24, 22.5, and 22 kDa) are detected in the
nucleus (Bugler et al., 1991). However, exogenous 18-kDa basic
FGF can enter the nucleus in the G phase of the cell cycle,
where it is localized in the nucleolus (Baldin et al., 1990).
The presence of FGF-1 and FGF-2 in the nucleus is likely to be
important in their biological function. It has been shown that
internalization of FGF-1 is essential for stimulation of cell division,
and a dual model of action has been proposed (Wiedlocha et
al., 1994). FGF-2 is bound to chromatin in the nucleus (Gualandris et al., 1993), and it can affect gene transcription in
cell-free systems (Nakanishi et al., 1992). FGF-2 taken into
the nucleolus appears to increase the transcription of genes encoding
ribosomal RNA (Bouche et al., 1987; Amalric et al.,
1994).
We report the production of specific antisera against the extracellular region of three FAF receptors and use these reagents to examine the intracellular localization of each receptor in breast epithelial cells. FGFR-1, -2, and -4 have a membrane-bound localization; however, in both breast cancer cells and normal breast epithelial cells, FGFR-3 was found in the nucleus.
Figure 1: Domains of FGFRs to which antisera were raised. A, structure of FGFR, position of PCR primers, and domains to which antisera bind. I, II, and III refer to immunoglobulin domains, TM to the transmembrane domain, JM to the juxtamembrane domain, TK to the tyrosine kinase domain, KI to the kinase insert domain, and CT to the carboxyl-terminal tail. B, Coomassie Blue-stained SDS-polyacrylamide gel of purified glutathione S-transferase fusion proteins.
Figure 2: Western analysis to show the cross-reactivity of anti-FGFR antisera. Lysates of COS cells transfected with FGFR-1, -2, -3, or -4 or untransfected (lanes M, 1, 2, 3, and 4, respectively) were run on a 7.5% polyacrylamide gel and transferred to nitrocellulose. The blots were probed with antisera against FGFR-2, FGFR-3, or FGFR-4 or a monoclonal antibody against FGFR-1 (UBI) by Western analysis.
Figure 3: Immunofluorescent staining of COS cells transfected with FGFR-1 (A), FGFR-2 (B), FGFR-3 (C), and FGFR-4 (D) and stained with antibodies against the transfected receptor. E and F show COS-7 cells transfected with FGFR-3 and treated with conditioned medium from breast epithelial cells (E) or 20 ng/ml acidic FGF (F) for 24 h before immunofluorescent detection of FGFR-3. G, H, I, and J show the basal levels of immunofluorescence of mock-transfected COS cells detected with antibodies against FGFR-1 (G), FGFR-2 (H), FGFR-3 (I), and FGFR-4 (J).
Figure 4: Immunofluorescent staining of breast epithelial cells with antisera to FGFR-1, FGFR-2, and FGFR-4. A, HBR-SV 161 cells probed with anti-FGFR-2; B, MCF-7 cells probed with anti-FGFR-2; C, HBR-SV161 cells probed with anti-FGFR-4; D, MCF-7 cells probed with anti-FGFR-4; E, HBR-SV-161 cells probed with anti-FGFR-1; F, MCF-7 cells probed with anti-FGFR-1.
Unexpectedly the antibody against FGFR-3 gave a nuclear staining pattern with no stain in the nucleoli but good stain over the rest of the nucleus (Fig. 5). The Ig2-glutathione S-transferase protein against which the anti-FGFR-3 antibody was raised was used to block specific binding of these antibodies by preincubating the diluted antibody with 10 µg/ml fusion protein. As shown in Fig. 5, B and D, such treatment could completely block the nuclear immunofluorescence of anti-FGFR-3. A second antibody against the C-terminal tail of FGFR-3 (Santa Cruz Biotechnology) was used to confirm the localization of FGFR-3. This antibody was reported to bind specifically to FGFR-3, and in our hands it bound to FGFR-3 but not to FGFR-1, -2, or -4 by Western analysis. The same staining pattern was found in both the normal and malignant cell lines, with anti-FGFR-3 giving nuclear staining with exclusion from the nucleoli (Fig. 5, C and F). Similar levels of staining for FGFR-3 were seen in normal and malignant breast cell lines.
Figure 5: Immunofluorescent staining of breast epithelial cells with antisera to FGFR-3. A, B, and C show staining of HBR-SV161 cells, while D, E, and F show staining of MCF-7 cells. A and D are probed with purified antisera against the second immunoglobulin domain of FGFR-3. B and E are probed with the same antibody but after preincubation with the glutathione S-transferase fusion protein to which it was raised; C and F are probed with a second purified antisera against the C-terminal tail of FGFR-3 (Santa Cruz).
Figure 6: Western blot of fractionated cells. Nuclear and cytoplasmic fractions of MCF-7 and HBR-SV161 cells were probed with antibodies against FGFR-1, FGFR-2, FGFR-3 (both N-terminal and C-terminal epitopes), and FGFR-4. Lane 1 was loaded with HBR-SV161 nuclei, lane 2 with HBR-SV161 cytoplasm, lane 3 with MCF-7 nuclei, and lane 4 with MCF-7 cytoplasm.
Figure 7:
An exon-deleted form of FGFR-3 is
transcribed in breast epithelial cells. A, diagrammatic
representation of PCR primers and the fragments generated. B,
partial nucleotide sequence of the 262-base pair PCR product, showing
the position of the exon deletion. C, Southern blot showing
the PCR fragments probed with a P-labeled internal
oligonucleotide
5`-CACCGGCCCATCCTGCAGGCG-3`.
Figure 8: Localization of FGFR-3 variants. Full-length FGFR-3 (R3) and variants of FGFR-3 missing the signal peptide (-SP), the transmembrane domain (-TM), or both the signal peptide and transmembrane domains (-SP & TM), were transiently expressed in COS-7X cells under an SV40 promoter. The cells were fixed and stained for FGFR-3 immunofluorescently.
We have raised specific antibodies to the second immunoglobulin-like domain of FGFR-2, FGFR-3, and FGFR-4 and have used these to examine the intracellular localization of FGFRs in normal breast epithelial cells and breast cancer cells. All the antibodies used were able to recognize native, glycosylated FGF receptors as seen by their ability to immunofluorescently stain transfected COS-7 cells. When used to stain breast cancer cells, the anti-FGFR-1 monoclonal gave levels of staining similar to background. However, Western analysis using the same antibody was able to detect FGFR-1 in these cells. These results indicate that FGFR-1 is present in breast epithelial cells but at relatively low levels at which the monoclonal antibody has difficulty at detecting it by immunofluorescence. Higher levels of FGFR-2, FGFR-3, and FGFR-4 were seen in immunofluorescence experiments. These results are consistent with reports of the amount of FGFR mRNA present in breast cancer cells where FGFR-3 and FGFR-4 could be detected easily by Northern analysis, whereas FGFR-2 and FGFR-1 could barely be detected (Lehtola et al., 1992; Ron et al., 1993). We notice higher expression of FGFR-4 and FGFR-2 in breast cancer cell lines than in normal breast epithelial cells. The higher levels of FGFR-4 expression are consistent with the report of FGFR-4 and FGFR-2 gene amplification in breast cancers (Jaakkola et al., 1993; Adnane et al., 1991).
The localization of
most of the receptors was as expected with FGFR-1, FGFR-2, and FGFR-4
giving a cytoplasmic vesicle staining pattern consistent with
internalized membranes and associating with the membrane rather than
the nuclear fraction of fractionated cells. However, by both of these
tests, FGFR-3 appears to be associated with the nucleus. Anti-FGFR-3
antisera give a blockable staining pattern in which it is excluded from
the nucleolus but present throughout the rest of the nucleus.
Confirmatory experiments showed that a second antibody against a
separate region of FGFR-3 gave an identical staining pattern. This
experiment also shows that the FGFR-3 isoform being detected will
contain both the growth factor binding domain and the C terminus of the
receptor, so it will have a complete tyrosine kinase domain as well as
an antibody binding domain. The staining pattern seen suggests that
much of the nuclear FGFR-3 may be soluble since confocal microscopy
showed FGFR-3 staining evenly distributed throughout the nucleus rather
than concentrated around the edge. Several nonspecific bands appear on
Western blots probed with antibodies against FGFR-3. However, we do not
believe that these are responsible for the nuclear staining pattern
seen in epithelial cells since untransfected COS-7 cells gave pale,
background immunofluorescent staining patterns with both cytoplasmic
and nuclear components and failed to give the clear nuclear stain seen
in breast epithelial cells, which expressed higher levels of FGFR-3. In
addition, we expect FGFR-3 to be present in breast epithelial cells due
to our Western data (Fig. 6) and reports of FGFR-3 mRNA in
breast epithelial cells (Lehtola et al. 1992; McLeskey et
al., 1994). Two forms of FGFR-3 are expressed in breast epithelial
cells as seen by immunoblotting of fractionated cells. These correspond
to the full-length 135-kDa form and a smaller form of 110 kDa. Of these
the 110-kDa form is predominantly associated with the nucleus of
fractionated cells. The 135-kDa form was found in different
compartments when different batches of cells were used. This could
reflect differences in the stimulatory state of those cells. There is
evidence that FGF2 expression is regulated by cell density (Bost and
Hjelmeland, 1993), so autocrine stimulation by FGFs may differ in the
different cell batches studied. There is evidence for FGFR-1 being
translocated to a perinuclear localization upon interaction with its
ligand during G period of the NIH 3T3 cell cycle (Prudovsky et al., 1994). In the case of the 135-kDa form of FGFR-3, the
nuclear and cytoplasmic localizations seen on Western blots of
fractionated cells could reflect differences of localization caused by
growth factor stimulation. However, such a migration to a perinuclear
position would not give the staining pattern throughout the nucleus
that we have observed in epithelial cells but would give a perinuclear
staining pattern (Prudovsky et al., 1994).
COS-7 cells transfected with the full-length FGFR-3 cDNA under an SV40 promoter gave a membrane staining pattern rather than the nuclear staining pattern seen in breast epithelial cells. There are several possible explanations for this discrepancy: (a) the COS cell transfection system leads to overexpression of proteins and the quantities of FGFR-3 produced are too high for correct localization; (b) only the full-length form of FGFR-3 is being expressed, whereas the 110-kDa form may be giving the nuclear localization in epithelial cells; or (c) there may be differences in growth factor production in the different cell types leading to translocation of the receptor. We have attempted to distinguish between these possibilities and find that growth factor stimulation is unlikely to account for differences in localization. Expression of an alternative variant of FGFR-3 is more likely to account for differences in localization since a smaller form of FGFR-3 was seen in the nucleus of breast epithelial cells. We used reverse transcriptase-PCR to amplify mRNAs transcribed from the FGFR-3 gene and detected an exon-deleted form of FGFR-3 that would lack the second half of the third immunoglobulin domain and the transmembrane domain. This splice variant of FGFR-3 would have the usual kinase domain and carboxyl-terminal tail, since no frameshift occurs as a consequence of the deletion. It would be missing 12 kDa of encoded protein sequence, including two N-linked glycosylation sites, which would account for much of the observed size decrease (Fig. 6). In addition, the loss of the transmembrane domain explains the soluble appearance of FGFR-3. It is not localized exclusively on the nuclear membrane but appears to be distributed throughout the nucleus. This would require the use of an alternative ATG codon at either codon 69 or codon 159 of FGFR-3. This would generate an intracellular rather than a secreted form of FGFR-3, which would be more consistent with our findings of 110-kDa FGFR-3 within the nucleus. Transfection of such a construct into COS-7 cells gave the nuclear staining pattern observed in epithelial cells, so this variant is a good candidate for the nuclear FGFR-3. In the case of FGFR-1, variants lacking a signal peptide and predicted to have an intracellular localization have been described (Jaye et al., 1992). The deletion of exons 7 and 8 represents a new splice variant of FGFR-3. FGFR-3 has been studied less fully than several other FGFRs, and the only splice variants described to date have used alternative forms of exon III (Avivi et al., 1993; Chellaiah et al., 1994).
The function of a nuclear FGFR can only be speculated on at present; however, both FGF1 and FGF2 have been found in the nucleus and probably have functions within it (Gualandris et al., 1993; Wiedlocha et al., 1994). FGFR-3 is able to bind both FGF1 and FGF2 (with lower affinity), so it could have a role in the nuclear function of both these growth factors (Chellaiah et al., 1994). A nuclear FGFR could have a role either in the transport of FGF to the nucleus or in the storage of FGF in an inactive form within the nucleus. Alternatively, it could have a more active role in mediating the nuclear function of FGF. The presence of a complete tyrosine kinase domain opens the question of whether the activated receptor can phosphorylate nuclear targets. Further experimentation will be required before a functional role for FGFR-3 in the nucleus of epithelial cells can be determined.