KGF RECEPTOR: TRANSFORMING POTENTIAL ON ... - Springer Link

289

Adenine Nucleotides in Cellular Energy Transfer and Signal Transduction s. Papa, A. Azzi & I.M. Tager (eds) © 1992 Birkhauser Verlag, Basel/Switzerland

KGF RECEPTOR: TRANSFORMING POTENTIAL ON FIBROBLASTS EPITHELIAL CELL-SPECIFIC EXPRESSION BY ALTERNATIVE SPLICING

AND

Toru Miki, Donald P. Bottaro, Timothy P. Fleming, Cheryl L. Smith, Jeffrey S. Rubin, Andrew M.-L. Chan, and Stuart A. Aaronson

Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, MD 20892, USA

SUMMARY' The mouse keratinocyte growth factor (KGF) receptor (KGFR) cDNA was isolated by an expression cDNA cloning strategy involving creation of a transforming autocrine loop. Characterization of the cloned.4.2 kb cDNA revealed a predicted membrane-spanning tyrosine kinase structurally related to the FGF receptor (FGFR). Structural analysis of the human KGFR cloned by the analogous procedure revealed identity with one of the fibroblast growth factor (FGF) receptors (bek/FGFR-2) except for a divergent stretch of 49 amino acids in their extracellular domains. Binding assays demonstrated that the KGFR was a high affinity receptor for both KGF and acidic FGF, while FGFR-2 showed high affinity for basic FGF and acidic FGF but no detectable binding by KGF. Analysis of the bek gene revealed two alternative exons responsible for the region of divergence between the two receptors. The KGFR transcript was specific to epithelial cells, and it appeared to be differentially regulated with respect to the alternative FGFR-2 transcript.

INTRODUCTION Growth factor signalling pathways play critical roles in normal development and in the genetic alterations associated with the neoplastic process. Our laboratory has recently identified (Rubin et al., 1989) and molecularly cloned (Finch et al., 1989) a growth factor, designated keratinocyte growth factor (KGF) ,

290 which has potent mitogenic activity for a wide variety of epithelial cells but lacks detectable activity on fibroblasts or endothelial cells.

Its synthesis by stromal fibroblasts of a

large number of epithelial tissues has suggested its likely role as an important paracrine mediator of normal epithelial cell proliferation.

Recent studies have further indicated specific

KGF binding to keratinocytes but not fibroblasts (Bottaro et al., 1990).

We reasoned that the ectopic expression of the KGF

receptor (KGFR) in the cells that secrete KGF might result in the transformed phenotype by creation of a transforming autocrine loop.

We have recently developed a highly efficient expression

cDNA cloning system with capability of plasmid rescue from stably-transfected cells

(Miki et al.,

1991b).

Using this

system, we prepared a cDNA expression library from epithelial cells that express the KGFR and introduced the library DNA into fibroblasts that express KGF but not its receptor.

By this

approach, it was possible to identify foci induced by expression of the KGFR cDNA and to molecularly clone the receptor.

MATERIALS AND METHODS Construction of CDNA libraries· in ApCEV27

cDNA libraries were constructed

(Miki et al., 1991b) by the automatic directional

cloning method (Miki et al., 1989).

Amplification of the library

and preparation of the DNA were performed by standard procedures. DNA transfection and cel] culture: carrier DNA (40

~g/plate)

Library DNA (5

~g/plate)

were introduced into cells using

calcium phosphate transfection (Wigler et al., 1977). maintained

in

Dulbecco's

and

modified Eagle's

Cells were

medium

(DMEM)

containing 5% calf serum. PCR assays:

The cDNA preparation (1

~l)

synthesized from each

RNA or human placenta genomic DNA (0.5

~g)

reaction buffer (Boehringer Mannheim), 200

~

0.2

~

was added to the each of four dNTPs,

each of the upstream and downstream primers, and 0.025

unit/~l

of Taq DNA polymerase

(Boehringer Mannheim).

The

291 reaction was cycled 30 times at 94° C for 1 min, at 60° C min and at 72° C

for 3

for 3 min.

Growth factor binding assays and cross1inking experiments' Recombinant KGF and bovine brain aFGF were purified and labeled with 125I-Na as described (Bottaro et a1., 1990). bFGF was obtained from R&D Systems.

Bovine brain

Bovine brain 125I-bFGF was

obtained from Amersham.

Specific actvities of all three tracers

were approximately 0.1

~Ci/ng.

Binding assays and covalent

affinity crosslinking experiments were performed as described (Bottaro et al., 1990).

RESULTS AND PISCUSSION Expression CPNA cloning of the mouse KGF receptor:

We prepared

a cDNA library from BALB/MK epidermal keratinocytes in ApCEV27 (Miki et al., 1991a).

The scheme for expression cloning of the

KGFR is shown in Fig. 1.

Transfection of NIH/3T3 mouse embryo

fibroblasts by the library DNA led to detection of several transformed foci.

Each focus was tested and shown to be

resistant to G418, indicating that it contained integrated vector sequences.

Three representative transformants were chosen for

more detailed characterization based upon differences in their morphologies.

When

we

performed plasmid

rescue,

each

transformant gave rise to at least 3 distinct cDNA clones as determined by physical mapping.

To examine their biological

activities, each clone was subjected to transfection analysis on NIH/3T3 cells.

A single clone rescued from each transformant was

found to possess high-titered transforming activity.

To screen

for cells expressing the KGFR, we performed binding studies with recombinant

125I-KGF as the tracer molecule.

One of the

transformants demonstrated specific high affinity binding of 125I-KGF, implying that the cDNA clone rescued from the focus encoded the KGFR,

whose introduction into NIH/3T3 cells had

completed an autocrine transforming loop.

292

b C>~

tOt to

Qtm;dQ

01

I~I

T

!

I ,...,.

Focus

Soft agar assay G-418 selection DNA extraction

DNA

T

C'>

C'>

I

0

" E. coli qQTranSformation

~~ -OCDO Digestion and ligation

Fig 1 Strategy for expression cDNA cloning of the KGFR. N1H/3T3 cells are transfected by ApCEV27-BALB/MK cDNA library DNA and scored at 14 to 17 days for transformed foci. Transformed cells are assayed for G-418 resistance to examine the presence of integrated vector sequence. Following expansion to mass culture, genomic DNA is isolated and subjected to plasmid rescue by digestion with either Not1, Xho1 or Mlu1, followed by ligation at low DNA concentration and transformation to a suitable bacterial strain. Bacterial colonies resistant to both the ampicillin and kanamycin are isolated. Plasmid DNA extracted from each colony is tested by transfection analysis on N1H/3T3 cells to identify the transforming cDNA clone. KGE receptor is a membrane-spanning t¥rosine kinase'

The rescued

transforming plasmid contained a 4.2kb cDNA insert.

We observed

a single KGFR transcript of around 4.2 kb in BALB/MK cells (Miki et al., 1991a).

Thus, our cDNA clone represented essentially the

complete transcript.

We next determined the nucleotide sequence

of the 4.2kb cDNA insert. Analysis of the sequence revealed that it contained a long open reading frame for a deduced protein of 707 amino acids with a size of 82.5 kd.

The amino acid sequence

of the KGFR predicted a transmembrane tyrosine kinase closely related to the bFGF receptor

(Fig.

2).

The putative KGFR

extracellular portion contained two immunoglobulin-like loops (1g loops).

The bFGF receptor contains a series of eight consecutive

293

o

2

3

4

1LI_ _L-_-'--_---'_ _-'1_ _..L-_---'-1_ _"'-_---L

EcoRl

NOli

I

BamHl1

EcoRl BgIIi

I

(kb)

I

I

BgIIi

I

L.

111 W~

1'l"/0I

30r1a11 n 1781

60

bilk

I

142~

II

74

88 #

KGFR 92

172 (%homology)

~~W-"~~--v.~n7.~lmr---r----lnl-------.--,1 ~

fG2

IG3

TM

JM

TKI

IK

TK2

bFGFR

C

ITZIl IGI Fig 2 Structural comparison of the predicted KGF and bFGF receptors. The region homologous to the published bek sequence is also shown . The schematic structure of the KGFR is shown below the restriction map of the cDNA clone. Amino acid sequence similarities with the smaller and larger bFGF receptor variants are indicated. S, signal peptide; IG1, IG2, and IG3, immunoglobulin-like domains; A, acidic region;, TM, transmembrane domain; JM, juxtamembrane domain; TKl and TK2, tyrosine kinase domains; IK, interkinase domain; C, C-terminus domain. acidic residues between the first and second IgG-like domains (Lee et al., 1989) . However, the KGFR did not contain such an acidic domain. The intracellular portion of the KGFR was highly homologous to the bFGF receptor tyrosine kinase. A partial mouse cDNA, bek, isolated by bacterial expression cloning using phosphotyrosine antibodies (Kornbluth et al., 1988) was identical to the KGFR in the tyrosine kinase domain (Fig. 2), indicating that the KGFR is encoded by the bek gene. Fllnctional analysis of the cloned KGF receptor·

Because of the

existence of more than one receptor of the FGF family, we sought to characterize in detail the binding properties of the KGFR isolated by expression cloning. Scatchard analysis of 12SI-KGF binding to the NIH/3T3-mKGFR transfectant revealed expression of two similar high affinity receptor populations . Out of a total of -3.8 x 105 sites/cell, 40% displayed a dissociation constant (Kd) of 180 pM, while the remaining 60 % showed a Kd of 480 pM.

294 These values are comparable to the high affinity KGFRs displayed by BALB/MK cells (Bottaro et al., 1990). The pattern of KGF and FGF competition for 125I-KGF binding to NIH/3T3-mKGFR cells was also very similar to that observed with BALB/MK cells. When 125I-KGF crosslinking was performed with NIH/3T3-KGFR cells, we observed a single species of 137 kd, corresponding in size to the smaller species identified in BALB/MK cells. Detection of this band was specifically and efficiently blocked by unlabelled KGF. When glycosylation is considered, the size of the KGFR predicted by sequence analysis corresponds reasonably well with the corrected size (115kd) of the crosslinked KGFR in NIH/3T3-KGFR cells. Moreover, KGF stimulation of the transfectant rapidly induced tyrosine phosphorylation of several cellular proteins. These results suggested that the cloned KGFR was biologically active for signal transduction (Miki et al., 1991a). Structural comparison of the KGF receptor with related molecules· Several human or avian cDNAs closely related to the KGFR have been reported. The external portions of human bek and TK14 and chicken cek3 proteins contain 3 Ig loops (Dionne et al., 1990; Houssaint et al., 1990; Pasquale, 1990). Computer analysis of these molecules (designated FGFR-2) showed that amino acid sequences are nearly identical. These FGFR-2 molecules also closely related to the KGFR but differ in that each contains an acidic region and is completely divergent in the carboxy terminal half of its third Ig-like domain from the KGFR. A gene, designated K-sam, was identified as an amplified sequence in a human stomach carcinoma (Hattori et al., 1990). A cDNA clone corresponding to one of the overexpressed K-sam transcripts predicts a 2-1oop bek variant, whose Ig loops correspond to those of the KGFR. However, it differs in that it contains an acidic region and may be truncated at its carboxy terminus as well. To study the differences of these related molecules in detail, we cloned human KGFR and compared the structure with human K-sam and FGFR-2. Human KGF receptor is identical to FGFR-2 except the third Ig lOQP regiQn: We isolated the human KGFR cDNA from B5/589 mammary

295 epithelial cell cDNA expression library by a similar strategy to the cloning of the mouse receptor (Miki et al., 1991c). Sequence analysis of the 4.5 kb cDNA insert revealed an open reading frame encoding a membrane-spanning tyrosine kinase closely-related to the mouse KGFR. Comparison of the predicted protein with FGFR-2 showed essentially complete identity with the exception of a strikingly divergent 49 amino acid stretch spanning the second half of the third loop into the stem region (Fig. 3).

mKGFR .. -- ................... M.............................. . hKGFR LK--HSGINSSNAEVLALFNVTEADAGEYI~VSNYIGQANQSAWLTVLPKQQAP K-sam .................................................. . FGFR2 .. AAGVNTTDKEI ... YIR ... FE ..... T.LAG.S .. ISFH ........ --- .. Fig. 3. Comparison of the amino acid sequences of the second halves of the third Ig loops of mouse KGFR (amino acids 196-250), human KGFR (311-365), human K-sam (222-276), and human FGFR-2 (311-365). The sequence of the human KGFR is shown in the second line. In the case of other molecules, only the amino acid residues different from the human KGFR are shown. The residues identical to the human KGFR are shown by dots. The dashes represent that the residues are not present in the molecule. The cysteine residue which is the start site of the third Ig loop is underlined.

Third Ig loqp divergent region determines KGF-binding prqperties; The mouse KGFR has been shown to bind KGF and aFGF at similar high affinity and basic FGF at 20 fold lower affinity (Miki et al., 1991a). In contrast, FGFR-2 has been reported to bind both aFGF and bFGF at high affinity (Dionne et al., 1990), but there is no available evidence concerning its ability to interact with KGF. We isolated a human cDNA whose product closely resembled the mouse KGFR, and yet differed only by a stretch of 49 amino acids in its third Ig loop from FGFR-2 (Miki et al., 1991c). This allowed us to characterize its binding properties and compare them to those of FGFR-2. For those studies, we utilized NIH/3T3 transfectants overexpressing either protein and markerselected NIH/3T3 cells transfected with the vector alone as a

296 KGFR

FGFR·2 5 IGl

A IG2

IG3

TM JM

TKl

IK TK2

c Fig 4. Schematic comparison of the structure of human KGFR and FGFR-2 and domains responsible for binding of aFGF, bFGF and KGF.

control. The human KGFR transfectant demonstrated substantial 125I-KGF binding, while neither FGFR-2 or the vector transfectant showed detectable binding (Miki et al., 1991c). These findings established that the cDNA encoded a human KGFR and suggested further that KGF lacked high affinity for FGFR-2. Evidence that FGFR-2 was indeed functional was derived from binding analyses with 125I-aFGF and 125I-bFGF. Both demonstrated a substantially greater number of binding sites on the FGFR-2 transfectant than on NIH 3T3 cells, which are known to express bFGFR (Ruta et al., 1989) and to show mitogenic response upon stimulation by either growth factor (unpublished results) . Whereas the human KGFR transfectant also bound increased amounts of 125I-aFGF, we

297 observed no increase in 125I-bFGF binding over the level observed with control NIH/3T3 cells (Miki et al., 1991c). All of these results demonstrated striking differences in the patterns of FGF and KGF binding by these two closely related human receptors (Fig. 4). Determination of ligand-binding specificity by alternative splicjng' The high degree of sequence identity of the FGFR-2 and KGFR strongly suggested that both the receptor species were encoded by the same gene. We reasoned that their divergent regions were encoded by different exons (exons K and B for KGF and bFGF, respectively) located between common upstream and downstream exons (U and P, respectively). To map these putative exons within the genomic sequence, we first compared nucleotide sequences of the divergent region. Two possible alternative locations of such exons could be postulated, and PCR analysis was performed to investigate the locations of these exons. The intron/exon map of this region was determined as shown in Fig. 5. Some of the PCR products were cloned and sequenced, and consensus sequences for intron/exon junctions were found in the expected positions. All of these results established that two receptors with different ligand-binding specificities were encoded by the same human gene (bek) and messages for the two receptors were generated by alternative splicing (Miki et al., 1991c) .

KGFR-type splicing

u

o

Fig. 5. The intron/exon structure of a part of the human bek gene which gives rise to the divergence. Exons and introns are shown by boxes and thick lines, respectively, with the approximate sizes in kb. The splicing events specific to generate the two receptors are also shown.

298 Tissue-specific expression of KGFR and FGFR-2 alternative transcrjpts·

To examine the level at which

expre~sion

of the two

receptor species might be regulated in various cell types, we used the PCR primers specific for the unique alternative exons of the KGFR and FGFR-2.

cDNA was synthesized from RNAs of different

human cell types using a primer specific to both the alternative transcripts.

The source of the RNAs include mammary epithelial

cells (B5/589), fibroblasts (M426), vesicular endothelial cells, melanocytes, and monocytes, as well as several tumor cell lines. The synthesized cDNA was then used to amplify KGFR or FGFR-2 specific sequences from the exon-specific primers.

Fig. 6 shows

the striking contrast in patterns observed

with

only one of the

alternative transcripts demonstrated in

each

of

analyzed.

While

the

epithelial

cells

KGFR FGFR2

expressed

the

cells

transcripts

- 162 bp

...,

... --"--

.... ---

- 153 bp

Fig 6. Differential expression of the messages for KGFR and FGFR-2 in various cell lines. Poly (A)+ RNAs extracted from the cells shown at the top were reverse-transcribed by a primer which can hybridize with the messages for either of the receptor species. Segments specific to KGFR and FGFR-2 are amplified from aliquote of the synthesized cDNAs by PCR using the primers specific to KGFR or FGFR-2. Human placenta DNA was used as a positive control for PCR to show equal amplification of the segments of KGFR and FGFR-2 cDNAs (first lane). PCR assay was also performed without template for a negative control (second lane). In other lanes, cDNAs synthesized from human poly(A)+. RNA were used as templates. Normal cells; B5/589 (mammary epithelial cells), M426 (lung embryonic fibroblasts), umbilical cord endothelial cells, monocytes from peripheral blood, and melanocytes. Cell lines established from tumors; Al623 (anaplastic tumor groin node), OM431 (eye melanoma), A172 (glioblastoma), and Jurcat (lymphoma). PCR products were separated by electrophoresis on a 3% NuSieve-GTG/l% SeaKem-GTG agarose gel. Location of the expected PCR products are shown at right in base pairs.

299 containing only the KGFR specific exon sequence, each of the other cell types expressed transcripts to corresponding to FGFR-2 specific exon sequence. These findings are in complete accordance with the tightly restricted specificity of KGF for cells of epithelial derivation (Rubin et al., 1989), suggesting that target cell-specificity of KGF was determined by cell typespecific alternative splicing of the bek gene transcript. The evolution of increasingly complex multicellular organisms has been associated with significant expansion of gene families of growth factors and their receptors. In general, this has been associated with gene duplications. In our present studies, the KGFR alternative transcript was found to be specific to epithelial cells, while the FGFR-2 transcript was detected in cells of a variety of other tissue types. The strikingly different ligand-binding affinities of these two receptors encoded by a single gene combined with their different patterns of expression provides a new dimension to growth factor receptor diversity and may reflect a general mechanism for increasing the repertoire of these important cell surface molecules.

REFERENCES Bottaro, D.P., Rubin, J.S., Ron, D., Finch, P.W., Florio, C., and Aaronson, S.A. (1990) J. BioI. Chem. 2£5, 12767-12770. Dionne, C.A., Crumley, G., Bellot, F., Kaplow, J.M., Searfoss, G., Ruta, M., Burgess, W.H., Jaye, M., and Schlessinger, J. (1990) EMBO J. ~, 2685-2692. Finch, P.W., Rubin, J.S., Miki, T., Ron, D. and Aaronson, S.A. (1989) Science 215, 752-755. Hattori, Y., Odagiri, H., Nakatani, H., Miyagawa, K., Naito, K., Sakamoto, H., Katoh, 0., Yoshida, T., Sugimura, T., and Terada, M. (1990) Proc. Natl. Acad. Sci. USA~, 5983-5987. Houssaint, E., Blanquet, P.R., Chanpion-Arnaud, P., Gesnel, M.C., Torriglia, A., Courtois, Y., and Breathnach, R. (1990) Proc. Natl. Acad. Sci. USA~, 8180-8184. Kornbluth, S., Paulson, K.E., and Hanafusa, H. (1988) Mol. Cell. BioI. ~, 5541-5544. Lee, P.L., Johnson, D.E., Cousens, L.S., Fried, V.A., and Williams, L.T. (1989) Science 215, 57-60. Miki, T., Matsui, T., Heidaran, M.A. and Aaronson, S.A. (1989) Gene H3, 137-146. Miki, T., Fleming, T.P., Bottaro, D.P., Rubin, J.S., Ron, D. and Aaronson, S.A. (1991a) Science 251, 72-75.

300 Miki, T., Fleming, T.P., Crescenzi, M., Molloy, C.J., Blam, S.B., Reynolds, S.H., and Aaronson, S.A. (1991b) Proc. Natl. Acad. Sci. USA ~, 5167-5171. Miki, T., Bottaro, D.P., Fleming, T.P., Smith, C.L., Burgess, W.H., Chan, A.M.-L. , and Aaronson, S.A. (1991c) Proc. Natl. Acad. Sci. USA (in press) . Pasquale, E.B. (1990). A distinctive family of embryonic protein-tyrosine kinase receptors. Proc. Natl. Acad. Sci. USA .al, 5812-5816. Rubin, J.S., Osada, H., Finch, P.W., Taylor, W.G., Rudikoff, S., and Aaronson, S.A. (1989) Proc. Natl. Acad. Sci. USA ~, 802806. Ruta, M., Burgess, W., Givol, D., Epstein, J., Neiger, N., Kaplow, J., Crumley, G., Dionne, C., Jaye, M., and Schlessinger, J. (1990) Proc. Natl. Acad. Sci. USA~, 87228726. Wigler, M., Silverstein, S., Lee, L.-S., Pellicer, A., Cheng, Y.C., and Axel. R. (1977) Cell,~, 223-232.