©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification and Characterization of a Functional Murine FLT3 Isoform Produced by Exon Skipping (*)

(Received for publication, September 21, 1994)

Chrystel Lavagna (§) Sylvie Marchetto Daniel Birnbaum Olivier Rosnet

From the Laboratoire d'Oncologie Moléculaire, INSERM U.119, 27, Bd. Leï Roure, 13009 Marseille, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The FLT3 gene encodes an hematopoietic receptor related to the receptors for colony-stimulating factor 1, FMS, and for Steel factor, KIT. The extracellular part of these molecules is exclusively composed of five immunoglobulin (Ig)-like domains, designated 1 to 5, from the amino terminus to the carboxyl terminus of the extracellular region. We have isolated a unique murine FLT3 cDNA that codes for a variant isoform of FLT3, devoid of the fifth Ig-like domain, by comparison with the prototypic form. The corresponding mRNA is the result of a splicing event that leads to the elimination of two coding exons. mRNA coding for this variant was detected in almost all the tissues expressing the mRNA coding for the prototypic molecule, although at a lower level. Ligand-induced tyrosine phosphorylation of the two isoforms was equivalent in COS-1 transfected cells, indicating that the fifth Ig-like domain is not strictly necessary for either ligand-binding or kinase activation.


INTRODUCTION

Cell growth and/or differentiation frequently involve activation of receptor-type tyrosine kinases at the cell surface. Receptor-type tyrosine kinases constitute a large family of related molecules grouped into several classes, mainly defined by structural similarities in the extracellular domains of these proteins. Stimulation of these molecules by their specific ligands induces receptor dimerization and intermolecular phosphorylation(1, 2, 3, 4, 5) . This leads to the appearance of phosphotyrosines which are docking sites for various cytoplasmic substrates and/or adaptor molecules implicated in regulatory events and signal transduction(6, 7, 8) . Among these are phospholipase C, phosphatidylinositol-3-kinase, GTPase activating protein, and GRB2/SEM5.

Hematopoiesis is a highly complex system in which a small number of self-renewing stem cells continuously generate mature progeny in the myeloid, erythroid, megacaryocytic, and lymphoid lineages. Three receptor-type tyrosine kinases that belong to a specific class, class III, participate in the regulation of hematopoietic progenitor development and mature cells activity. They are FMS, a receptor for the myelomonocytic specific factor CSF-1(^1)(9) , KIT, whose ligand, Steel factor, is involved in the development and function of several hematopoietic cell types, and, in addition, in the migration and maturation of some neural crest-derived cells(10, 11, 12) , and FLT3, (^2)also named FLK2, a receptor expressed in lympho-hematopoietic tissues, the central nervous system, and the placenta(13, 14, 15) . At the cellular level, FLT3/FLK2 transcripts have been detected in hematopoietic stem cell-enriched human and murine subpopulations(15, 16) , suggesting a role in very early hematopoiesis. Using a FMS/FLT3 chimera approach, we and others have identified cytoplasmic substrates which inducibly bind to and/or are phosphorylated by FLT3(17, 18) . Those events are qualitatively similar to those following either KIT or FMS stimulation. The more striking difference is that a binding site for phosphatidylinositol-3-kinase has been found in the carboxyl terminus of FLT3 instead of residing in the kinase insert domain, as seen for KIT, FMS, and the nonhematopoietic receptors for platelet-derived growth factors, which also belong to class III(18, 19) .

A gene encoding a ligand for the FLT3 receptor (FLT3 ligand or FL) has been recently isolated and characterized(20, 21) . Multiple RNAs code for various membrane-bound and secreted FL isoforms. Recombinant soluble FL can stimulate recruitment and proliferation of human and mouse stem cells or progenitor-enriched populations, in combination with various cytokines, such as interleukin (IL)-3, IL-6, IL-7, and granulocyte macrophage-CSF.

Class III receptor-type tyrosine kinases comprise five immunoglobulin (Ig)-like domains, designated 1 to 5, from the amino terminus to the carboxyl terminus of the extracellular region. The Ig-like domains 1 to 3 of FMS (24) and KIT (22, 23) appear sufficient for ligand binding. The role of the fourth and fifth Ig-like domains is not precisely known.

We describe here the isolation of a variant form of the mouse FLT3 receptor devoid of the fifth Ig-like domain and its characterization at both the structural and functional levels.


EXPERIMENTAL PROCEDURES

cDNA Screening and Sequencing

An oligo(dT) cDNA library was constructed from poly(A) RNAs extracted from day 19.5 postcoitum mouse placentas using the Amersham cDNA synthesis and cloning kits(13) . A unique composite murine Flt3 cDNA was deduced from overlapping clones designated MP14, -37, and -58(13) . A subsequent screening was performed, using as a probe the 5` extremity of another clone, MP47, corresponding to nucleotides 94 to 460. A single full-length 3.2-kb-long cDNA was obtained, designated MP61, that differs from previous clones by a deletion of nearly 300 bp, located in a PstI-BglII fragment. This fragment was subcloned in appropriate Bluescript vector (Stratagene, La Jolla, CA) and sequenced by the dideoxynucleotide method, using double-stranded templates and T7 Sequencing Kit (Pharmacia Biotech). Sequence analysis was carried out using PC-Gene software (Intelligenetics).

Expression Analysis in Mouse Tissues Using RT-PCR

cDNA was prepared from 2 µg of total RNA, using random hexamers as primers, and the SuperScriptII reverse transcriptase (Life Technologies, Inc.). PCR was performed in a PREM apparatus (Wessex International, Andover Hampshire, UK), using an equivalent of 500 ng of reverse-transcribed RNA (or 1 ng for the beta(2)-microglobulin control) and 50 pmol of each nucleotide as primers. Reactions were performed in a total volume of 50 µl, using 1 unit of Taq polymerase (Promega) in the recommended buffer. The first cycle was run as follows: denaturation at 94 °C for 2 min, annealing at 55 °C for 2 min, and synthesis at 72 °C for 3 min. The next 28 cycles were run using the same parameters except that the denaturation step was only 1 min. For the last cycle, synthesis time was 7 min. Oligonucleotides for Flt3 expression analysis were: sense primer 5`-TTAAAGCGTACCCACGAATCCG-3` and antisense primer 5`-CTGACTCTCGTACCTAAATTGC-3`. Sense primer 5`-TGGTGCTTGTCTCACTGACC-3` and antisense primer 5`-ATAGAAAGACCAGTCCTTGC-3` were used for the beta(2)-microglobulin control. One-fifth of the amplified products was run in 2,5% agarose gels in 1 times TBE and transferred onto Nytran membranes (Schleicher and Schuell, Dasel, Germany) in 20 times SSC. Hybridization was performed at 42 °C in 6 times SSC and 1 times Denhardt's, and washings were performed at 42 °C in 2 times SSC and 0.1% SDS. The probes were internal oligonucleotides (-P labeled with T4 polynucleotide kinase): 5`-TGTTGTCTTGGATGAAAGG-3` for Flt3 and 5`-ATTTCAATGTGAGGCGGGTGG-3` for beta(2)-microglobulin.

As a control experiment (see ``Results''), the 320-bp Flt3 amplification product from birth placenta was excised from gel and DNA-eluted by centrifugation on a nylon wool cushion in a 1.5-ml bottom pierced tube. 1 µl of this DNA was amplified again as described above, as well as 1 µl of the library-derived truncated cDNA. 5 µl of the resulting amplification products were digested by frequent cutter restriction enzymes and run on a 3% agarose gel.

Construction and Screening of a Murine Genomic Library

A cosmid library was constructed from EcoRI partial digest of DNA extracted from NIH3T3 cells. DNA fragments 35-45 kb long were purified by velocity centrifugation in a 15-40% sucrose gradient and ligated to EcoRI-cleaved, phosphatase-treated pHC79 DNA vector (Boehringer Mannheim, Mannheim, Germany). Ligated material was packaged into phage particles using Gold Gigapack Packaging Extracts (Stratagene) and introduced into 490A Escherichia coli cells. The nonamplified library was screened under stringent conditions (6 times SSC, 1 times Denhardt's, 5 µg/ml denatured salmon sperm DNA at 68 °C for 20 h) with a mix of three P-labeled cDNA probes corresponding to the 5`, central, and 3` parts of the Flt3 cDNA.

DB-6 Cosmid Mapping and Subcloning

Exon locations in the DB-6 cosmid were determined by Southern blot hybridization as described (25) , using, as a P-labeled probe, the 608-bp DNA obtained after PCR amplification of the Flt3 cDNA, using primers described above. Hybridizing restriction fragments were subcloned and sequenced as described above.

Expression of FLT3 and FLT3-Ig in COS-1 Cells

cDNAs coding for FLT3 (26) and FLT3-Ig were cloned in the SV40 promoter-based expression vector pECE(27) . COS-1 cells were grown in Dulbecco's modified Eagle's medium, 10% heat-treated fetal calf serum and supplemented with penicillin and streptomycin. For transient transfections, 3 times 10^5 cells were seeded in 90-mm culture plates, grown for 18 h, and transfected using 5 µg of pECE-FLT3 or pECE-FLT3Ig plasmids and 25 µl of Lipofectin (Life Technologies, Inc., Grand Island, NY), following recommendations of the furnisher. Cells were either analyzed by metabolic labeling or ligand-stimulated at day 3 post-transfection.

Metabolic Labeling with [S]Methionine, Immunoprecipitation, and Analysis on Polyacrylamide Gels

Transfected COS-1 cells were incubated for 30 min in modified Eagle's medium supplemented with Earle's salts without methionine (Life Technologies, Inc.) supplemented with 3% fetal calf serum. Cells were then labeled in some medium with [S]methionine (2 MBq/µl) for 3-4 h. After labeling, cells were washed twice in phosphate-buffered saline and lysed in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate, 100 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Lysates were cleared by centrifugation and incubated for 1 h with protein A coupled to Sepharose and preimmune rabbit serum to reduce nonspecific binding. Immunoprecipitations were performed on precleared supernatants with a 1:100 dilution of anti-FLT3 antiserum (26) and protein A-Sepharose. Immune complexes were washed three times in lysis buffer, heated in SDS/2-mercaptoethanol-containing loading buffer, and separated by electrophoresis on 6.8% polyacrylamide gels. Gels were treated with AMPLIFY (Amersham) prior to drying and exposure to x-ray films (Kodak XAR).

Cell Stimulation with FLT3 Ligand

At day 3 post-transfection, 2 ml of culture medium were removed from COS-1 plates and prewarmed with or without 20 units of E. coli-produced murine FLT3 ligand (21) and added for 5 min to the plates from which the remaining medium had been removed. Cells were lysed in the same lysis buffer as above, supplemented with 100 µM sodium orthovanadate.

Western Blot Analysis

Immune complexes were separated by polyacrylamide gel electrophoresis (PAGE) on a Mini-V 8-10 vertical gel electrophoresis apparatus (Life Technologies, Inc.) and transferred to Immobilon membrane (Millipore, St-Quentin-Yvelines, France) using the same apparatus. The membrane was probed with the anti-phosphotyrosine monoclonal antibody 4G10 (UBI, Lake Placid, NY) at a 1:1000 dilution, stripped, and probed again with the anti-FLT3 antibody (26) at a 1:500 dilution. Washing and probing were performed in Tris-buffered saline, 0,5% Tween 20. Horseradish peroxidase-coupled anti-mouse or anti-rabbit immunoglobulin antibodies (Immunotech S.A., Marseille, France) were used for detection. Revelation was performed with an ECL kit (Amersham, Les Ullis, France). Scanning was carried out using an OmniMedia Scanner (XRS, Torrance, CA) and a Bio Image Application System (Millipore).


RESULTS

Cloning of a Variant Mouse FLT3 cDNA

We screened a cDNA library constructed from day 19.5 postcoitum placenta to clone cDNA sequences corresponding to the major 3.5-3.7-kb-long FLT3 transcript expressed in this tissue. Among several clones representative of this mRNA, a 3.2-kb clone, MP61, had an internal sequence deletion of 300 bp when compared to the others (see Fig. 1A). Further characterization of this clone by restriction analysis showed that a missing sequence contained within a BglII-PstI restriction fragment (Fig. 1A) was the only difference with the prototypic Flt3 cDNA. This fragment was subcloned and sequenced. The missing part consisted of a 288-bp sequence coding for the fifth Ig-like domain and adjacent amino acids. The original reading frame was preserved (Fig. 1, B and C) so that this cDNA codes for a FLT3 isoform in which the sequence corresponding to residues 438 to 534, according to (13) , is replaced by a unique serine residue (Fig. 2A). The truncated FLT3 isoform is hereafter referred to as FLT3-Ig (Fig. 2B).


Figure 1: The Flt3 cDNA. A, schematic representation and restriction map of the murine Flt3 cDNA. The box represents the coding region. The shaded part is the sequence missing in the variant MP61 cDNA. Restriction enzyme sites are indicated. B, nucleotide sequences at the boundaries of the alternative region, in the prototypic Flt3 cDNA. Six codons are presented, along with the encoded amino acids. C, sequence found in the MP61 cDNA clone and resulting amino acids.




Figure 2: Structure of the FLT3 protein and variable region. A, juxtamembrane variable regions of FLT3 and FLT3-Ig. The fifth Ig-like domain sequence is detailed (top) along with the unique serine residue present in FLT3-Ig (bottom). Putative intramolecular disulfide bonds are shown, and putative N-glycosylation sites are indicated by brackets. B, schematic representation of the FLT3 and FLT3-Ig protein. Loops represent the Ig-like domains. Shaded and black boxes represent the transmembrane and the split tyrosine kinase domains, respectively. Putative N-glycosylation sites are indicated by black circles. Ig-like domains are numbered from the amino to the carboxyl terminus.



Genomic Organization of the Murine Flt3 Gene in the Region Coding for the Fifth Ig-like Domain

To better define the mechanism leading to the generation of such a variant Flt3 mRNA, we examined the exon organization in the region coding for the missing Ig-like domain and the surrounding sequences. Cosmids corresponding to the mouse Flt3 gene were isolated. One of them, called DB-6, contained coding sequences that corresponded to the Ig-like domains, as well as the 3`-untranslated sequences of Flt3, and was chosen for further study.

Four exons were found to code for amino acids 404 to 569, corresponding to a sequence of the FLT3 protein stretching from the amino terminus of the fourth Ig-like domain to the end of the transmembrane segment. The location and size of these exons, labeled A to D, are shown in Fig. 3and Table 1. Intron sizes and typical splice acceptor and donor sequences, matching canonical sites defined by Mount(49) , are also indicated (Table 1). Two of these exons are absent in the mRNA coding for FLT3-Ig. Introns on the 5` side of exon B and 3` side of exon C, are, respectively, phase II and phase I introns so that the original reading frame is preserved.


Figure 3: Restriction map, exon-intron structure, and protein correspondence of the Flt3 gene in the region coding for the variable FLT3 portion. Lower panel, restriction map of the genomic region hybridizing to the probe corresponding to the Flt3 cDNA variable region (see ``Experimental Procedures''). Coding exons are shown as black boxes. Broken lines indicate the DB-6 cosmid sequences that were not checked thoroughly in this study. Restriction enzyme sites used in the subcloning strategy are shown. Upper panel, schematic representation of the FLT3 protein in the region coded by exons A, B, C, and D. Correspondence between protein subdomains and their respective coding exons is shown by the thin stippled lines.





Previous studies have demonstrated that the FMS and KIT genes, which code for class III receptor-type tyrosine kinases highly related to FLT3, show striking similarities in their genomic structures, not only in the region coding for the evolutionary conserved kinase domain but also in that coding for the extracellular portion of the receptor(28, 29, 30, 31) . This observation was recently extended to the human FLT3 gene, in the region that codes for the cytoplasmic domain(32) . The portion of the extracellular coding region of the murine Flt3 gene which we analyzed shows a similar conservation with FMS and KIT, as shown by the organization and sequences of the corresponding protein subdomains depicted in Fig. 4. Exons A, B, C, and D of Flt3 correspond to exons 7, 8, 9, and 10 of KIT(28) and exons 8, 9, 10, and 11 of FMS(33) , respectively.


Figure 4: Comparison of the mouse FLT3 receptor with human KIT and FMS proteins in the extracellular juxtamembrane and transmembrane regions: conservation of exon-encoded subdomains. Below the three sequences, the amino acid positions identical in the three molecules are indicated by stars, whereas identities between any two molecules are indicated by dots. Gaps were introduced to maximize the alignment. The position of the amino terminus of the fourth Ig-like domain (IG4), the fifth Ig-like domain (IG5), and the transmembrane region (TM) are indicated by heavy lines above the sequences. The amino acid numbers of the first and last residues of each sequence are shown and defined according to Refs. 13, 46, and 47. Boxes indicate FLT3 sequences encoded by exons A, B, C, and D and sequences encoded by homologous exons of KIT and FMS.



mRNA Expression of Flt3 and Flt3-Ig in Mouse Tissues

We have designed oligonucleotide primers that could detect, in RT-PCR experiments, the two transcripts coding for FLT3 and FLT3-Ig (see ``Experimental Procedures''). Incidentally, we confirmed that RT-PCR products corresponding to the major 3.5-3.7-kb mRNA, coding for the prototypic FLT3 protein, were predominantly detected in developing placenta, in the central nervous system and in lymphohematopoietic organs (Fig. 5, A and B). It could also be detected, albeit at a lower level, in the other tissues tested. An amplification product corresponding to the FLT3 variant was consistently observed in the tested tissues, together with the larger product, but at a much lower level, with the exception of ovary where it was undetectable (Fig. 5B). To confirm that the lower PCR product corresponded to the FLT3-Ig-coding mRNA, the corresponding band was excised from the gel, amplified again, and digested with frequent cutting restriction enzymes, together with an amplification product using the library-derived truncated cDNA as a template. Restriction patterns showed total identity (Fig. 6).


Figure 5: Detection of FLT3 and FLT3-Ig mRNAs by RT-PCR in mouse tissues. Reverse-transcribed RNAs from tissues were amplified by PCR (30 cycles, annealing at 55 °C) for a 608- or 320-bp sequence, respectively, corresponding to mRNAs coding for the FLT3 and FLT3-Ig isoforms, using Flt3 specific oligonucleotide primers (see ``Experimental Procedures''). A negative control was performed, using reverse-transcribed water as a template. Positive controls used cloned cDNA coding for FLT3 or FLT3-Ig as templates in PCR amplifications. Amplification of beta(2)-microglobulin mRNA was used as an internal control and yielded a 221-bp product. One-fifth of the resulting amplification products was run on a 2.5% agarose gel. Panel A, ethidium bromide stained resulting gels. O, negative control; FLT3 and FLT3-Ig, positive controls; PL.X, placenta at X days p.c.; PL.B., placenta at birth; , DNA molecular weight markers (X174/HaeIII DNA fragments). beta(2)M, beta(2)-microglobulin control. Panel B, same gels as in panel A were blotted on nylon membranes and hybridized with radiolabeled oligonucleotides internal to the amplified sequence (see ``Experimental Procedures'').




Figure 6: Identity in restriction enzyme pattern between products obtained from PCR amplification of a library-derived cloned cDNA and tissue-derived total cDNA. PCR amplifications were performed using as templates the 320-bp band excised from the gel (lanes 1, see Fig. 5) or a library-derived cloned cDNA (lanes 2, see ``Experimental Procedures''). Aliquots of each PCR product were digested with the indicated restriction enzymes.



Transient Expression of FLT3-Ig in COS-1 Cells

In order to demonstrate that the variant cDNA was able to code for a protein of expected characteristics of mobility in polyacrylamide gel, it was cloned in the SV40-based eucaryotic expression vector pECE (see ``Experimental Procedures''), and expressed in COS-1 monkey cells. The cDNA drove the synthesis of two polypeptides of apparent molecular masses 115 and 130 kDa (Fig. 7). These two polypeptides are likely to be qualitatively equivalent to the 132- and 155-kDa forms of the prototypic FLT3 receptor (Fig. 7). They are, respectively, high mannose type and complex carbohydrate glycosylated proteins, the latter being expressed at the cell surface(26) . Amino acid sequences of FLT3 and FLT3-Ig predict core proteins of 110,500 and 100,000 Da, respectively, so that the protein sizes observed on the gel suggest that one or more functional glycosylation sites are present on the sequence missing in FLT3-Ig. Three such putative sites have been found in this region, one between the fourth and fifth Ig-like domains, and two in this latter domain itself (see Fig. 2A).


Figure 7: Expression of FLT3-Ig and FLT3 in COS-1 cells. COS-1 cells were transfected with SV40-based expression vectors for FLT3-Ig (lane 2), FLT3 (lane 3), and FLT4 (lane 1), a related receptor-type tyrosine kinase(48) , labeled with [S]methionine, lysed, and submitted to immunoprecipitation using an anti-FLT3 antiserum, as described under ``Experimental Procedures.'' Autoradiogram of SDS-PAGE analysis of the precipitated proteins is shown. Specific bands are indicated by arrowheads. Molecular size markers are shown on the left.



Ligand-induced Phosphorylation of FLT3 and FLT3-Ig

Ligand stimulation of class III receptor-type tyrosine kinases induces dimerization and transphosphorylation of the receptors on tyrosine residues, unmasking binding sites for SH2 domains containing molecules involved in intracellular signal transduction. We used recombinant FLT3 ligand to assess the responsive capacity of FLT3-Ig expressed in Cos-1 cells. In spite of a high constitutive phosphorylation state of both glycosylated forms of FLT3 (as observed by others(34) ) and FLT3-Ig, a clear increase in the phosphotyrosine content of the mature upper forms was induced upon stimulation with the FLT3 ligand (Fig. 8). This increase was estimated at 2.3-fold by densitometric analysis.


Figure 8: Effect of the FLT3 ligand on tyrosine phosphorylation of FLT3 and FLT3-Ig in transiently transfected COS-1 cells. COS-1 cells were transfected to express FLT3 or FLT3-Ig. Three days post-transfection, cells were incubated for 5 min with 20 units/ml FLT3 ligand (lanes labeled +) or with medium alone (lanes labeled -) and then lysed and immunoprecipitated with a specific FLT3 antiserum. Immunoprecipitated proteins were run on an SDS-6.8% polyacrylamide gel and blotted onto a nylon membrane as described under ``Experimental Procedures.'' Membranes were subsequently incubated with an anti-phosphotyrosine monoclonal antibody (leftmost panel) or an anti-FLT3 antiserum (rightmost panel). Molecular size markers are shown on the left.




DISCUSSION

Cell surface recognition molecules are usually composed of different domains which are structural and/or functional elements. Among receptor-type tyrosine kinases, extracellular ligand binding domains are juxtapositions of typical peptidic sequences such as cysteine-rich, immunoglobulin-like, or fibronectin type III domains. Class III, IV, and V receptor-type tyrosine kinases possess structurally homogeneous extracellular regions exclusively made of, respectively, two or three, five, and seven immunoglobulin (Ig)-like domains.

A striking feature of FGF receptor genes, which code for class IV receptor-type tyrosine kinases, is the expression of a multitude of structural variants, most of which are generated by alternative splicing(35, 36) . Such events can lead to the expression of isoforms devoid of the first amino-terminal Ig-like domain, without affecting ligand binding.

The existence of an isoform containing four Ig-like domains, such as that reported here for FLT3, has not been reported for other class III receptor-type tyrosine kinases. This variant results from a splicing event which lead to the elimination of two exons, both the length and location of which are conserved in the FMS and KIT genes. The transcripts of the two FLT3 isoforms, detected by RT-PCR, appear consistently co-expressed, although at different levels, the larger one being predominantly expressed. In accordance with the low level of expression of the shorter transcript, we could not detect a 3.3-kb Flt3 mRNA in Northern blots experiments, except in testis. Ligand stimulation of FLT3-Ig expressing Cos-1 cells induced tyrosine phosphorylation of the receptor to the same level as the five Ig-like domain-containing molecule.

The role of the fifth Ig-like domain in these receptor-type tyrosine kinases remains unknown. The availability of a variant isoform of FLT3 devoid of this domain allows us to test whether such a molecule is still functional. We found that the missing fifth domain is dispensable for both ligand binding and for the conformational change inducing kinase activation. It has been previously hypothesized that the corresponding platelet-derived growth factor receptor alpha Ig-like domain, classified as a V-like domain(37) , could be involved in stabilization of dimers after ligand fixation(38) . As ligand-induced receptor phosphorylation occurs in trans in the receptor dimer, our data could indicate that dimer instability would not preclude an intersubunit phosphorylation. Several groups have described the occurrence of two KIT isoforms which differ by the absence or presence of an asparagine-rich tetrapeptide amino-terminal to the transmembrane domain, as the result of alternative use of two splice donor sites(40, 41, 42) . The KIT isoform lacking the insert is more efficient in transduction of a mitogenic signal and is constitutively activated, in vitro, at a basal level(41) . The insert could thus negatively modulate dimerization. Whether the absence of the fifth FLT3 domain mimics this latter situation will await the generation of FLT3 ligand growth-responsive stably transfected cell lines. However, Blechman et al.(39) recently suggested that a putative dimerization site may be located on the fourth rather than on the fifth Ig-like domain.

Alternatively, the fifth Ig-like domain could play a role in the induction of specific intracellular signaling events. Indeed, extracellular cytokine receptor domains have been shown to participate in the specificity of cytokine-induced cellular tyrosine phosphorylations(43) . In addition to classical pathways initiating from the interaction of SH2-containing molecules with receptor phosphotyrosines, other transmembrane signaling molecules could interact with FLT3 via the fifth Ig-like domain.

Finally, the fifth domain could be involved in ligand specificity. Deletion of one of the two Ig-like domains of the B7 surface molecule can alter its binding to CTLA4 but not to CD28(45) , indicating that Ig-like domain cooperation can bring additional ligand binding specificity. Moreover, we cannot exclude an interaction of this particular domain with an alternative unidentified ligand or a particular isoform of the recently identified FLT3 ligand. In the case of FGFR2, the second and third Ig-like domains have been shown to be, individually, sufficient for binding to different FGFs(44) .

In conclusion, it is likely that the prototypic FLT3 protein is the major effector molecule, whereas the FLT3-Ig isoform could play a regulatory or accessory role. Delineation of signal transduction events, interacting proteins, and receptor modulation for both FLT3 and FLT3-Ig will bring more information about FLT3 regulation and function.


FOOTNOTES

*
This work was supported by INSERM and by grants from the ``Association pour la Recherche sur le Cancer'' and the ``Comité des Bouches du Rhône de la Ligue Nationale Contre le Cancer.'' 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.

§
Recipient of a fellowship from the ``Ministère de l'Enseignement Supérieur et de la Recherche.''

(^1)
The abbreviations used are: CSF-1, colony-stimulating factor 1; RT-PCR, PCR amplification of reverse-transcribed RNAs; bp, base pair(s); kb, kilobase(s); FGF, fibroblast growth factor; FGFR, FGF receptor; PAGE, polyacrylamide gel electrophoresis.

(^2)
The nomenclature for genes and proteins is as follows: FLT3, protein; FLT3, human gene for FLT3; Flt3, mouse gene for FLT3.


ACKNOWLEDGEMENTS

We are grateful to our colleagues of the Molecular Oncology group for helpful discussions and to F. Birg, C. Mawas, and P. Dubreuil for critical reading of the manuscript. We also thank S. Sealand (Schering Plough, Lyon, France) and F. Lee (DNAX Research Institute, Palo Alto, CA) for providing the FLT3 ligand.


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