(Received for publication, September 21, 1994)
From the
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.
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()(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, (
)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.
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.
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.
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.
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
-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).
M,
-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.
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.
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.
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 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.