Stem Cell Factor Presentation to c-Kit
IDENTIFICATION OF A BASOLATERAL TARGETING DOMAIN*
Bernhard
Wehrle-Haller
and
Beat A.
Imhof
From the Department of Pathology, Centre Medical Universitaire, 1 Rue Michel-Servet, 1211 Geneva 4, Switzerland
Received for publication, September 12, 2000, and in revised form, December 28, 2000
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ABSTRACT |
Stem cell factor (also known as mast cell
growth factor and kit-ligand) is a transmembrane growth factor with a
highly conserved cytoplasmic domain. Basolateral membrane expression in
epithelia and persistent cell surface exposure of stem cell factor are
required for complete biological activity in pigmentation, fertility,
learning, and hematopoiesis. Here we show by site-directed mutagenesis
that the cytoplasmic domain of stem cell factor contains a monomeric leucine-dependent basolateral targeting signal. N-terminal
to this motif, a cluster of acidic amino acids serves to increase the
efficiency of basolateral sorting mediated by the leucine residue.
Hence, basolateral targeting of stem cell factor requires a
mono-leucine determinant assisted by a cluster of acidic amino acids.
This mono-leucine determinant is functionally conserved in
colony-stimulating factor-1, a transmembrane growth factor related to
stem cell factor. Furthermore, this leucine motif is not capable of
inducing endocytosis, allowing for persistent cell surface expression
of stem cell factor. In contrast, the mutated cytoplasmic tail found in
the stem cell factor mutant MgfSl17H
induces constitutive endocytosis by a motif that is related to signals for endocytosis and lysosomal targeting. Our findings therefore
present mono-leucines as a novel type of protein sorting motif for
transmembrane growth factors.
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INTRODUCTION |
Stem cell factor (SCF)1
belongs to the family of cell surface-anchored growth factors with
highly conserved cytoplasmic domains, which includes the related
colony-stimulating factor-1 (CSF-1) (1). SCF is expressed as two
alternatively spliced membrane-bound forms (M1 and M2), distinguished
by an exon containing a proteolytic cleavage site in the M1 form. This
site is used to generate soluble growth factor from the M1
membrane-bound precursor. The membrane anchor of SCF is required for
its biological activity in vivo because the expression of
only the extracellular receptor binding domain leads to the loss of
SCF-dependent cells affecting skin pigmentation, sterility,
hematopoiesis, and learning (2-4). Furthermore, a point mutation,
which results in the skipping of the exon coding for the cytoplasmic
tail of mouse SCF (MgfSl17H), leads to an
altered cytoplasmic sequence that abrogates coat pigmentation and male
fertility and reduces hematopoiesis (5-7). In this mouse mutant, cell
surface expression of SCF is reduced, and basolateral sorting in
epithelial tissues is lost (8). Hence, the cytoplasmic tail of SCF
harbors information required for efficient cell surface presentation
and basolateral targeting of SCF, functions that are absolutely
required to fulfill its function in vivo.
Polarized epithelial cells exhibit an apical and basolateral surface
with distinct protein compositions. Basolateral sorting of
transmembrane proteins takes place in the trans-Golgi
network (TGN) or endosomal compartments and is mediated by
clathrin-coated vesicles (9). Selective incorporation of proteins into
these transport vesicles is accomplished by adaptor complexes (10). Short cytoplasmic targeting sequences frequently containing either a
tyrosine or di-leucine motif have been identified in the sorted proteins and are required for the interaction with adaptor complexes and for basolateral transport of the proteins (11). Recently a
tyrosine-based targeting motif has been shown to bind to an epithelial
specific AP1 subunit that is required for basolateral transport (12).
When the tyrosine or the di-leucine sorting domains are removed from
the proteins, apical instead of basolateral sorting occurs, mediated by
N-linked carbohydrates or by association with lipid rafts
(13-15). Some basolateral sorting signals resemble endocytic signals
used to incorporate membrane proteins into clathrin-coated pits at the
plasma membrane, suggesting that basolateral sorting and endocytosis
are regulated by similar mechanisms. For example, the macrophage Fc
receptor and the invariant chain of the class II major
histocompatibility complex contain a di-leucine-based determinant that
is used for basolateral sorting as well as endocytosis (16, 17).
Furthermore, many membrane proteins carry several different targeting
determinants, which enables them to shuttle between the basolateral
plasma membrane and endosomes (18, 19).
Although SCF does not have typical tyrosine or di-leucine sorting
sequences in its cytoplasmic tail, it is delivered directly to the
basolateral cell surface in epithelial cells and does not accumulate in
an intracellular compartment (8). Consequently, SCF remains at the cell
surface until the extracellular domain is proteolytically shed within
0.5 (M1) to 5 (M2) h depending on the respective splice form (20).
Because basolateral sorting is critical for the proper biological
function of SCF, we tried to identify the possibly novel basolateral
targeting determinant in the cytoplasmic tail of SCF. To do so, we used
reporter constructs consisting of extracellular green fluorescent
protein (GFP)-tagged SCF or chimeras of the extracellular domain of the
interleukin-2 receptor
-chain (Tac) fused to the transmembrane and
cytoplasmic sequences of SCF. In these chimeras the normal
intracellular domain of SCF is left intact, allowing optimal
interaction of the latter with the sorting machinery of polarized cells
and the identification of critical targeting domains by mutagenesis.
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EXPERIMENTAL PROCEDURES |
SCF Chimeras and Site-directed Mutagenesis--
cDNA for SCF
and Tac were kindly provided by Drs. John Flanagan (Harvard Medical
School, Boston) and Pierre Cosson (University of Geneva, Switzerland),
respectively. Mouse CSF-1 and mouse tyrosinase cDNAs were kindly
provided by Drs. Willy Hofstetter (MMI, Bern, Switzerland) and
Friedrich Beermann (ISREC, Lausanne, Switzerland), respectively.
SCF-GFP chimeras were constructed in pcDNA3 (Invitrogen, Groningen,
The Netherlands) by inserting the enhanced GFP sequence (CLONTECH Laboratories, Palo Alto, CA) together
with a Myc tag 5' into the exon 5/6 junction of SCF (SSTLGPEK/DSRV),
which resulted in the following sequence:
SSTLGPEQKLISEEDLGQS··IV ... (enhanced GFP) ... YK··TGPEK/DSRV (single letter amino acid code;
the sequence of the Myc tag is underlined). To prevent translation at
internal start sites producing cytoplasmic GFP, we replaced the start
codon of GFP with nucleotides coding for a ClaI site. A
unique PinAI site was introduced C-terminal to the GFP
sequence to swap wild-type and mutant cytoplasmic tail sequences at
this site.
To generate the Tac-SCF chimera (all in pcDNA3), the transmembrane
and cytoplasmic domains of SCF were swapped at a unique BglII site in Tac located at a homologous leucine (L) and
glutamine (Q) residue upstream of the transmembrane sequences of SCF
and Tac. This resulted in the sequence: ...
SIFTTDLQWTAMALP ... at this position
(conserved LQ is bold and transmembrane residues of SCF are underlined).
The Tac-CSF-1 and Tac-tyrosinase (Tac-tyr) chimeras were constructed in
a similar way. CSF-1 and tyrosinase transmembrane and cytoplasmic
sequences were polymerase chain reaction amplified with a
BglII site containing the forward primers (CSF-1:
AACAGATCTCCAGATCCCTGAGTCTG; tyrosinase: AACAGATCTCCAAGCCAGTCGTATCTGG)
at a common glutamine residue (Q) and swapped with the Tac sequence of
this region creating the respective junctional sequences:
Tac-CSF-1: ... SIFTDLQIPESVFHLLV ...
and Tac-tyr: ...
SIFTTDLQASRIWPWLL ... (the common glutamine residue (Q) is bold, and the respective transmembrane region is underlined). Tac-EGFP was cloned by polymerase chain reaction amplification of EGFP with a HindIII-containing primer and
inserted at a unique HindIII site at the extreme C terminus
of Tac (TIQASSstop) resulting in the new junctional sequence
(TIQASTMV ... (EGFP)).
Site-specific mutagenesis of the cytoplasmic tail of SCF was performed
using polymerase chain reaction overlap extension. Two overlapping
polymerase chain reaction fragments containing a specific mutation were
amplified with external primers (containing either the PinAI
or BglII site for SCF-GFP or Tac-SCF chimeras, respectively)
and swapped with the wild-type sequence of the cytoplasmic tail. All
constructs were verified by dideoxy sequencing. A list of primers used
to generate the different constructs listed in Fig. 2 can be provided
upon request.
Cell Culture, Live Fluorescence Microscopy, and
Immunocytochemistry--
MDCK II cells were kindly provided by Dr.
Karl Matter (University of Geneva, Switzerland) and cultured in
Dulbecco's modified Eagle's medium (Life Technologies,
Paisley, Scotland) supplemented with 10% fetal calf serum (Inotech,
Dottikon, Switzerland). Cells at 60% confluence were transfected using
calcium phosphate as described (21), and stable clones were selected
with 0.6 mg ml
1 G418 (Life Technologies). For
each construct, at least two different clones were analyzed for the
steady-state distribution of SCF-GFP fluorescence or anti-Tac
immunohistochemistry. To visualize the GFP fluorescence, cells were
grown to confluence on glass coverslips. Prior to observation, the
culture medium was exchanged with F-12 medium (Life Technologies)
supplemented with 10% fetal calf serum to reduce autofluorescence,
which is higher in Dulbecco's modified Eagle's medium. Cells were
mounted on an inverted confocal microscope (LSM-410, Zeiss, Oberkochen,
Germany) and visualized with standard fluorescein isothiocyanate
optics. To reveal the localization of transfected Tac-SCF chimeras or
endogenous E-cadherin in SCF-GFP-transfected MDCK II cells, monolayers
of stable transfected clones grown on glass coverslips were fixed with
4% paraformaldehyde in phosphate-buffered saline for 5 min. Cells were
washed with phosphate-buffered saline, permeabilized with 1% Triton
X-100 (Sigma Chemical Co., St. Louis, MO) in phosphate-buffered saline
and blocked with 1% bovine serum albumin (Sigma) in phosphate-buffered
saline. Cells were then stained as indicated with either anti-Tac
monoclonal antibody 7G7 (22) or with anti-Arc-1 monoclonal antibody
(23), which is directed against canine E-cadherin. After washing, bound
antibodies were revealed with Texas Red-coupled anti-mouse antibodies
(Southern Biotechnoloy Associates Inc., Birmingham, AL). Fluorescence
was subsequently analyzed on a confocal microscope as indicated above. Contrast enhancement was performed in Photoshop (Adobe Systems Inc.,
San Jose, CA).
Endocytosis Assay--
Wild-type and mutant Tac-SCF constructs
were transfected into SV40-transformed African green monkey kidney
cells (COS-7) using Fugen 6 according to the manufacturer's
recommendation (Roche, Basel, Switzerland). 2 days after transfection,
cells were cooled on ice, and anti-Tac antibodies were added for 1 h at 1 µg ml
1. Prior to warming, unbound
antibodies were washed away, and internalization was allowed for 30 min
at 37 °C in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum. Antibodies that remained cell surface-bound were
subsequently removed with ice-cold acidic glycine buffer (0.1 M, pH 2.5). Cells were then fixed with 4% paraformaldehyde
for 5 min, washed, permeabilized, blocked, and stained with Texas
Red-conjugated anti-mouse antibodies (Southern Biotech) to reveal
internalized anti-Tac·Tac-SCF complexes (see above). Cells were
viewed on an Axiovert 100 microscope (Zeiss) equipped with a digital
camera (C4742-95, Hamamatsu Photonics, Shizuoka, Japan) and the Openlab
software (Improvision, Oxford, UK). Contrast enhancement was done in
Photoshop (Adobe). The experiment was performed three times with
qualitatively similar results, and representative examples of cells
from one experiment were chosen for Fig. 7.
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RESULTS |
Leucine 26 Is Required for Basolateral Targeting of SCF--
GFP
was inserted into the alternatively spliced extracellular domains of
both membrane-bound variants of SCF and transfected into polarized
epithelial MDCK II cells (Fig. 1).
Confocal microscopy revealed that both wild-type constructs accumulated
in basal and lateral membranes where they colocalized with E-cadherin,
a marker for the lateral membrane compartment in polarized epithelial
cells (shown for M2 variant; Fig. 1).

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Fig. 1.
SCF-GFP chimeras localize to the basolateral
aspect of polarized MDCK II cells. Confocal microscopy of
wild-type membrane-bound (M2) SCF-GFP proteins (A,
fluorescein isothiocyanate channel) and anti-E-cadherin staining
(B, Texas Red channel) of fixed MDCK II is shown. A
corresponding Z-scan of the monolayer is shown below. Note the overlap
of staining in lateral regions of individual cells. Above the
panels, a schematic view of the two differentially spliced
wild-type forms of SCF (SCF-M1, cleavable and SCF-M2, noncleavable) and
the chimera of SCF with GFP (SCF-GFP) is shown. SP, signal
peptide; RBD, SCF receptor binding domain; PCS
(arrow), proteolytic cleavage site; MD, membrane
domain; CT cytoplasmic tail. The bar in
B corresponds to 24 µm.
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To identify the motif in the cytoplasmic tail of SCF responsible for
basolateral sorting, we created various cytoplasmic SCF mutants of the
membrane-bound (M2) form of GFP-tagged SCF (SCF-GFP) (Fig.
2A). Mutants lacking the last
eight C-terminal amino acids (d36, d29) still localized to the
basolateral membrane. However, when 15 or more amino acids were deleted
(d22, d12), SCF-GFP was located on the apical membrane and showed no
basolateral expression. The critical region for basolateral sorting was
demonstrated to reside within the sequence
21NEISMLQQ28 because an internal deletion
mutant (d21-28) also localized to the apical membrane. Interestingly,
to be functional, it appeared that this sequence must be considerably
separated from the membrane; deletion of intervening amino acids
proximal to the membrane (d5-20) interfered with basolateral sorting.
Increasing the distance of the 21NEISMLQQ28
motif from the membrane by reinserting amino acids 5-11 (d12-20) only
partially rescued basolateral targeting, suggesting that other amino
acids important for basolateral targeting are present N-terminal to the
21NEISMLQQ28 motif (see below).

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Fig. 2.
Cytoplasmic tail sequences of wild-type and
mutant SCF. A, alignment of sequences of wild-type and
cytoplasmic tail mutants of mouse SCF and their respective steady-state
distribution in MDCK II cells. Construct 1, the name of the
constructs represents the site of amino acid deletions (marked with a
dotted line) or point mutations (bold and
underlined). Polarity 2, steady-state
localization of GFP and Tac SCF chimeric proteins in polarized MDCK II
cells (wt, wild-type; BL, basolateral;
AP, apical). B, Clustal W alignment of different
SCF cytoplasmic tail sequences. GenBank accession numbers are M59964
(human), M57647 (mouse), D13516 (chicken), and AF119044
(salamander).
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Sequence comparison among human, mouse, chicken, and salamander SCF
(24) revealed the residues 24SML26 as being
completely conserved within the 21NEISMLQQ28
motif (Fig. 2B). This sequence encompasses a serine at
position 24 as well as a di-hydrophobic methionine-leucine at positions 25 and 26, respectively (see above). To test whether a portion of this
motif was required for basolateral sorting of SCF, we created various
point mutations encompassing these conserved residues (Fig.
2B and Fig. 3). The
modification of serine 24 to either an alanine (S24A) or to an aspartic
acid (S24D) resembling a phosphoserine, as well as the replacement of
the conserved glutamic acid 19 by lysine (E19K) had no effect on
basolateral targeting (Fig. 3, C, E, F). In contrast, the
modification of leucine 26 to either alanine (L26A) or the replacement
of methionine 25 and leucine 26 by a double-alanine (M25A/L26A) led to
apical accumulation of the mutant SCF-GFP constructs (Fig. 3,
B and D). The analysis of the SCF-GFP chimeric
mutant proteins thus suggests that the leucine at position 26 of the
cytoplasmic tail of SCF is critical for basolateral sorting of SCF.
However, it is not known whether this putative basolateral signal
requires the context of dimerized SCF molecules or whether it can
provide intracellular targeting information independently.

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Fig. 3.
Leucine 26 is required for basolateral
targeting of SCF-GFP constructs in polarized MDCK II cells.
Confocal microscopy (fluorescein isothiocyanate channel) of live
wild-type (A) and mutant SCF-GFP-M2 (B-F)
expressing confluent MDCK II cells. Basolateral staining is lost upon
mutation of leucine 26 to alanine (B), or methionine 25 and
leucine 26 to a double alanine (D). Replacement of serine 24 by alanine (C) or aspartic acid (E), as well as
the change of glutamic acid 19 to lysine (F), did not alter
basolateral localization of the constructs. Below each panel
a corresponding Z-scan is shown. The bar in F
corresponds to 24 µm.
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Extracellular SCF Sequences Are Not Required for Basolateral
Targeting--
Dimer formation involving the extracellular domain of
SCF or lateral association of the extracellular and/or intracellular portions of SCF with other proteins that contain targeting information may in fact be responsible for the polarized expression of SCF. Therefore, to test the ability of the cytoplasmic targeting sequence of
SCF to mediate polarized expression independently of the extracellular domain, we replaced the latter with the extracellular domain of Tac
(Fig. 4) (22). Wild-type Tac as well as
Tac with a C-terminally fused EGFP accumulated apically when expressed
in MDCK II cells (Fig. 4A). In contrast, Tac-SCF chimeras
expressing the wild-type cytoplasmic domain of SCF localized to
basolateral membranes in a manner identical to the SCF-GFP wild-type
constructs (Fig. 4B). Likewise, constructs involving
extracellular Tac with deletion mutations of the cytoplasmic tail of
SCF (d29, basolateral, Fig. 4C; d22, apical, Fig.
4D; d21-28, apical, Fig. 4E; d5-20, apical (not shown); and d12-20, basolateral/apical, Fig. 4F),
showed identical basolateral sorting behaviors compared with the mutant SCF-GFP constructs. This indicates that the extracellular domain of SCF
is not required for basolateral targeting and that the basolateral
targeting motif of SCF contained within its cytoplasmic portion is
sufficient to direct the Tac extracellular domain basolaterally. Moreover, sequences N-terminal to methionine 25 and leucine 26 removed
in the d12-20 mutation influence the efficiency of basolateral targeting (Fig. 4F).

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Fig. 4.
The basolateral targeting determinant in SCF
acts independently of the extracellular domain. Confocal
microscopy of anti-Tac antibody stained and fixed MDCK II cells stable
transfected with Tac-EGFP (A) and wild-type (B)
or mutant (C-F) Tac-SCF chimeric constructs is shown. A
scheme representing the Tac-SCF chimera is shown above the
panels. The fusion protein consists of the extracellular
domain of Tac and the transmembrane and cytoplasmic sequence of SCF.
A, unmodified Tac with C-terminal EGFP fusion of which the
anti-Tac is antibody-stained. B, Tac-SCF chimera with
wild-type SCF sequences. C, deletion of the last 8 amino
acids from the cytoplasmic tail of SCF does not alter basolateral
targeting of the Tac hybrid (d29). However, removal of the last 15 amino acids (d22) (D) or amino acids 21-28 (E)
resulted in an apical localization of Tac-SCF. The deletion of amino
acids N-terminal to the leucine 26-containing region (d12-20) resulted
in basolateral as well as apical localization of the chimeric proteins
(F). Below each panel, a corresponding Z-scan is
shown. SP, signal peptide of SCF and Tac, respectively;
ED, extracellular domain. The bar in F
corresponds to 24 µm.
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Efficient Basolateral Targeting Is Mediated by an Acidic Cluster
N-terminal to the Monomeric Leucine Determinant--
Although it is
evident that the leucine residue at position 26 is critical for
basolateral targeting it is not known whether a second hydrophobic
residue (methionine 25) as found in all di-leucine-like determinants is
equally required for basolateral sorting of SCF. Moreover, the region
N-terminal to the ML motif which is also required for efficient
basolateral targeting (12ENIQINEED20) bears a
domain important for SCF sorting as well.
To address the first question, we replaced methionine 25 with an
alanine residue and analyzed the distribution of the Tac-SCF construct
at steady-state conditions. In contrast to the leucine 26 to alanine
mutation, the change of methionine 25 to alanine did not affect
basolateral targeting of Tac-SCF (Fig. 5,
A and B). Moreover, replacement of methionine by
leucine in an attempt to create a classical di-leucine determinant led
to intracellular and apical localization of Tac-SCF (not shown).
Therefore, this finding revealed the existence of a novel type of
leucine-based basolateral targeting signal in SCF, which does not
require a second hydrophobic amino acid to be functional.

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Fig. 5.
An acidic cluster-assisted monomeric
leucine-dependent basolateral targeting determinant.
Confocal microscopy of anti-Tac antibody-stained fixed MDCK II cells
stable transfected with Tac-SCF point mutations of hydrophobic and
acidic amino acids is shown. A, apical localization of the
Tac-SCF chimera carrying a double alanine substitution of methionine 25 and leucine 26 (M25A/L26A). B, the single point mutation at
methionine 25 to alanine did not alter basolateral targeting.
Similarly, the point mutation of glutamic acid 22 to alanine (E22A) did
not influence basolateral targeting (C). Alanine
substitution of the acidic cluster 18EED20
(E-D18A-A) resulted in basolateral as well as apical accumulation of
Tac chimeric proteins (D). Below each panel, a
corresponding Z-scan is shown. The bar in D
corresponds to 24 µm.
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Analysis of sequences N-terminal to leucine 26 which are absent in the
d12-20 mutation reveal an unusually high concentration of acidic amino
acids. An acidic cluster N-terminal to an FI motif has recently been
identified as a basolateral targeting signal in the furin protease
(25). To test whether the acidic cluster in SCF contributes to
basolateral sorting or whether other acidic amino acid residues
localized closely to the leucine residue are critical, we mutated
glutamic acid 22 (22EXXML26) to
alanine (Fig. 5C). In addition, we replaced glutamic acid 19 with a
lysine to destroy the acidic cluster formed by residues 18EED20 (Fig. 3F). Neither
modification had any effect on basolateral targeting of Tac or of the
GFP chimeric SCF constructs. However, the replacement of all three
acidic residues 18, 19, and 20, with alanine residues (E-D18A-A) did
alter basolateral sorting of the Tac-SCF chimeras. In clones
expressing relatively low amounts of the Tac-E-D18A-A chimera,
basolateral targeting was still efficient; however, in clones
expressing higher amounts of mutant Tac-SCF, both basolateral and
apical surface staining was detected (Fig. 5D). Anti-Tac
staining of these clones strongly resembled the phenotype already seen
with the d12-20 mutation (Fig. 4F). These data suggest
that the removal of the acidic cluster
(18EED20) is the cause of the phenotype of the
d12-20 mutation which results in a reduced efficiency of basolateral
transport mediated by the monomeric leucine determinant.
Comparison of the Basolateral Targeting Domain of SCF with That of
CSF-1--
SCF belongs to a large family of transmembrane growth
factors that play important roles during development, tissue
homeostasis, and hematopoiesis. Based on sequence and functional
homologies, SCF is most closely related to CSF-1 (26). The similarities between the two factors extend to their respective receptor tyrosine kinases, c-Fms, the receptor for CSF-1, and c-Kit, the
receptor for SCF, which are structurally conserved and which have
evolved by chromosomal duplication (27). Sequence comparison (Fig.
6E) of the cytoplasmic domain
of CSF-1 with that of SCF reveals in addition to the most C-terminal
valine residue, a leucine-containing motif at a position comparable to
the basolateral targeting domain of SCF. However, the cluster of acidic
amino acids N-terminal to this leucine motif is not conserved in CSF-1.
To determine the basolateral sorting activities of CSF-1, we expressed
the transmembrane and the cytoplasmic tail domains fused to the
extracellular domain of Tac and studied its steady-state distribution
in confluent monolayers of MDCK II cells (Fig. 6). The wild-type
Tac-CSF-1 chimeric construct was expressed on the basolateral surface
of MDCK II cells (Fig. 6C). However, a considerable amount
of Tac-CSF-1 was also detected on the apical surface of confluent MDCK
II cells (Fig. 6C'), a situation unlike the one observed
with wild-type Tac-SCF chimeras (Fig. 6A and A').
The distribution of Tac-CSF-1 on basolateral as well as apical surfaces
gave the impression that this construct is not sorted. To determine
whether the homologous leucine in CSF-1 can interact with the sorting
machinery of the cell, we mutated leucine 24 of CSF-1 to alanine. The
respective Tac chimera (Tac-CSF-L24A) accumulated apically (Fig.
6D'), similar to Tac-SCF-L26A (Fig. 6D'),
suggesting that the leucine at the respective position in CSF-1 is
nevertheless recognized as a basolateral sorting signal but that the
efficiency of basolateral transport is lower compared with that of
wild-type SCF. This difference may depend on the presence of the acidic
cluster in SCF which is absent from CSF-1.

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Fig. 6.
Functional conservation of the leucine
determinant in CSF-1. Confocal microscopy of anti-Tac
antibody-stained, fixed MDCK II cells stable transfected with wild-type
and leucine to alanine (L26A, SCF; L24A, CSF-1) mutation of Tac-SCF
(A and B) and Tac-CSF-1 (C and
D) chimeras is shown. A confocal section at the level of the
nucleus (A-D) and the apical cell surface
(A'-D') is shown to appreciate the differences between
basolateral and apical expression of wild-type versus mutant
chimeric constructs at steady-state levels. Below each
panel, a corresponding Z-scan is shown. The bar
in D' corresponds to 24 µm. E, comparison of
the cytoplasmic tail sequences of mouse SCF with mouse CSF-1. The
basolateral targeting sequences for SCF identified in this study
(acidic cluster and leucine 26) and the functionally conserved leucine
24 in CSF-1 are underlined.
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In Contrast to Wild-type, the Mutant Cytoplasmic Tail of
MgfSl17H SCF Induces Constitutive Endocytosis--
Many
basolateral sorting determinants have been shown to induce endocytosis,
for example the basolateral targeting motif (ML) in the invariant chain
of the major histocompatibility complex II also mediates endocytosis of
the respective proteins (11, 28). Therefore, we tested whether the
wild-type cytoplasmic tails of SCF, expressed as a Tac chimera
(Tac-SCF), are able to internalize Tac-SCF·anti-Tac complexes in
nonpolarized COS-7 cells. Anti-Tac antibodies were bound to transfected
cells in the cold. Subsequently, Tac-SCF·anti-Tac antibody complexes
were allowed to internalize at 37 °C and visualized after acid
removal of cell surface remaining anti-Tac antibodies. Wild-type
Tac-SCF-expressing cells (Fig.
7B) as well as cells
expressing various C-terminal deletions encompassing the basolateral
sorting signal showed a similar low amount of internalized anti-Tac
antibodies (not shown). This suggests that the mono-leucine determinant
in SCF does not induce endocytosis, a finding that is consistent with
the persistent cell surface expression of membrane-bound SCF.

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Fig. 7.
Endocytosis of MgfSl17H mutant
Tac-SCF by a lysosomal targeting signal. Confocal microscopic
sections at the level of the nucleus or apical surface of anti-Tac
antibody-labeled confluent MDCK II stable transfected with different
Tac-SCF constructs (A, D, J) and
Tac-tyrosinase (Tac-tyr, G) are shown. Only a weak staining
of intracellular Tac chimeras is detected in wild-type
Tac-SCF-expressing cells (A). In cells transfected with the
Tac-SCF-17H (D) construct, extensive intracellular vesicular
anti-Tac staining can be observed, which resembled cells transfected
with the Tac-tyr chimera (G). Mutation of the di-leucine of
the putative internalization motif of Tac-SCF-17H to a di-alanine
(17H-LLAA) resulted in a loss of intracellular but led to apical
localization (J). Standard fluorescence microscopy of
endocytosed anti-Tac antibody bound to wild-type and mutant Tac-SCF or
Tac-tyr constructs transiently transfected into COS-7 cells is also
shown. After 30 min at 37 °C, internalized Tac-SCF (or tyr)/anti-Tac
antibodies complexes were visualized with (B, E,
H, K) or without (C, F,
I, L) acid removal of cell surface-bound
noninternalized antibodies. Wild-type Tac-SCF proteins were not
internalized during the 30-min incubation period (B). In
contrast, Tac-SCF-17H mutant proteins accumulated in large
intracellular vesicles (E). Likewise, Tac-tyr constructs
were internalized efficiently (H). However, the di-leucine
mutation in Tac-SCF-17H (17H-LLAA) abolished the capacity to
internalize cell surface-bound anti-Tac antibodies (K).
Comparable levels of the different Tac-SCF constructs were initially
expressed on the COS-7 cell surfaces as illustrated by staining of
parallel cultures from which the anti-Tac antibody was not removed from
the cell surface (C, F, I,
L). The bar in L corresponds to 24 µm.
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In contrast to wild-type SCF, GFP and Tac-SCF chimeras containing the
cytoplasmic tail of the MgfSl17H mutation
accumulated in intracellular vesicular structures (Tac-SCF-17H, Fig. 7D; see also Ref. 8). This intracellular accumulation of the mutant constructs could be the result of retention of newly synthesized chimeric proteins in the endoplasmic reticulum as suggested
by Briley and colleagues (29) or of endocytosis of cell surface SCF. To
determine, whether the intracellular steady-state localization of
Tac-SCF-17H in polarized MDCK II was the result of endocytosis (Fig.
7D), we compared the localization with that of Tac-tyr.
Tyrosinase is a protein that carries an established signal for
endocytosis and lysosomal/melanosomal targeting and is therefore
constitutively internalized from the cell surface (30). Interestingly,
in polarized MDCK II cells, Tac-tyr localized to intracellular
vesicular structures (Fig. 7G), resembling the staining seen
for the Tac-SCF-17H construct (Fig. 7D). Sequence analysis
of the cytoplasmic domain of MgfSl17H
(KYAATERERISRGVIVDVSTLLPSHSGW; Ref. 5) revealed
a sequence homologous to the signal for endocytosis and
lysosomal/melanosomal targeting, identified in tyrosinase, LIMP II and
CD3
(D17XXXLL22) (30-32).
Furthermore, mutation of the leucine residues
(Leu21-Leu22), which are part of this putative
motif in MgfSl17H to alanines, resulted in the
loss of intracellular but led to apical accumulation of Tac-17H-LLAA in
polarized MDCK cells (Fig. 7J). In addition, using the
anti-Tac internalization assay in COS-7 cells, we tested whether the
intracellular localization of the MgfSl17H
mutant was caused by increased endocytosis of surface-expressed MgfSl17H Tac chimeras. Indeed, compared with
wild-type Tac-SCF, significantly more Tac-SCF-17H·anti-Tac complexes
were internalized (Fig. 7E), and a similar intracellular
staining pattern was observed as for the Tac-tyr construct (Fig.
7H). Furthermore, internalization of the Tac-SCF-17H chimera
was blocked by the di-leucine mutation (17H-LLAA; Fig. 7K).
This suggests that the reduced amount of cell surface SCF observed in
the MgfSl17H mutation (8, 33) is caused by
constitutive removal of the MgfSl17H mutant SCF
from the cell surface by endocytosis. Therefore, the MgfSl17H mutation represents a molecular gain of
function mutation with respect to the endocytosis of SCF.
 |
DISCUSSION |
An Acidic Cluster Assists the Leucine-dependent
Basolateral Targeting Signal in SCF--
We identified here a novel
motif in a transmembrane growth factor that is used for basolateral
targeting but not for endocytosis. Both an acidic cluster and a
critical leucine residue are required for efficient basolateral
targeting of SCF. Although the leucine is indispensable for basolateral
transport, the presence of the acidic cluster enhances the efficiency
of basolateral sorting. Because the acidic cluster is not absolutely
required for basolateral targeting, it is unlikely that the two motifs
form a single sorting determinant. Interestingly, a basolateral
targeting motif has been described for the polymeric Ig receptor, which
does not belong to the family of tyrosine or di-leucine determinants
and which does not mediate endocytosis. This critical targeting domain
consists of a single valine located in a
-turn and two critical
residues 3 and 4 amino acids N-terminal to it. Mutation of valine to
alanine reduces basolateral targeting and destabilizes the
-turn
(34). In addition, the amino acids N-terminal to the valine which do not participate in the
-turn are also required for efficient basolateral sorting and form a second, valine-independent, functional targeting domain (35). Based on these similarities, it is possible that
leucine 26 of SCF is part of a
-turn or loop, exposing its hydrophobic side chain in such a way that it could bind to the adaptor
complex of clathrin-coated vesicles. In addition, many di-leucine
sorting motifs have been described which require critical N- or
C-terminally located acidic residues as described for the furin
protease (25), the low density lipoprotein receptor (16) and the
invariant chain of major histocompatibility complex class II (17). In
contrast to these determinants, in which the acidic amino acids are
essential for basolateral targeting, the acidic cluster in SCF is
partially dispensable serving however to increase the fidelity of the
basolateral sorting process. Members of the recently identified
phosphofurin acidic cluster-sorting (PACS) family of adaptor proteins,
which bind to clusters of acidic amino acids, are involved in directing
TGN localization and plasma membrane sorting (18, 19). Interestingly,
intracellular sorting of the furin protease by PACS is regulated by the
phosphorylation of critical serine residues adjacent to a cluster of
acidic amino acids. The same PACS binding, acidic cluster which directs
TGN localization, is also required for basolateral sorting of furin (25). Although PACS may bind to the acidic amino acid cluster in SCF
and thereby increasing the fidelity of the basolateral sorting process
in the TGN, there is no indication that this is a
phosphorylation-dependent interaction involving the
conserved serine residue at position 24. However, in the absence of
such acidic clusters as in the cytoplasmic tail of CSF-1, reduced
protein recognition at the level of the TGN could affect the fidelity of basolateral targeting compared with SCF. Consequently this inefficiency of basolateral sorting might lead to the apical
accumulation of CSF-1 by an N-glycan-dependent
apical targeting pathway (13).
MgfSl17H, a Gain of Function Mutation Leading to
Constitutive Endocytosis of Mutant SCF--
Many basolateral targeting
signals resemble those for coated pit localization and endocytosis. In
contrast to the protease furin or the invariant chain, wild-type SCF is
expressed at the cell surface and is not endocytosed. Interestingly,
the cytoplasmic tail of SCF found in the
MgfSl17H mutation has a high capacity for
inducing endocytosis when expressed as a Tac chimera. Analysis of the
cytoplasmic tail of the MgfSl17H mutant reveal a
match of sequence between
KYAATERERISRGVIVDVSTLLPSHSGW (5), and the
signal for endocytosis or lysosomal/melanosomal/vacuolar targeting
(DXXXLL). This sequence was found in CD3
(DXXXLL) (31) and in related form in the invariant chain
(DDQXXLI; EXXXML) (17, 28, 36), Vam3p
(EXXXLL) (37), LIMP II (EEXXXLL) (32), and
tyrosinase (D/EEXXXLL) (30). In all these proteins, the endocytotic activity is critically dependent on the presence of the
di-leucine motif and is lost after alanine mutagenesis similar to our
observations for the MgfSl17H mutation.
Therefore the MgfSl17H mutation may represent a
gain of function in respect to endocytosis and lysosomal targeting of
SCF. As a consequence, only a limited amount of mutant SCF would be
available on the cell surface to stimulate responsive,
c-Kit-expressing neighboring cells. This could be the cause for
the reduced amount of peripheral SCF-dependent mast cells
and a limited capacity to support hematopoiesis as observed in
MgfSl17H mutant animals (6, 7). In contrast,
based on our results, wild-type SCF lacks a signal for endocytosis, and
this is consistent with the role of the cytoplasmic tail of SCF for
continuous presentation and signaling of the noncleavable form of SCF
toward responsive cells.
The Biological Role of Intracellular Targeting of SCF and Related
Transmembrane Growth Factors--
Our results suggest multiple roles
for the cytoplasmic tail of SCF. First, SCF is targeted to the cell
surface in a polarized fashion, being expressed basolaterally and not
at the apical surface. Second, after reaching the surface, SCF is
retained at the plasma membrane. The first function of the cytoplasmic
tail would be important in cells within polarized tissues, such as
keratinocytes, Sertoli cells, and neurons, whereas the second function
would be relevant to all SCF-expressing cells (Fig.
8). We suggest that the absence of
basolateral delivery of SCF leads to the death of melanocytes and male
germ cells, which normally require basal delivery of SCF from polarized
keratinocytes and Sertoli cells, respectively, as illustrated by the
MgfSl17H mutation (8). In addition to the loss
of pigmentation and fertility, the absence of spatial learning has been
demonstrated in a mouse mutant of SCF (MgfSld),
which lacks the transmembrane and cytoplasmic domain (4). In contrast
to wild-type SCF, such mutant forms of SCF are secreted from the apical
surfaces of polarized epithelia (8). Cell surface expression of SCF in
unpolarized stromal cells of the bone marrow is required for
hematopoiesis (8). Consequently, constitutive endocytosis resulting in
reduced cell surface expression of SCF would lead to a hematopoietic
defect comparable to that of the MgfSl17H mutant
mice (6, 7).

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|
Fig. 8.
Multiple biological effects of cytoplasmic
mutations in SCF. Illustration of the polarized expression of SCF
in basolateral and dendritic aspects of basal keratinocytes
(A), Sertoli cells (C), and neurons
(E), respectively. Cell surface expression of SCF is also
found in nonpolarized stromal cells of the bone marrow or dermal
fibroblasts in the skin (G). Cell surface SCF protein is
represented by gray shading and c-Kit-expressing
(SCF-dependent) cells by dark shading
(A, C, E, and G). The
mutation of the cytoplasmic targeting determinants of SCF leads to
apical or axonal accumulation as well as reduced cell surface
expression (light shading in B, D,
F, and H). Consequently, pigmentation defects
(B), sterility in males (D), and learning
(F) and hematopoietic defects are observed in the respective
tissues (H) (affected cells are indicated by reduced size,
numbers, and gray shading; B, D,
F, and H). Note, dendritic and axonal
localization of wild-type and cytoplasmic mutant SCF protein in neurons
is extrapolated from the polarized expression patterns in epithelial
cells reported in this paper. The loss of spatial learning has so far
only been demonstrated in mice lacking transmembrane and cytoplasmic
sequences of SCF (MgfSld) (4), a mutant form of
SCF which is secreted from apical aspects of polarized epithelia (8).
wt, wild-type tissue; mutant, tissue expressing
cytoplasmic tail mutants of SCF.
|
|
Based on functional similarities and sequence comparison with other
transmembrane growth factors such as CSF-1, we propose that the
basolateral determinant and associated functions are not unique to SCF.
Because of the absence of mouse mutations affecting the cytoplasmic
tail of CSF-1 it is not known whether the role of its cytoplasmic tail
is equally important as that of SCF. In op/op mice that lack CSF-1,
CSF-1-dependent macrophages are absent from epithelial as
well as mesenchymal tissues. Intravenous injection of soluble CSF-1
rescues only mesenchymal macrophages, suggesting a specific requirement
for epithelial derived CSF-1 in promoting the survival of epithelial
macrophages in vivo (38). Moreover, neurological defects
have been reported in op/op mice (39), and accumulation of microglia in
the brain is induced by amyloid-
peptide-stimulated neuronal release
of CSF-1 in Alzheimer's disease (40).
Clearly defined targeting determinants in SCF and other transmembrane
growth factors may offer possibilities for altering polarized
presentation and cell surface expression of these factors. This may
lead to new therapeutic approaches for treatment of pathological conditions such as allergies, chronic inflammation, osteoporosis, or
hyperpigmented lesions caused by overexpression or mutations of these factors.
 |
ACKNOWLEDGEMENTS |
We thank Marie-Claude Jacquier for excellent
technical assistance and Caroline Johnson-Léger, Monique
Wehrle-Haller, Claes Wollheim, Pierre Cosson, and James Weston for
discussions and critical reading of the manuscript. We give special
thanks to Robert Kelsh for suggesting a gain of function mutation as
the cause of the MgfSl17H phenotype. We thank
Willy Hofstetter, Friedrich Beermann, and Karl Matter for
providing cDNAs for CSF-1, tyrosinase, and MDCK II cells.
 |
FOOTNOTES |
*
This work was supported by the Swiss National Science
Foundation Grants 31-52727-97 (to B. W.-H.) and 31-49241-96 (to
B. A. I.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
41-22-702-5735; Fax: 41-22-702-5746; E-mail:
Bernhard.Wehrle-Haller@medecine.unige.ch.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M008357200
 |
ABBREVIATIONS |
The abbreviations used are:
SCF, stem cell
factor;
CSF-1, colony-stimulating factor-1;
TGN, trans-Golgi
network;
GFP, green fluorescent protein;
EGFP, enhanced green
fluorescent protein;
Tac, interleukin-2 receptor
-chain;
MDCK, Madin-Darby canine kidney;
PACS, phosphofurin acidic
cluster-sorting.
 |
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