J. Exp. Med.
Volume 187, Number 9, May 4, 1998 1451–1461
The R ockefeller University Press • 0022-1007/98/05/1451/11 $2.00
Role of Dimerization of the Membrane-associated
Growth Factor Kit Ligand in Juxtacrine Signaling:
T he Sl Mutation Affects Dimerization and
Stability—Phenotypes in Hematopoiesis
By Youichi Tajima,
Eric J. Huang,
Malcolm A.S. Moore, and Peter Besmer
Center , and
Molecular Biology Program and
Sloan-Kettering Division, Graduate School of Medical Sciences , Cornell University, New
Cell Biology Program , Mem orial Sloan-Kettering Cancer
The Kit ligand (KL)/Kit receptor pair functions in hematopoiesis, gametogenesis, and melano-
genesis. KL is encoded at the murine
steel (Sl) locus and encodes a membrane growth factor
which may be proteolytically processed to produce soluble KL. The membrane-associated
form of KL is critical in mediating Kit function in vivo. Evidence for a role of cytoplasmic do-
main sequences of KL comes from the
Sl mutation, a splice site mutation that replaces the
cytoplasmic domain with extraneous amino acids. Using deletion mutants and the
we have investigated the role of the cytoplasmic domain sequences of KL in biosynthetic pro-
cessing and cell surface presentation. The normal KL protein products are processed for cell
surface expression, where they form dimers. Both
of KL were processed to the cell surface; however, the rate of transport and protein stability
were affected by the mutations. Deletion of cytoplasmic domain sequences of KL did not affect
dimerization of KL. In contrast, dimerization of the
addition, we have characterized the hematopoietic cell compartment in
Sl mutation has only minor effects on hematopoiesis. Tissue and peritoneal mast cell
numbers were reduced in mutant mice as well as in myeloid progenitors. Interestingly, long-
term bone marrow cultures from
Sl mice did not sustain the long-term production of he-
matopoietic cells. In addition, homing of normal hematopoietic progenitors to the spleen of ir-
Sl/Sl recipient mice was diminished in transplantation experiments, providing
evidence for a role of Kit in homing or lodging. These results demonstrate that the membrane
forms of KL exist as homodimers on the cell surface and that dimerization may play an impor-
tant role in KL/Kit-mediated juxtacrine signaling.
and the cytoplasmic deletion mutants
protein was reduced substantially. In
number of growth factors that derive from membrane-
anchored precursors and growth factors which function
both as soluble and as membrane growth factors have been
described in the past several years. They include members
of the epidermal growth factor family, e.g., epidermal
growth factor, TGF-
, and c-erb/HER 2 ligand (1), several
hematopoietic growth factors, including the c
CSF-1, IL-1, and TNF (2, 3). Transmembrane
olypeptides which transmit extracellular signals are
most often secreted as soluble factors. An increasing
growth factors have also been identified in the nematode
Caenorhabditis elegans (lin-3) (4) and in
(boss) (5, 6). Several of these transmembrane
growth factors release soluble growth factors by proteolytic
processing or shedding (7–9). The nonprocessed mem-
brane-anchored growth factors themselves possess biologi-
cal activities and promote cell adhesion and juxtacrine
stimulation in adjacent cells expressing cognate receptors
on their cell surface (3). Furthermore, the recently de-
scribed ligands of the eph-family receptor tyrosine kinases
with roles in axon guidance are obligate membrane growth
The protooncogene c
-kit encodes a receptor tyrosine ki-
nase which is a member of the platelet-derived growth fac-
tor receptor subfamily (11). c-
KL, also called steel factor, are encoded at the murine
and its cognate ligand,
BMMC, bone marrow–derived mast cell; CFU-GEMM, CFU-granulo-
cyte erythroid megakaryocyte macrophage; CFU-S, CFU-spleen; endo,
endoglycosidase; ER , endoplasmic reticulum; KL, Kit ligand;
VLA, very late antigen;
W, white-spotting; WBC, white blood cell.
Abbreviations used in this paper:
BFU-E, burst-forming unit–erythroid;
Dimerization of Kit Ligand in Juxtacrine Signaling
The phenotypes of mice with mutations at the
loci imply functions of Kit/KL in germ cell development,
melanogenesis, and hematopoiesis (17–21). In target cells,
KL promotes cell proliferation, survival, cell adhesion/mi-
gration, and differentiation. The
protein products, KL-1 and KL-2, produced by alternative
splicing (2, 22). Both the KL-1 and KL-2 proteins are syn-
thesized as transmembrane proteins and are expressed on
the cell surface (2). Both membrane-bound KL-1 and KL-2
can mediate direct cell–cell contact with c
cells, e.g., bone marrow–derived mast cells (BMMCs) (22,
23) and primordial germ cells (24, 25). Proteolytic process-
ing of both membrane-bound KL-1 and KL-2 releases bio-
logically active soluble KL proteins, although with different
efficiencies and through distinct proteolytic cleavage mech-
anisms (2, 9, 26). Molecular characterization of various
alleles has provided important insight into the role of the
soluble and membrane-anchored forms of KL (14, 15, 22,
27). Homozygotes for the
able and less severely affected, implying some residual func-
tional activity of KL, but they display all of the pleiotropic
effects normally associated with
Molecular analysis showed that the
of an intragenic deletion including the transmembrane do-
main and COOH terminus, generating a secreted KL pro-
tein product with normal biological activity (2, 22, 27).
The biological characteristics of mice carrying the
tation imply that the
Sl KL protein sustains some activity
but is largely defective in facilitating proliferation and sur-
vival of target cells, indicating that the membrane-anchored
forms of KL play pivotal roles in c-
allele contains a splice donor site mutation resulting in the
substitution of amino acids 239–273 in the cytoplasmic do-
main with 27 extraneous amino acids (28). The phenotypes
of mice carrying the
Sl allele include white spotting
with residual pigmentation on the ears and macrocytic ane-
mia (28, 29). Interestingly, the
not female fertility.
The importance of the cell membrane–anchored forms
of KL revealed by the phenotypes of the
the intriguing phenotypes of mice carrying the
raise questions about the role of the cytoplasmic domain se-
quences of KL. Comparison of the cytoplasmic domain se-
quences of murine and human KL reveals 91% shared iden-
tity. In contrast, the extracellular domain sequences of
murine and human KL are 79% identical. The strong con-
servation of the cytoplasmic domain sequences of KL
throughout evolution suggests a function for these se-
quences. The cytoplasmic domain sequences of KL may in-
teract with itself or other molecule(s), and these interac-
tions may play a role in processing to the cell surface, cell
surface presentation, and juxtacrine signaling. We have in-
vestigated the role of cytoplasmic domain sequences of
wild-type KL and the
Sl protein maturation and cell sur-
face presentation. We have also investigated the character-
istics of hematopoietic cells in mice carrying the
) (12, 13) and
ste e l
) loci (14–16), respectively.
gene encodes two KL
) are vi-
mutations (17, 18).
allele arose as a result
allele affects male but
lele. The results obtained in this study are discussed in the
context of mechanisms governing juxtacrine signaling.
Materials and Methods
Construction of KL Deletion Mutants.
the cytoplasmic domain of KL were created by the primer-
directed PCR mutagenesis method. One 5
initiation codon of KL (5
) and several 3
primers containing a termination
codon located at the desired position were used in the PCR reac-
tion. A plasmid containing the KL cDNA clone 19.1.1 (2) was
used as template. The 3
primers used to create the following
mutants were 5
, for KL-L263; 5
, for KL-N254; 5
, for KL-V248; and 5
of the internal deletion mutation, KL-
an M13 phage DNA containing the KL cDNA clone 19.1.1 and
the following oligonucleotides were used: for KL-
. All mutations were confirmed by sequence
analysis. The mutant KL cDNAs were subsequently cloned into
pCDM8 for expression in COS-1 cells.
Transie nt Expre ssio n o f KL c DNAs in COS-1 Ce lls.
were transfected with the DEAE-dextran method described pre-
viously (2, 30) with minor modifications. Briefly, COS-1 cells
were grown to subconfluence 1 d before use, trypsinized, and re-
seeded on 150-mm petri dishes at a density of 6
dish. After 24 h, the cells had reached
transfected with 5
g of plasmid DNA in the presence of 10%
DEAE-dextran (Sigma Chemical Co., St. Louis, MO) for 6–12 h.
The medium containing the plasmid DNA was removed, and the
cells were shocked with 10% DMSO/PBS
DMSO was removed by washing the cells with PBS
Transfected COS-1 cells were grown in DME containing 10%
FCS, 100 mg/ml L-glutamine, and antibiotics.
Pulse Chase and Im m unoprecipitation Analyses of KL Proteins.
At 72 h after transfection, COS-1 cells expressing KL proteins
were used for pulse chase experiments. Cells were incubated with
methionine-free DME containing 10% dialyzed FCS for 30 min
and labeled with
S-methionine (DuPont-NEN, Boston, MA) at
0.5 mCi/ml. At the end of the labeling period, the labeling me-
dium was replaced with regular medium containing an excess
amount of methionine. Cell lysates and supernatants were col-
lected at the indicated times. Cell lysates were prepared as de-
scribed previously (31) in 1% Triton X -100, 20 mM Tris (pH
7.5), 150 mM NaCl, 20 mM EDTA, 10% glycerol, and protease
inhibitors phenylmethyl sulfonyl chloride (1 mM) and leupeptin
g/ml). For immunoprecipitation analysis of KL protein
products, equal volumes of cell lysates or supernatants were im-
munoprecipitated with excess anti-KL antiserum. KL antiserum
was obtained by immunization of rabbits with soluble native and
recombinant murine KL, and both antisera were used with indis-
tinguishable results. The anti-KL serum was conjugated to pro-
tein A–Sepharose (Pharmacia Biotech, Inc., Piscataway, NJ) and
washed three times with wash A (0.1% Triton X -100, 20 mM
Tris [pH 7.5], 150 mM NaCl, 10% glycerol). Anti-KL serum—
protein A–Sepharose conjugate was incubated with supernatants
and cell lysates at 4
C for at least 2 h. The immunoprecipitates
Deletion mutations in
primer covering the
for KL-Q241. For generation
241/254, and KL-
70% confluence and were
for 1 min. R esidual
Tajima et al.
were then washed once in wash B (50 mM Tris, 500 mM NaCl,
5 mM EDTA, 0.2% Triton X-100), three times in wash C (50 mM
Tris, 150 mM NaCl, 0.1% Triton X -100, 0.1% SDS, 5 mM
EDTA), and once in wash D (10 mM Tris, 0.1% Triton X -100).
For gel analysis, immunoprecipitates were solubilized in SDS
sample buffer by boiling for 5 min, and analyzed by SDS-PAGE
(12%) and autoradiography. For endoglycosidase (endo) H treat-
ment, digestion with endo H (Boehringer Mannheim Biochemi-
cals, Indianapolis, IN) was performed by incubating immunopre-
cipitates for 16 h at 37
C with 5 mU of endo H in 50 ?l of 0.1 M
sodium phosphate buffer, pH 6.1, containing 0.1% Triton X -100,
0.03% SDS, and 20 mM EDTA.
Im m unofluorescence Microscopy.
COS-1 cells were trypsinized and reseeded on 12-mm round
coverslips. After 32 h, the cells were fixed for 15 min at 25?C
with 2% (vol/vol) formaldehyde in PBS and rinsed twice with
PBS. Cells were then either used directly for immunofluores-
cence staining or permeabilized by incubating for 15 min at 25?C
with 0.1% (wt/vol) saponin in PBS. For staining, cells were incu-
bated at 25?C for 30 min consecutively with 100 ?l of normal
goat serum, anti-KL serum (1:100 dilution) (32), or an ER
marker antibody recognizing the signal sequence receptor (1:100
dilution) (33) and FITC-conjugated goat anti–rabbit antibody,
sequentially. The unbound antibody was removed by washing in
PBS, and the coverslips were mounted on microscope slides using
Covalent Cross-linking of KL Protein, Im m unoprecipitation, and
Western Blot Analysis. At 72 h after transfection of COS-1 cells
expressing KL proteins, the transfected cells were washed with
HBSS?? and then incubated with 1 mmol/liter bis(sulfosuccin-
imidyl) substrate (BS3) (Pierce, R ockford, IL) for 1 h at 37?C.
The reaction was terminated with 10 mmol/liter Tris-HCl (pH
7.5) for 10 min. Cell lysates were prepared by incubating equiva-
lent amounts of cell lysate with anti–mouse stem cell factor rat
mAb (2 ?g per 107 cells) (Genzyme Corp., Cambridge, MA) for
3 h at 4?C. Protein G–Sepharose beads (Pharmacia Biotech, Inc.)
were used to collect the antigen–antibody complexes. For gel
analysis, immunoprecipitates were solubilized in SDS sample
buffer by boiling for 5 min, and analyzed by SDS-PAGE (10%).
Proteins were electrophoretically transferred onto nitrocellulose
membrane (Bio-R ad Laboratories, Hercules, CA). After transfer,
the membrane was incubated with 4% skim milk/TBST (50
mmol/liter Tris-HCl, pH 7.5, 150 mmol/liter NaCl, 0.05%
Tween 20) for 12 h at room temperature, and Western blot anal-
ysis using anti–mouse stem cell factor polyclonal antibody (1:500
dilution; Genzyme Corp.) and enhanced chemiluminescence de-
tection (Pierce Chemical Co., R ockford, IL) were used for iden-
Mast Cell Adhesion Assay. Transfected COS cells were
grown in 24-well plates (7.5 ? 104 per well). BMMCs, obtained
as described previously (34), were washed three times with R PMI
complete medium and incubated for 1 h with transfected COS cells
(1.5 ? 105 cells per well). Nonadherent BMMCs were then removed
by washing three times with medium, and the BMMCs attached
to the COS cells were counted by visual inspection in the micro-
scope. BMMCs were counted as total number in each well or the
number attached to individual COS cells. To compete the adhesion
of BMMCs to COS cells, the c-kit antagonistic antibody ACK2 (a
gift from Dr. Nishikawa, Kyoto University, Kyoto, Japan) was used.
Analysis of Peripheral Blood Param eters and Hem atopoietic Progeni-
tor Assays. Blood samples for platelet and white blood cell
(WBC) count were drawn from the retroorbital plexus or the tail
vein with a capillary pipette (Unopette; Becton Dickinson Lab-
At 40 h after transfection,
ware, R utherford, NJ). Platelet and WBC numbers were deter-
mined using a hemacytometer and phase–contrast microscopy.
The hematocrit was determined using heparinized microhemat-
ocrit tubes (Fisher Scientific Co., Pittsburgh, PA).
For in vitro progenitor assays, bone marrow was harvested, and
the cellularity was determined as described previously (35). 105
bone marrow cells were resuspended in 1 ml of IMDM/0.8%
methylcellulose (Fisher Scientific Co.)/30% FBS (Hyclone Labo-
ratories, Inc., Logan, UT)/0.2 mM hemin (Sigma Chemical Co.)
containing 50 ng/ml mouse IL-3 (BioSource International, Ca-
marillo, CA), 3 U/ml human erythropoietin (Amgen, Thousand
Oaks, CA), and 20 ng/ml recombinant mouse KL. Cultures were
incubated at 37?C in a 5% CO2 atmosphere. After 7 d of incuba-
tion, colonies were scored on the basis of gross morphology as
erythroid burst (BFU-E), granulocyte/macrophage (CFU-GM),
and mixed colonies (CFU-GEMM).
Splenetic CFU (CFU-S)–day 12 were assessed by injection of
105 donor bone marrow cells suspended in MEM supplemented
with Mops and 10% fetal bovine serum into the lateral tail vein of
lethally irradiated (9.5 Gy) recipient mice. Mice were killed 12 d
later, their spleens were fixed in Bouin’s solution and 10% forma-
lin, and macroscopically visible colonies on the surface of the
spleens were enumerated.
For long-term bone marrow cultures, bone marrow cells from
one femur were flushed into a T-25 flask in Fisher’s medium
containing 20% horse serum and dexamethasone. Cultures were
incubated at 33?C and semidepopulated at weekly intervals.
Determ ination of Mast Cell Num bers in the Skin and Peritoneal
Cavity. For the determination of mast cell numbers in the skin,
pieces of dorsal skin were removed from different parts of the
back, smoothed onto a piece of thick filter paper to keep them
flat, and fixed in Carnoy’s solution. Tissues were embedded in
paraffin, and 5-?m sections were stained with 0.1% acidified
toluidine blue (pH 4.0). In skin sections, mast cells between the
epithelium and the paniculus carnosus were counted under the
microscope. The number of mast cells per centimeter length of
skin was determined by dividing the mast cell number by the
length of each section of skin counted. For each sample, measure-
ments were made in two separate histological sections and aver-
aged to provide the sample mean.
Peritoneal mast cells were obtained from SlKL2/SlKL2 mice and
control mice by lavage of the peritoneal cavity with 5 ml PBS.
Mast cells were identified by staining with 0.1% toluidine blue (34).
Construction of KL-1 Cytoplasm ic Dom ain Variants and
Analysis of Their Turnover Characteristics in COS-1 Cells.
The cytoplasmic domain sequences of KL are highly con-
served in evolution. To study roles for cytoplasmic domain
sequences of KL, COOH-terminal deletion mutations of
varying lengths were constructed (Fig. 1). The wild-type
KL-1 cDNA plasmid was used as a template for mutagene-
sis. The deletion mutations were named according to the
position of the COOH-terminal amino acid, e.g., KL-
L263, KL-N254, KL-V248, and KL-Q241. In addition, an
internal deletion mutation, KL-?241/254, and KL-Sl17H
were constructed. All constructs were subcloned into the
eukaryotic expression vector pCDM8 suitable for transient
expression in COS-1 cells.
The primary KL translation products are progressively
Dimerization of Kit Ligand in Juxtacrine Signaling
processed primarily by carbohydrate modification as they
are transported through the endoplasmic reticulum (ER )
and Golgi compartments to the cell surface (2). Biosyn-
thetic processing of the protein products of the KL cyto-
plasmic domain variants and KL-Sl17H was investigated by
performing pulse chase experiments in transiently trans-
fected COS-1 cells by using 35S-methionine. Immunopre-
cipitation analysis using anti-KL antiserum and SDS-PAGE
indicated that the biosynthetic processing pattern of all cy-
toplasmic domain mutants and KL-Sl17H was similar to that
of wild-type KL-1 (Fig. 2). Smaller molecular mass (Mr)
proteins representing the unglycosylated forms of KL were
synthesized: KL-1 (35,000), KL-L263 (33,000), KL-N254
(31,000), KL-V248 (33,000, not shown), KL-?241/254
and KL-Sl17H (33,000), and KL-Q241 (30,000). These pro-
teins were progressively modified by glycosylation to form
more mature products representing the cell surface forms.
The respective Mr for the mature proteins were as follows:
KL-1, 45,000 daltons; KL-L263, 43,000 daltons; KL-
N254, 41,000 daltons; KL-V248 (42,000 daltons, not
shown), KL-?241/254 and KL-Sl17H (43,000 daltons); and
KL-Q241 (40,000 daltons) (Fig. 2). Previous cell surface
I125-iodination experiments have shown that the 45-kD
KL-1 protein represented the mature KL-1 protein on the
cell membrane (2). Quantitation of the different KL pro-
tein products during the chase indicated that after 15 min,
?50% of the immature 35-kD KL-1 protein was processed
to the mature 45-kD form. With KL-Sl17H, this process was
somewhat delayed, in that 30–45 min was required for 50%
of the smaller 33,000-Mr protein products to be processed
to their higher 43,000-Mr forms. KL-L263, KL-N254, KL-
V248, and KL-?241/254 were also delayed in their matu-
ration to a similar extent as KL-Sl17H. KL-Q241 displayed
the most striking delay of maturation, as only a small frac-
tion of the KL-Q241 protein products was processed to the
mature 40-kD protein. In agreement with these findings,
stromal cells derived from homozygous Sl17H/Sl17H mice
express a reproducibly reduced level of cell surface KL
(?60% of normal levels) as determined by FACS analysis
compared with the low levels of cell surface KL seen in
normal controls (Fig. 3).
We then determined whether the wild-type KL-1 and
the mutant KL protein products follow the same matura-
tion process through the ER and the Golgi complex. Upon
reaching the medial Golgi complex, N-acetylglucosamine
is added to glycoproteins, rendering the carbohydrates re-
sistant to the cleavage by endo H (36, 37). To characterize
the maturation pathway of mutant KL proteins, we used
sensitivity to endo H treatment. COS-1 cells transfected with
various constructs were pulse labeled with 35S-methionine
and then chased in the presence of excess nonradioactive
methionine for 0, 1, and 2 h, and KL protein products
were immunoprecipitated from cell lysates and digested
with endo H. The wild-type KL-1 protein became resistant
to endo H treatment at 1 and 2 h of chase, consistent with
rapid transit through the ER Golgi complex. The KL-L263
and KL-N254 protein products displayed a slight persis-
tence of endo H sensitivity at 1 h of chase compared with
KL-1, indicating that passage of these proteins through the
Golgi complex was delayed (Fig. 4 A). This is consistent
with the delayed maturation of these proteins to their cell
surface forms. Most of the KL-Q241 protein remained sen-
sitive to endo H treatment at 1 h of chase, indicating that
amino acid sequence of KL-Sl17H. SP, Signal peptide. ECD, Extracellular
domain. THS, Transmembrane segment. CD, Cytoplasmic domain.
Schematic representation of KL cytoplasmic variants and
variants and KL-Sl17H. COS-1 cells were transfected with various mutant
constructs, KL-1, KL-L263, KL-N254, KL-?241/254, KL-Sl17H, and
KL-Q241, labeled with 35S-methionine for 30 min, and chased in regular
medium. Cell lysates were collected at designated time points, immuno-
precipitated with anti-KL antibody, and analyzed by SDS-PAGE (12%).
Arrowheads, Mature glycosylated KL proteins. *Immature forms. Molecu-
lar mass (Mr) markers are indicated in kilodaltons.
Biosynthetic characteristics of KL-1 cytoplasmic domain
Tajima et al.
the protein was not properly transiting the Golgi complex.
A more thorough time course comparing KL-1 and KL-
Sl17H showed that at 45 and 60 min of chase, KL-Sl17H dis-
played some persistence of endo H sensitivity, whereas KL-1
had become fully resistant (Fig. 4 B). Thus, KL-Sl17H is de-
layed in exiting the ER . Immunofluorescence staining of
KL-1 expressing COS-1 cells revealed a pattern character-
istic of a membrane protein which outlined the shape of
the cytoplasm (Fig. 5). The KL-Sl17H staining pattern was
indistinguishable from that of KL-1. KL-Q241 staining was
observed at the cell surface as well, but appeared less in-
tense than KL-1 staining. Upon permeabilization, staining
patterns of all KL-expressing cells included a reticular pat-
tern surrounding the nucleus. R elative to cell surface stain-
ing, KL-Q241 immunofluorescence was more pronounced
in this juxtanuclear region, and this intracellular staining
pattern appears to overlap with that of an E.R . marker an-
tibody. Therefore, these results are consistent with the idea
that the delay in maturation of the KL-Q241 protein and
to a lesser extent of the SL17H protein is a consequence of a
delay in passage through a pre-Golgi compartment.
Efficient Form ation of Hom odim ers of Cell Mem brane–associ-
ated KL and Cytoplasm ic Dom ain Deletion Variants, and Di-
m inishe d Fo rm atio n o f Dim e rs o f the Sl17H Pro te in.
tors and cytokines commonly activate their receptors by
mediating receptor dimerization. R ecent studies (see refer-
ences 50 and 51) with the Kit receptor suggest that bivalent
binding of KL dimers is driving Kit receptor dimerization.
Soluble KL is predominantly in a dimeric state. However,
studies of KL dimerization kinetics imply that in vivo, 90%
of KL circulating in serum exists as monomers. It is not
known if the membrane-associated KL protein on the cell
surface is monomeric or dimeric. To address this question,
we set out to identify KL dimers by using chemical cross-
linking methodology. COS-1 cells transiently transfected to
express KL-1 and KL-2 were treated with a hydrophilic and
a hydrophobic cross-linker (DSS and BS3), cell lysates were
then fractionated by SDS-PAGE, and KL protein products
were identified by Western blotting (Fig. 6). Both cross-
linkers efficiently cross-linked the normal KL-1 and KL-2
transmembrane proteins to produce dimeric molecules of
80–85 kD (Fig. 6). These results suggest that a major frac-
tion of the cell-associated KL-1 and KL-2 molecules are in
a dimeric form. It was next of interest to determine
whether the cytoplasmic domain sequences of KL and/or
the Sl17H cytoplasmic domain sequences affect the formation
of cell-associated KL dimers. Whereas deletion of cytoplas-
mic domain sequences did not affect KL dimer formation
as determined by cross-linking (Fig. 6), importantly, Sl17H
dimer formation was diminished significantly (Fig. 7), and
removal of the Sl17H cytoplasmic domain sequences by de-
letion restored the ability to form dimers (Fig. 7).
Cell Adhesion Properties of KL Cytoplasm ic Dom ain Vari-
ants.In an earlier study, Cheng and Flanagan found a sig-
nificant reduction in binding of a Kit ectodomain alkaline
phosphatase fusion protein to COS cells expressing the KL
Sl17H protein compared with COS cells expressing the nor-
mal KL protein (26). The reduction in KL Sl17H dimeriza-
tion we observed in COS cells may contribute to the re-
duced binding of Kit ectodomain. KL/Kit interactions may
also be important in cell–cell adhesion. Therefore, we ex-
plored the functional consequences of the cytoplasmic do-
main deletion and Sl17H mutations on cell–cell adhesion.
cell surface expression of KL in
Sl17H/Sl17H fibroblasts by FACS.
of KL-1, KL cytoplasmic domain
variants, and KL-Sl17H. COS-1
cells were transfected with differ-
ent KL constructs and labeled
with 35S-methionine. Cell lysates
collected at different time points
were immunoprecipitated and
treated in the presence or ab-
sence of endo H. The reaction
products were analyzed by SDS-
PAGE (12%). Arrowheads, Imma-
ture KL protein products.
Endo H treatment
Dimerization of Kit Ligand in Juxtacrine Signaling
Cell adhesion between kit receptor–expressing mast cells
and KL-expressing COS-1 cells or fibroblasts had been
demonstrated previously (22, 23). In 24-well dishes, BMMCs
(1.5 ? 105 cells per well) were plated on top of COS-1–
expressing KL-1, KL-N254, KL-?241/254, KL-Q241,
and KL-Sl17H. 72 h after transfection, 1.5 ? 105 BMMCs
were plated per well. After an incubation period of 1 h at
37?C, nonattached BMMCs were removed, and adherent
BMMCs were counted by visual inspection in a micro-
scope. COS cells expressing KL-1, KL-N254, and KL-
?241/254 had equivalent numbers of BMMCs attached to
the cell surface, and attachment was inhibited completely
by the antagonistic anti–c-kit mAb ACK2 (38) (Fig. 8).
Adhesion of BMMCs to COS-1 cells expressing KL-Q241
was reduced dramatically (Fig. 8). Similar numbers of BMMCs
attached to COS-1 cells transfected with either KL-Q241,
the secretory KL-1S (2), or a control plasmid. Interestingly,
the number of BMMCs attached to COS cells expressing
KL-Sl17H was only about half of that attached to COS cells
expressing KL-1 (Fig. 8).
Effects of the Sl17H Mutation on Hem atopoiesis In Vivo and In
Vitro. Previous analysis of mutant Sl17H/Sl17H mice sug-
gested mild effects in the hematopoietic system, i.e., a mild
macrocytic anemia was observed (29). We have reexam-
ined the effects of the Sl17H mutation on the hematopoietic
system in vivo as well as in vitro. Careful analysis of the in
vivo hematopoietic parameters indicates that hematocrit
values, WBC, neutrophil, and platelet numbers, as well as
bone marrow cellularity are normal in homozygous mutant
mice (Table 1). In contrast, the numbers of skin mast cells
were reduced by 45% and peritoneal mast cells by 85%, in-
dicating an effect of the Sl17H mutation on mast cell devel-
opment and/or number (Table 1). In agreement with a
lack of an erythroid deficiency, in vitro analysis of hemato-
poietic progenitors revealed normal numbers of BFU-E.
However, mixed colonies (CFU-GEMM) and CFU-GM
were reduced slightly, by ?33%. We also established long
term bone marrow cultures and followed output of he-
COS-1 cells expressing wild-type and mutant KL protein products. COS-1
cells transfected with KL-1, KL-Q241, and KL-Sl17H grown on coverslips
were fixed with 2% formaldehyde (NP, nonpermeabilized control) and
permeabilized with 0.1% saponin (P). The cells were incubated with anti-
KL antibody or an ER marker antibody and FITC-conjugated goat anti–
rabbit antibody. Photographs were taken with fluorescent microscopy.
KL protein expression detected by immunofluorescence in
cells. COS-1 cells were transfected with various normal and mutant KL-1
and KL-2 constructs: (A) KL-1, KL-2, and KL-S; (B) KL-1: L263, V248,
?241/254; KL-2: L263, N254, Q241. 72 h after transfection, they were
treated with the cross-linkers DSS and BS3 for 1 h at 37?C. Cell lysates
were collected and fractionated by SDS-PAGE (12%), and KL protein
products were identified by Western blotting. Arrowheads, KL dimers.
*Monomers. Molecular mass (Mr) markers are indicated in kilodaltons.
Demonstration of KL-1, KL-2 dimers in transfected COS-1
face. COS-1 cells were transfected with normal KL-1 constructs and mu-
tant Sl17H constructs containing various COOH-terminal deletions: Sl17H-
T257, Sl17H-R 249, and Sl17H-A240. 72 h after transfection, cell surface
proteins were biotinylated, and cells were treated with the cross-linkers
DSS and BS3 (as indicated) for 1 h at 37?C. Cell lysates were fractionated
on streptavidin-sepharose columns, the biotinylated fraction was electro-
phoretically fractionated by SDS-PAGE (12%,) and KL protein products
were identified by Western blotting. Arrowheads, KL dimers. *Monomers.
Molecular mass (Mr) markers are indicated in kilodaltons.
The Sl17H mutation affects dimer formation on the cell sur-
Tajima et al.
matopoietic cells at weekly intervals. These experiments
showed that cultures from Sl17H/Sl17H bone marrow were
not able to sustain long-term production of hematopoietic
cells (Fig. 9). This result is reminiscent of the defect ob-
served in cultures derived from bone marrow of Sl/Sld
mice, which produce only the soluble form of KL and no
Kit-mediated adhesion of hematopoietic cells is known
to be mediated by at least two distinct mechanisms: (a)
tethering via the membrane growth factor–receptor inter-
action, and/or (b) by inside-out activation of integrin-
mediated adhesion (very late antigen [VLA]-4 and VLA-5)
(22, 23, 39, 40). Furthermore, it has been established that
VLA-4–mediated adhesion is critical for homing or lodging
of early hematopoietic progenitors to the bone marrow in
transplantation experiments (41, 42). Therefore, we deter-
mined whether Sl17H/Sl17H mice were suitable recipients in
spleen colony assays using normal donor bone marrow. In-
terestingly, we observed a reduced number of spleen colo-
nies (65% of normal) in Sl17H/Sl17H recipient mice (Table
2). This may suggest that the Sl17H mutation impairs lodg-
ing/homing of transplanted CFU-S progenitors to the
spleen of irradiated mice.
The Sld allele arose as a result of an intragenic deletion
including the transmembrane domain and COOH termi-
nus, generating a secreted KL protein product with normal
biological activity (2, 22, 27). Analysis of the Sld phenotype
has been of great value in understanding the differential bi-
ological roles of membrane-associated and soluble forms of
KL. The biological characteristics of homozygous Sld/Sld
mice and of Sl/Sld mice indicate that the Sld protein sup-
ports some level of KL function. However, Sld mice display
major defects in facilitating proliferation and survival of tar-
tory KL-1S, different KL cytoplasmic variants, and KL-Sl17H. COS-1 cells
were transferred to 24-well plates 24 h after transfection at a density of 7.5 ?
104 cells per well. BMMCs (1.5 ? 105 cells per well) were incubated with
transfected COS-1 cells for 1 h in the presence and absence of anti–c-kit
antibody. Nonattached BMMCs were washed away three times with me-
dium, and the number of attached BMMCs was counted by microscopic
inspection. The number of BMMCs attached to individual COS cells is
Adhesion of BMMCs to COS-1 cells expressing KL-1, secre-
mice. Nonadherent cells were counted at weekly intervals. R esults from
three separate experiments are shown.
Long-term culture of bone marrow cells from Sl17H/Sl17H
T able 1. Mast Cells and Hem atopoietic Progenitors in Sl17H/Sl17H and C3H Control Mice
Genotype Skin mast cells Peritoneal mast cells
cellularity CFU-GEMMCFU-GM BFU-E
162.5 ? 4.549.5 ? 9.9 11.54 ? 0.77 2,626 ? 335
770 ? 191
29,003 ? 2,871
9,717 ? 1,215
1,955 ? 430
2,677 ? 204
91.6 ? 12.88.0 ? 1.6 12.42 ? 1.671,634 ? 608
583 ? 210
19,923 ? 3,163
10,266 ? 1,811
1,925 ? 515
2,391 ? 657
Dimerization of Kit Ligand in Juxtacrine Signaling
get cells. Therefore, the cell-associated form of KL plays a
critical role in c-kit function, and the cytoplasmic domain
of KL is potentially important to the processes mediated by
juxtacrine signaling. This notion is supported by the mu-
tant phenotypes of the Sl17H allele, a splice site mutation
that results in the substitution of amino acids 239–273 in
the KL cytoplasmic domain with 27 extraneous amino ac-
ids (28). Therefore, the Sl17H allele provided an opportu-
nity to analyze the in vitro and in vivo consequences of cy-
toplasmic domain modification.
The cytoplasmic domain of KL is highly conserved in
evolution (Fig. 1), yet very little is known about its func-
tion. We have attempted to elucidate the roles of KL cyto-
plasmic domain sequences as they relate to biosynthetic
processing, cell adhesion, and juxtacrine signaling by using
in vitro and in vivo genetic approaches. The major conclu-
sions of our study are (a) that cytoplasmic domain se-
quences are important for biosynthetic processing of KL
through the ER and Golgi complex and to the cell surface;
(b) that the membrane forms of KL exist as homodimers on
the cell surface and that dimerization may be an essential
step in KL/Kit-mediated juxtacrine signaling; and (c) that
analysis of in vivo phenotypes of Sl17H/Sl17H mice revealed
Kit-dependent processes in hematopoiesis in which mem-
brane KL is limiting, and they suggest a role for Kit in
homing of hematopoietic progenitors to spleen.
Our findings that the KL cytoplasmic domain is required
for normal processing to the cell surface are consistent with
reports on a variety of secreted or membrane-anchored
proteins in which cytoplasmic domain mutations disrupted
intracellular trafficking and maturation. For example, mu-
tations removing the four COOH-terminal cytoplasmic
residues of ?-1 proteinase inhibitor or the COOH-termi-
nal 22 amino acids of thyroxine-binding globulin caused
nascent protein to be retained in the ER with resultant lack
of secretion (43, 44). Additionally, single point mutations
of glycines in the cytoplasmic domain of P-glycoprotein or
a single point mutation in the cytoplasmic kinase domain of
the Kit receptor caused these proteins which are normally
membrane-anchored to be inefficiently glycosylated and
retained in the ER (45, 46). Cytoplasmic domain mutant
proteins may not interact properly with molecules involved
with transport from the ER to the cell surface. Also, incor-
rect folding as a consequence of cytoplasmic domain muta-
tions may be responsible for the maturation defects (47).
Soluble KL forms noncovalent dimers in solution (15,
48, 49), and it is thought that dimerization of the ligand as
well as of the receptor are essential steps for receptor activa-
tion. Therefore, it seemed reasonable that juxtacrine signal-
ing by cell-associated forms of KL may involve membrane
KL dimers. Our finding of efficient dimer formation of
cell-associated KL-1 and KL-2 molecules on transfected
COS cells supports this conjecture. Importantly, the appar-
ent large fraction of KL dimers on the cell surface in the
absence of cognate receptor implies that dimer formation
may not be a rate-limiting event in juxtacrine signaling be-
tween KL and Kit. This is in contrast to receptor tyrosine
kinases such as Kit which primarily exist as monomers on
the cell surface and which form dimers in response to en-
gagement with the cognate ligand (50, 51). Our analysis of
various cytoplasmic domain deletion mutations suggests
that the cytoplasmic domain of KL does not have an active
role in dimer formation. The decreased efficiency of dimer
formation of the SL17H protein suggests that the nonsense
cytoplasmic domain sequence of SL17H prevents normal KL
dimerization (possibly by steric hindrance), and this may
contribute to the phenotypic defects of SL17H mice.
Cell adhesion assays indicated that Kit-mediated attach-
ment of BMMCs to COS cells expressing KL-Sl17H was
?50% of the KL-1 control. This was in contrast to KL-
N254 and KL-?241-254, where no significant reduction in
adhesion was seen. Interestingly, while a similar delay in
the processing of Sl17H, KL-N254, and KL-?241-254 to
the cell surface is observed, Sl17H specifically displays re-
duced cell surface dimerization. This may suggest that the
adhesion defect of Sl17H is a consequence of reduced Sl17H
cell surface dimer formation. Therefore, the KL-Sl17H pro-
tein is expressed on the cell surface, but the abnormal cyto-
plasmic domain sequences appeared to interfere with mem-
brane KL–mediated adhesion of BMMCs and juxtacrine
The Sl17H mutation affects melanogenesis and gametoge-
nesis (28, 29). A mild effect on hematopoiesis of Sl17H has
also been reported. We have now investigated the hemato-
poietic parameters in Sl17H/Sl17H mice in detail. Although
bone marrow cellularity, hematocrit, and WBC, neutro-
phil, and platelet counts appeared to be normal, the num-
ber of tissue mast cells was appreciably affected by the Sl17H
mutation. In addition, hematopoietic progenitor numbers
in the bone marrow of Sl17H/Sl17H mice did not deviate sig-
nificantly from normal controls, although slightly reduced
numbers of mixed colonies (CFU-GEMM) and slightly in-
creased numbers of CFU-S were observed. Evidence that
the hematopoietic microenvironment is compromised in
Sl17H/Sl17H mice comes from two different experiments.
First, reciprocal transplantation of normal bone marrow
into Sl17H/Sl17H and normal mice revealed a reduced num-
ber of spleen colonies in the Sl17H/Sl17H recipients, suggest-
ing that homing or lodging to the spleen is affected by the
mutation. Second, the bone marrow from Sl17H/Sl17H mice
T able 2.
Sl17H/Sl17H and Control C3H Mice into Irradiated Control and
Reciprocal Transplantation of Bone Marrow from
Donor R ecipientCFU-S/105 cells
25.3 ? 2.1
36.9 ? 2.0 P ? 0.0008
25.1 ? 1.7
16.5 ? 1.0 P ? 0.0024
Tajima et al.
does not support long-term culture of hematopoietic pro-
genitors. The biochemical defects we observed for the
Sl17H protein, including delayed processing and reduced
dimer formation, may contribute to these hematopoietic
deficiencies. The observation that homing or lodging of
progenitors to the spleen of irradiated mice is affected is of
great interest. KL had been shown to mediate adhesion
processes in hematopoietic cells and mast cells by means of
an inside-out activation of the integrins VLA-4 and VLA-5
(39, 40, 52, 53). Also, VLA-4–mediated adhesion has been
demonstrated to play a role in homing or lodging of he-
matopoietic progenitors to the spleen (41). In addition,
tethering via the membrane growth factor–receptor inter-
action is thought to provide a mechanism for KL/Kit-
mediated cell–cell adhesion (2, 22). Although our results
provide evidence that supports a role for KL/Kit in homing
or lodging of hematopoietic progenitors to the spleen pos-
sibly by an integrin-mediated mechanism, a role for KL/
Kit in homing to the bone marrow as suggested by Papay-
annopoulou and Craddock (54) remains to be investigated.
The number of germ cells in neonatal gonads is reduced
similarly in male and female Sl17H/Sl17H mice as a result of
an effect on primordial germ cells; however, the Sl17H allele
affects male but not female fertility (28, 55). In postnatal
spermatogenesis, KL-2 is more abundant than KL-1, whereas
in oogenesis, KL-1 and KL-2 are equally abundant (2, 32).
Since the KL-2 protein is more resistant to proteolytic pro-
cessing and therefore is more stable on the cell surface, the
preferred expression of KL-2 in testis development implies
a more selective role for transmembrane KL in spermatogen-
esis. Therefore, the defect in dimerization of Sl17H would
more severely affect spermatogenesis and may in part ac-
count for the sterility of Sl17H males, while females remain
fertile. In addition, immunohistochemical studies showed
that the transmembrane KL protein accumulates in the ba-
solateral region of the seminal epithelium in Sertoli cells at
the time when c-kit–expressing spermatogonia begin to
mature (32). KL-expressing Sertoli cells supporting KL/c-
kit–dependent spermatogonial proliferation have the mor-
phology of a polarized epithelium. Possibly, the cytoplas-
mic domain of KL may direct sorting of this protein to the
basolateral region of Sertoli cells and/or affect its stability in
the basolateral region of these cells. In the adult ovary, fol-
licle cells surrounding the oocyte are less polarized than
Sertoli cells. Therefore, although a 50% decrease in Sl17H
protein may be tolerated in oogenesis, misdirected expres-
sion of KL-Sl17H in polarized Sertoli cells may affect sper-
matogonial proliferation and survival and result in male ste-
We would like to thank Harry Satterwhite and Maureen Sullivan for expert assistance with hematological as-
says, and Dr. Jeffrey R avetch for the use of his cell sorter and for helpful discussions. We would also like to
thank Drs. Martin Wiedmann, Marcus Bosenberg, Atanasio Pandiella, Joan Massague, and R osemary Bach-
varova for many discussions, and Dr. Shin-ichi Nishikawa for generously providing ACK2 antibody. ER
marker–specific antibody was generously provided by Tom R apoport (Harvard Medical School, Boston,
MA) and Martin Wiedmann (Memorial Sloan-Kettering Cancer Center, New York).
Support by grants from the American Cancer Society and the National Institutes of Health is acknowledged.
Address correspondence to Peter Besmer, Memorial Sloan-Kettering Cancer Center, 275 York Ave., New
York 10021. Phone: 212-639-8188; Fax: 212-717-3623; E-mail: email@example.com E.J. Huang’s
current address is the Pathology Department, University of California San Francisco Medical School, San
Received for publication 21 Novem ber 1997 and in revised form 9 February 1998.
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